U.S. patent application number 13/685327 was filed with the patent office on 2013-07-25 for methods and compositions for promoting renal repair and regeneration.
This patent application is currently assigned to KINTAN PTY LTD.. The applicant listed for this patent is Kintan Pty Ltd.. Invention is credited to David Arthur Hume, Christina Victoria Jones, Melissa Helen Little, Sharon Denise Ricardo.
Application Number | 20130189223 13/685327 |
Document ID | / |
Family ID | 39584860 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130189223 |
Kind Code |
A1 |
Ricardo; Sharon Denise ; et
al. |
July 25, 2013 |
Methods and Compositions for Promoting Renal Repair and
Regeneration
Abstract
Methods and compositions comprising CSF-1 are provided for
regenerating, repairing or otherwise treating renal cells, tissues
and/or organs, and more particularly, in prophylactic or
therapeutic treatment of diseases or conditions associated with
renal damage and/or dysfunction. In particular, CSF-1 may be
particularly efficacious in treating acute renal failure. CSF-1
protein or an encoding nucleic acid may be administered to
suppress, ameliorate or otherwise treat an existing renal disease
or condition or to prevent, inhibit, suppress or otherwise protect
against subsequent renal damage and/or renal failure. CSF-1 may
also be used to regenerate or repair renal cells, tissue and/or
organs ex vivo, the treated cells, tissue and/or organs then being
suitable for subsequent transplantation to an animal, such as a
human. Compositions and methods are provided for promoting organ
development in warm blooded animals, and in particular in certain
aspects a premature infant or foetus. Compositions and methods are
also provided for the administration of at least one colony
stimulating factor-1 protein (CSF-1), precursor, variant, analogue,
derivative thereof, or combinations thereof, or otherwise, at least
one nucleic acid molecule encoding colony stimulating factor-1
protein (CSF-1), precursor, variant, analogue, derivative thereof,
or combinations thereof.
Inventors: |
Ricardo; Sharon Denise;
(Mornington, AU) ; Hume; David Arthur; (Roslin,
GB) ; Little; Melissa Helen; (Brisbane, AU) ;
Jones; Christina Victoria; (Wheelers Hill, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kintan Pty Ltd.; |
South Yarra |
|
AU |
|
|
Assignee: |
KINTAN PTY LTD.
South Yarra
AU
|
Family ID: |
39584860 |
Appl. No.: |
13/685327 |
Filed: |
November 26, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13236177 |
Sep 19, 2011 |
8338370 |
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13685327 |
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11902062 |
Sep 18, 2007 |
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13236177 |
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PCT/AU2007/001372 |
Sep 17, 2007 |
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11902062 |
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PCT/AU2006/000357 |
Mar 17, 2006 |
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PCT/AU2007/001372 |
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Current U.S.
Class: |
424/85.1 ;
424/93.7; 514/44R; 530/395 |
Current CPC
Class: |
A61K 35/12 20130101;
A61P 1/00 20180101; A61P 11/00 20180101; A61P 19/00 20180101; A61K
31/70 20130101; A61K 31/7088 20130101; A61K 38/18 20130101; A61P
13/12 20180101; A61K 38/193 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/85.1 ;
514/44.R; 424/93.7; 530/395 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 35/12 20060101 A61K035/12; A61K 31/7088 20060101
A61K031/7088 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2005 |
AU |
2005901346 |
Sep 15, 2006 |
AU |
2006905099 |
Sep 18, 2006 |
AU |
2006905156 |
Claims
1-17. (canceled)
18. A method of treating an existing renal disease or condition in
an animal, comprising administering CSF-1 protein, or an encoding
nucleic acid, to an animal in need of such treatment, to thereby
suppress, ameliorate or otherwise treat said existing renal disease
or condition.
19. The method of claim 18, wherein the CSF-1 protein is
bacterially expressed, non-glycosylated human recombinant
CSF-1.
20. The method of claim 19, wherein the CSF-1 protein is a
C-terminal 150 amino acid fragment of CSF-1 protein.
21. The method of claim 18, wherein the animal is a human.
22. The method of claim 18, wherein the existing renal disease or
condition is acute renal failure.
23. A method of prophylactically treating a renal disease or
condition in an animal including the step of administering CSF-1
protein, or an encoding nucleic acid, to an animal in need of such
treatment, in the absence of a therapeutically effective amount of
G-CSF, to thereby prevent, inhibit, suppress or otherwise protect
against subsequent renal damage and/or renal failure.
24. The method of claim 23, wherein the CSF-1 protein is
bacterially expressed, non-glycosylated human recombinant
CSF-1.
25. The method of claim 24, wherein the CSF-1 protein is a
C-terminal 150 amino acid fragment of CSF-1 protein.
26. The method of claim 23, wherein the animal is a human.
27. The method of claim 23, wherein the renal disease or condition
is acute renal failure.
28. A method of regenerating or repairing renal tissue in an animal
including the step of administering a CSF-1 protein, or an encoding
nucleic acid, to an animal in need of such treatment, to thereby
regenerate or repair renal tissue in said animal.
29. The method of claim 28, wherein the CSF-1 protein is
bacterially expressed, non-glycosylated human recombinant
CSF-1.
30. The method of claim 29, wherein the CSF-1 protein is a
C-terminal 150 amino acid fragment of CSF-1 protein.
31. The method of claim 28, wherein the animal is a human.
32. A method of regenerating, repairing or otherwise treating renal
tissue ex vivo including the step of exposing one or more isolated
renal cells, tissues or organs to a CSF-1 protein to thereby effect
proliferation and/or regeneration of said cells, tissues or
organs.
33. The method of claim 32, wherein the CSF-1 protein is
bacterially expressed, non-glycosylated human recombinant
CSF-1.
34. The method of claim 32, wherein the CSF-1 protein is a
C-terminal 150 amino acid fragment of CSF-1 protein.
35. The method of claim 32, wherein the renal tissue is human,
renal tissue.
36. A method of renal transplantation, including the step of
administering to an animal one or more renal cells, tissues or
organs exposed ex vivo to a CSF-1 protein or encoding nucleic acid
according to claim 32.
37. The method of claim 36, wherein the CSF-1 protein is
bacterially expressed, non-glycosylated human recombinant
CSF-1.
38. The method of claim 37, wherein the CSF-1 protein is a
C-terminal 150 amino acid fragment of CSF-1 protein.
39. The method of claim 36, wherein the animal is a human.
40. A pharmaceutical composition for use in treating an existing
renal disease or condition, said pharmaceutical composition
comprising a CSF-1 protein or an encoding nucleic acid and a
pharmaceutically acceptable carrier, diluent or excipient.
41. A pharmaceutical composition for use in regenerating or
repairing human renal tissue, said pharmaceutical composition
comprising a CSF-1 protein or an encoding nucleic acid and a
pharmaceutically acceptable carrier, diluent or excipient.
42. A pharmaceutical composition for use in prophylactically
treating a renal disease or condition, said pharmaceutical
composition comprising a CSF-1 protein or an encoding nucleic acid
and a pharmaceutically acceptable carrier, diluent or excipient, in
the absence of a therapeutically effective amount of G-CSF.
43. The pharmaceutical composition of any one of claims 40-42,
wherein the CSF-1 protein is bacterially expressed,
non-glycosylated human recombinant CSF-1.
44. The pharmaceutical composition of claim 43, wherein the CSF-1
protein is a C-terminal 150 amino acid fragment of CSF-1
protein.
45. The pharmaceutical composition of claim 41, for regenerating or
repairing human renal tissue in vivo.
46. The pharmaceutical composition of claim 41, for regenerating or
repairing human renal tissue ex vivo.
47. Use of CSF-1 in the manufacture of a medicament for treating an
existing renal disease or condition in an animal.
48. Use of CSF-1 in the manufacture of a medicament for
prophylactically or therapeutically treating acute renal failure in
an animal.
49. Use according to claim 47 or claim 48, wherein the animal is a
human.
50. Use according to claim 49, wherein CSF-1 is native CSF-1
obtained from human urine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/236,177, filed Sep. 19, 2011, which is a continuation of
U.S. application Ser. No. 11/902,062 filed Sep. 18, 2007, which is
a continuation-in-part of a PCT application filed in the Australian
Receiving Office on Sep. 17, 2007, (which claims priority to
Australian Provisional Application No. AU 2006905099, filed on Sep.
15, 2006, entitled "Method For Promoting Organ Development" and
Australian Provisional Application No. AU 2006905156, filed Sep.
18, 2006, entitled "Method For Promoting Organ Development"), and
is also a continuation-in-part of International Patent Application
No. PCT/AU2006/000357, filed Mar. 17, 2006, entitled "Renal Repair
and Regeneration" (which claims priority to Australian Provisional
Application No. AU 2005901346, filed Mar. 18, 2005, entitled "Renal
Repair and Regeneration"). Each of these documents, including those
in parenthesis, are incorporated herein by reference in its
entirety. In addition, each of the following documents are
incorporated herein by reference in its entirety: [0002] Bertram J
F (1995) Analyzing renal glomeruli with the new stereology;
International Review of Cytology; 161: 111-172. [0003] Dressier, G.
R. (2002). Development of the Excretory System. Mouse
Development--Patterning, Morphogenesis and Organogenesis. [0004] J.
Rossant and P. P. L. Tam. Houston, Academic Press: 395; Goldenring
J (2004). Respiratory Distress Syndrome in Infants. MedlinePlus
Medical Encyclopedia. [Available at
http://wwvv.nlm.nih.gov/medlineplus/ency/article/001563.htm].
[0005] Hayashi M. Zhu K. Sagesaka T. Fukasawa I. Inaba N. Elevation
of amniotic fluid macrophage colony-stimulating factor in
normotensive pregnancies that delivered small-for-gestational-age
infants. American Journal of Reproductive Immunology. 57(6):488-94,
2007 June [0006] Hinchliffe, S., Sargent, P., et al. (1991). "The
effect of intrauterine growth expressed in absolute number of
glomeruli assessed by the "disector" method and Cavalieri
principle." Lab Investigator, 64: 777-784. [0007] Horster, M.,
Braun, G., et al. (1999). "Embryonic renal epithelia: Induction,
nephrogenesis and cell differentiation." Physiological Reviews,
79(4): 1157-1191. [0008] Hume, D., Monkley, S., et al. (1995).
"Detection of c-fms protooncogene in early mouse embryos by whole
mount in situ hybridisation indicates roles for macrophages in
tissue remodelling." British Journal of Haematology, 90(4):
939-942. [0009] Kett M M, Alcorn D, Bertram J F, Anderson W P
(1996). Glomerular dimensions in spontaneously hypertensive rats:
effects of ATI antagonism. Journal of Hypertension; 14: 107-113.
[0010] Keith J C Jr. Pijnenborg R. Luyten C. Spitz B. Schaub R. Van
Assche F A. Maternal serum levels of macrophage colony-stimulating
factor are associated with adverse pregnancy outcome. European
Journal of Obstetrics, Gynecology, & Reproductive Biology,
89(1):19-25, 2000. [0011] Wei S. Lightwood D. Ladyman H. Cross S,
Neale H. Griffiths M. Adams R. Marshall D. Lawson A. McKnight A J.
Stanley E R. Modulation of CSF-1-regulated post-natal development
with anti-CSF-1 antibody, Immunobiology. 210(2-4):109-19, 2005.
[0012] Dai X M. Zong X H. Sylvestre V. Stanley E R. Incomplete
restoration of colony-stimulating factor 1 (CSF-1) function in
CSF-1-deficient Csflop/Csflop mice by transgenic expression of cell
surface CSF-1. Blood, 103(3): 1114-23, 2004. [0013] Seckl J R,
Holmes M C. Mechanisms of disease: glucocorticoids, their placental
metabolism and fetal `programming` of adult pathophysiology. Nat
Clin Pract Endocrinol Metab., 3(6):479-88, 2007. [0014] Gennaro,
Alfonso, Remington's Pharmaceutical Sciences, 18.sup.th edition,
Mack Publishing Co. (1990). [0015] University of the Sciences in
Philadelphia (editor) Remington: The Science and Practice of
Pharmacy 21.sup.st edition (2005). [0016] Rae F, Woods K, Sasmono
T, Campanale N, Taylor D, Ovchinnikov D, Grimmond S M, Hume D A,
Ricardo S D, and Little M H. Characterisation and trophic functions
of murine embryonic macrophages based upon the use of a CSF-1R-EGFP
transgenic reporter. Developmental Biology (In Press, accepted May
24) 2007.
FIELD
[0017] THIS INVENTION relates to use of colony stimulating factor 1
(macrophage colony stimulating factor) in relation to the kidney.
More particularly, this invention relates to use of colony
stimulating factor 1 for treating a renal disease or condition
associated with renal damage or dysfunction.
[0018] The present disclosure relates to embodiments for promoting
organ development in warm blooded animals, and in particular in
certain aspects a premature infant or foetus. Compositions and
methods are provided for the administration of colony stimulating
factor-1 protein (CSF-1), or a precursor, variant, analogue or
derivative thereof, or otherwise, a nucleic acid molecule encoding
colony stimulating factor-1 protein (CSF-1), or a precursor,
variant, analogue or derivative thereof.
BACKGROUND
[0019] Development of the kidney is a process involving branching
morphogenesis. The metanephros, or permanent kidney, is first
observed at E10.5 in the mouse. Reciprocal inductive interactions
between the ureteric bud (UB), an outgrowth of the Wolffian duct,
and the metanephric mesenchyme (MM) result in branching of the UB
to form the collecting duct system of the mature kidney and the
differentiation of the mesenchyme into the glomeruli and
uriniferous tubules (Saxen, 1987, Organogenesis of the kidney.
Cambridge University Press., Cambridge). In both embryonic and
adult kidney, most epithelial structures are surrounded by renal
interstitium. Interstitial cells are responsible for the production
of extracellular matrix components and development and support of
the functional units of the kidney, including feedback control of
the glomerular capillary blood flow.
[0020] The adult (Hume et al., 1983, J Exp Med., 158 1522-36) and
embryonic mammalian renal interstitium contains resident
macrophages. The phenotype and potential tissue specific function
of renal macrophages, and tissue macrophages in general, is not
well defined. A little is known about their growth factor
production and receptor profile. The expression of Cxcr4 on renal
macrophages allows them to respond to the production of Cxcl12 by
the comma and S-shaped bodies of the kidney (Grone et al., 2002,
JASN, 13, 957-67). Conversely, renal macrophages express Cxcl10
(IP-IO), allowing them to signal to the Cxcr3 receptor in the
developing kidney mesenchyme (Grone, et al., 2002, supra).
[0021] The importance of macrophage infiltration in development is
mirrored in adult tissue repair. In numerous examples of tissue
repair, including models of acute damage to muscle, liver, lung,
gastrointestinal tract and peripheral nervous system, infiltration
by macrophages and production of macrophage-derived trophic factors
appears to be absolutely essential for regeneration (Kluth, et al.,
2004, Kidney International., 66 542-57).
[0022] But macrophages are the classical two-edged sword.
[0023] In systems where the damage is severe or progressive and
where the damage stimulus remains, including chronic inflammation,
macrophages are the dominant cell type in the inflammatory exudates
and they are implicated directly in cell death and tissue damage.
Indeed, conventional wisdom in both renal disease and allograft
rejection has been that macrophages are responsible for damage
(Eitner, et al., 1998, Transplantation, 66, 1551-7; Segerer, et
al., 2003, Curr. Opin. Nephrol. Hypertens., 12, 243-9) and many
therapeutic strategies have focused on ways in which to reduce
macrophage recruitment and activation. A reduction in the
production of chemokines involved in macrophage recruitment,
proliferation and activation has been shown to be potentially
beneficial not only in renal disease classically associated with
immune perturbations, such as glomerulonephritis and lupus
nephropathy, but also in unilateral ureteric obstruction and
diabetes (Naito, et al., 1996, Mol. Med., 2, 297-312; Utsunomiya,
et al., 1995, J. Diabetes Complications, 9, 292-5). However, this
is not always the case (Veilhauer, et al., 2004, Kidney Blood
Press. Res., 27, 226-38; Holdsworth, et al., 2000, Curr. Opin
Neprhol. Hypertens., 9, 505-11). Macrophage migration inhibitory
factor (MIF), while associated with renal injury in the rat, has
been shown to be independent of macrophage recruitment and renal
fibrosis in a unilateral ureteral obstruction (UUO) model in the
mouse (Rice et al., 2004, Nephrology 9 278-287).
[0024] CSF-1 (macrophage colony-stimulating factor; M-CSF) is the
major growth factor for cells of the macrophage lineage. Increased
CSF1 levels are associated with renal disease and allograft
rejection (Isbel, et al., 2001, Nephrol. Dial. Transplant., 16,
1638-47; Le Muer, et al., 2002, Leukoc. Biol., 72, 530-7; Le Muer,
et al., 2004, Nephrol. Dial. Transplant., 19, 1862-5). CSF-1 acts
on its target cells by binding to colony-stimulating factor 1
receptor (CSF-1R), a cell-surface tyrosine kinase receptor encoded
by the c-fins proto-oncogene, which is expressed in macrophage and
trophoblast cell lineages (Sasmono, et al., 2003, Blood, 101,
1155-1163). c-fms is critical for the proliferation, survival and
differentiation of macrophages as disruption of the gene results in
large depletions of macrophages in most tissues (Dai et al., 2002,
Blood, 99, 111-20).
[0025] Mutation of the CSF-1 gene, such as that present in op/op
mice, or blockade of CSF-1 function with either anti-CSF-1 or
anti-c-fms antibodies, greatly reduces renal damage in several
models including experimental glomerular nephritis, renal tubular
interstitial nephritis, autoimmune nephritis and ureteral ligation
(Lenda, et al., 2003, J. Immunology, 170, 3254-62; Jose, et al.,
2003, Am. J. Transplant, 3, 294-300). In each of these model
systems, CSF-1 is produced locally, and probably also systemically
(although this is seldom measured), and the interpretation has been
that CSF-1 acts to recruit and activate macrophages to cause tissue
damage.
[0026] Administered granulocyte colony stimulating factor (G-CSF)
has been shown to protect mouse kidneys from subsequent cisplatin
damage. Cisplatin is a widely-used anticancer drug that can induce
acute renal failure due to renal tubular injury. The protective
effect provided by G-CSF was enhanced by CSF-1 (i.e., M-CSF;
Iwasaki, et al., 2005, JASN, 16, 658). However, the administration
of CSF-1 alone prior to the induction of cisplatin damage showed no
protective effect.
[0027] CSF-1 has been reported to impair the progression of
lipid-induced nephrotoxiocity in streptozotocin-induced diabetic
rats, by modulating the recruitment of macrophages to the
glomerulus (Utsunomiya, et al., 1995, supra). However, this
contradicts Miyazaki, et al., 1997, Clin. Exp. Immunol., 108, 318,
who showed that increased M-CSF production is associated with an
increase in recruitment of macrophages to the glomerulus in
lipid-induced nephrotoxicity.
[0028] In humans, renal disease is a severe and debilitating
ailment that is broadly classified as "chronic" or "acute".
[0029] Chronic renal disease (CRD) refers to the gradual decline in
renal function. This ultimately progresses to end stage renal
disease (ESRD) when the renal filtration rate falls below 10%. CRD
prevalence is rising at 6-8% per annum worldwide. Subsequently the
incidence of ESRD is also increasing. Currently, the only available
treatment options for ESRD are renal transplantation and dialysis.
Transplantation extends survival over dialysis, but is associated
with surgical morbidity and faces a shortage of viable organs.
Dialysis replaces solute clearance but does not replace all renal
functions, such as endocrine or metabolic functions. For those
receiving dialysis treatment, the quality of life is poor and
mortality rates are high (16% pa). Acute renal failure (ARF) is a
common outcome in the postoperative patient, due to nephrotoxic or
ischaemic insult during treatment for another condition. ARP
patients receive dialysis treatment, but the lack of adjunct
therapy to dialysis is thought to contribute to the high mortality
rate of 50-75%. For both acute and chronic renal conditions, there
is an urgent need for more advanced therapeutic approaches.
[0030] Compared to infants who have born following a normal, full
term pregnancy, premature infants, particularly babies born before
32 weeks of gestation, are at a considerably greater risk of
developing a number of serious health problems including, for
example, renal and lung disorders.
[0031] For instance, the low birth weight and insufficient physical
development of premature infants predisposes them to respiratory
complications such as respiratory distress syndrome (RDS) and
chronic lung disease (also known as bronchopulmonary dysplasia).
RDS is associated with irregular breathing difficulties and occurs
in approximately 60 to 80 percent of infants born before 28 weeks
gestation, and in 15 to 30 percent of those born between 32 and 36
weeks of gestation. Treatment of such infants typically involves
supplemental oxygen, but in some cases, also requires the use of a
mechanical ventilator and continuous positive airway pressure.
Moreover, in severe cases, treatment will additionally involve the
administration of an artificial lung surfactant. While such
treatments are very successful, long-term ventilator treatment is
undesirable since this can lead to lung deterioration, which in
turn, can lead to bronchopulmonary dysplasia.
[0032] It is also known that premature infants are born with
reduced numbers of nephrons (filtration units of the kidney), an
outcome that may be associated with increased risk of developing
hypertension and reduced renal function following injury later in
life.
[0033] Lung development: analogies between human and mouse:
[0034] The human lung is derived from the foregut at about 4 weeks
gestation and begins as a diverticulum. The lung diverticulum is
covered with splanchnic mesoderm that gives rise to the tissue
components of the mature adult lung such as cartilage, smooth
muscle and blood vessels. Lung development is characterised by
branching morphogenesis that gives rise to the primary, secondary
and tertiary bronchi. The stages of foetal lung development are
classified into three distinct phases, namely; the pseudoglandular,
canalicular and saccular phases. Some aspects of alveolar lung
development including epithelial cell differentiation begin in the
canalicular phase. However, approximately 15-18% of alveoli form
late in gestation, with most of the alveoli formed after birth.
Shortly after birth, the surface area of the air-blood interface
increases with the formation of the alveolar ducts and sacs.
[0035] Premature infants can survive with lung development in the
late canalicular or early saccular phase. This is a phase when the
conducting airways have stopped branching and are enlarging at
their distal termini. There is a progressive loss of extracellular
matrix and mesenchymal cells that separate the capillaries from the
sites of alveoli. These premature infants survive without alveoli
by treatment involving mechanical ventililation and the
administration of an artificial lung surfactant, although, as
mentioned above, they are at risk of developing bronchopulmonary
dysplasia.
[0036] In mice, the lung also arises from the ventral foregut, but
at approximately embryonic day 9.5 (E9.5). Subsequently, the
respiratory tree develops through the pseudoglandular (E9.5-16.5),
canalicular (E16.5-17.5), and saccular (E17.5-postnatal day 5)
phase. While mouse and human lung development is highly analogous
from an embryological point of view and while the same genes are
critical in both organisms, in contrast to the human lung,
alveolarisation is not complete before birth in the mouse. At
birth, the mouse lungs consists of immature terminal saccules with
some secondary septa, with alveolarisation and alveolar separation
occurring during the during the first 1-3 postnatal weeks. The
alveolar surfaces increase through the enlargement of pre-existing
alveoli with formation of new alveoli.
[0037] Kidney development: analogies between humans and mice:
[0038] The development of the kidney is highly analogous between
human and mouse with respect to the embryo logical origin of the
tissues involved, the morphogenetic processes and the genes
regulating these processes.
[0039] In the human (as for the mouse), both the renal and genital
systems originate from the intermediate mesoderm. Development of
the kidney undergoes three distinct stages before resulting in the
mature adult kidney. The process begins with the formation of the
pronephros, then the mesonephros and finally the metanephros, after
which the pronephros and mesonephros regress, and the metanephros
remains to form the functional adult kidney. Metanephric
development begins with the outgrowth of ureteric bud, originating
from the Wollfian duct, invading the surrounding metanephric
mesenchyme. The functional units within the kidney responsible for
filtration of the blood, concentration of the filtrate to form
urine and reclamation of water and ions are the nephrons. The
formation of these functional units is referred to as
nephrogenesis. Human nephrogenesis (development of kidney nephrons)
is completed before birth. The number of nephrons in normal human
kidneys ranges from approximately 300,000 to more than one million.
After birth, the nephron number is complete and no new nephrons are
able to be formed. In humans, development of the permanent kidney
begins around gestational week 5. In the third trimester, 60% of
nephrons are formed and continue to form until approximately 36
weeks. No new nephrons are formed after this time.
[0040] In the mouse (as with humans), there are three embryonic
kidneys, the pronephros, mesonephros and metanephros, and the
development of the final permanent kidney, the metanephros, begins
with the outgrowth of ureteric bud, originating from the Wollfian
duct, invading the surrounding metanephric mesenchyme. This occurs
at around embryonic day 9-10.5 (E9-10.5) and requires inductive
signals from the metanephric mesenchyme to initiate bud
development. The induced mesenchyme sends reciprocal signals to
induce growth and branching of the ureteric bud. Nephron formation
(nephrogenesis) is induced when factors secreted by the ureteric
bud cause the induction, condensation and aggregation of the
mesenchyme. Each aggregate undergoes epithelialisation and then
proceeds through the developmental stages of the polarised vesicle
stage, the comma and the S-stage. There is continued branching with
new aggregates forming at the tips, and this process continues with
the induction of new nephrons. By the end of nephrogenesis, there
are more than 26 terminally differentiated cell types with distinct
location, morphology and function. Unlike the human, in the mouse
kidney development continues in mice until around 7-10 days after
birth.
[0041] Growth factors in kidney and lung development:
[0042] Growth factors, aside from their influence in cell growth,
contribute greatly to many processes including cell migration,
morphogenesis, differentiation and proliferation. The roles of
growth factors in branching morphogenesis in the lung and
nephrogenesis in the kidney are controlled by an array of inductive
and inhibitory signals. The crucial roles of factors including
insulin-like growth factor-I and II (IGF-I and IGF-II), hepatocyte
growth factor (HGF), and epithelial growth factor (EGF) have been
well established in the developing lung and kidney. It is, however,
considered that there may be numerous other growth factors which
play significant roles in development of the lung and kidney.
[0043] In has been found that in warm blooded animals, usings the
embodiments disclosed it is possible to promote organ development
(as reflected in, for some organs, an increase in organ weight),
and more particularly, increased growth and/or enhanced
nephrogenesis and lung maturation. It has also been found that
promoting organ development and/or maturation in a warm blooded
premature infant or foetus is possible.
SUMMARY OF THE INVENTION
[0044] Notwithstanding the typical association between elevated
CSF-1, macrophages and tissue and organ damage, the present
inventors have identified CSF-1 as having a hitherto unrealized
role in supporting and promoting renal tissue repair and
regeneration.
[0045] The invention is therefore broadly directed to use of CSF-1
for regenerating, repairing or otherwise treating renal cells,
tissues and/or organs, and more particularly, in prophylactic or
therapeutic treatment of diseases or conditions associated with
renal damage and/or dysfunction.
[0046] In a particular forms, the invention relates to use of CSF-1
for treatment of acute renal damage and/or dysfunction.
[0047] In a first aspect, the invention provides a method of
prophylactically or therapeutically treating a renal disease or
condition in an animal including the step of administering a CSF-1
protein or an encoding nucleic acid to an animal in need of such
treatment.
[0048] In one form, the method according to the first aspect may be
used to suppress, ameliorate or otherwise treat an existing renal
disease or condition.
[0049] In another form the method according to the first aspect may
be used as prophylaxis to prevent, inhibit, suppress or otherwise
protect against subsequent renal damage and/or renal failure.
[0050] Suitably, in embodiments relating to prophylactic or
protective administration of CSF-1 to prevent renal damage, CSF-1
is administered in the absence of a therapeutically effective
amount of G-CSF.
[0051] Preferably, the renal disease or condition is acute renal
failure.
[0052] In a second aspect, the invention provides a method of
regenerating, repairing or otherwise treating renal tissue in an
animal including the step of administering a CSF-1 protein or an
encoding nucleic acid to an animal in need of such treatment.
[0053] In a third aspect, the invention provides a method of
regenerating, repairing or otherwise treating renal tissue ex vivo
including the step of exposing one or more isolated renal cells,
tissues or organs to a CSF-1 protein or encoding nucleic acid.
[0054] In a fourth aspect, the invention provides a method of renal
transplantation, including the step of administering to the animal
one or more renal cells, tissues or organs exposed ex vivo to a
CSF-1 protein or encoding nucleic acid.
[0055] In a fifth aspect, the invention provides a pharmaceutical
composition for use in treating a renal disease or condition, said
pharmaceutical composition comprising a CSF-1 protein or an
encoding nucleic acid and a pharmaceutically acceptable carrier,
diluent or excipient.
[0056] Suitably, in embodiments relating to prophylactic or
protective administration of CSF-1 to prevent renal damage, said
pharmaceutical composition does not comprise a therapeutically
effective amount of G-CSF.
[0057] It will be appreciated from the foregoing that the renal
cells may be, or may include, isolated renal macrophages as well as
kidney cells.
[0058] In a sixth aspect, the invention provides use of CSF-1 in
the manufacture of a medicament for prophylactically or
therapeutically treating a renal disease or condition in an
animal.
[0059] In one embodiment, said medicament is for prophylactically
or therapeutically treating acute renal failure in an animal.
[0060] In another embodiment, said medicament is for treating an
existing renal disease or condition in an animal.
[0061] It will be appreciated that the present invention has broad
application to animals inclusive of human and non-human
mammals.
[0062] Preferably, the animal is a human.
[0063] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0064] Certain embodiments disclosed provide compositions for and
methods for treating complications arising from or related to low
birth weight in mammals, including for example, humans, pigs,
horses, dogs and other livestock. Low birth weight may be caused by
premature or preterm birth or by poor foetal growth, such as
intrauterine growth restriction. There are many causes of poor
foetal growth, some of which include chromosomal abnormalities,
placental dysfunction, placenta previa, smoking, drug or alcohol
abuse, amnionitis, abruptio placentae or preeclampsia, maternal
hypertension, maternal hypoxemia, maternal toxemia, polyhydramnios,
urinary tract infection, malnutrion, infection, anemia, diabetes,
inadequate maternal weight gain and various diseases. The
compositions and methods described herein may be used to treat
and/or prevent any of these causes of low birth weight and the
complications. In some embodiments, the compositions and methods
described herein may be used specifically to treat or prevent
causes of low birth weight such as foetal alcohol syndrome,
placental insufficiency, intrauterine growth retardation (IUGD),
foetal growth restriction as a result of infections, genetic
abnormalities such as mutations in the gene that encodes
11-.beta.-hydroxysteroid dehydrogenase type 2, maternal
hypertension, diabetes, alcohol and illicit drug abuse or
inadequate maternal weight gain.
[0065] Low birth weight in babies can result in a large number of
complications including immature organ growth, such as immature
lungs and kidneys, respiratory distress syndrome (RDS),
intraventricular hemorrhage (IVH), Patent ductus arteriosus (PDA),
necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP),
and osteopenia. The long term adverse effects of a low birth weight
include increased risk of heart disease and renal failure,
increased risk of diabetes and obesity and a possible consequence
on intelligence. The compositions and methods described herein may
be used to treat one or more complications arising from low birth
weight in mammals and may be administered prior to birth, such as
to the mother or to the foetus or after birth, such as to the
infant.
[0066] Certain embodiments disclosed provide methods of treating
complications arising from or related to low birth weight in
mammals, such as in humans, pigs, dogs, horses or other livestock,
such as in premature infants, in low birth weight infants or in
foetuses comprising administering to said mammals:
[0067] at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof or combination thereof, or
[0068] at least one nucleic acid molecule encoding said at least
one colony stimulating factor-1 protein (CSF-1), and/or at least
one precursor, variant, analogue, derivative thereof, or
combination thereof.
[0069] Certain embodiments disclosed include methods of treating
complications arising from or related to low birth weight in
mammals such as in humans, pigs, horses, dogs or other livestock,
such as in premature infants, in low birth weight infants or in
foetuses comprising administering to said mammals:
[0070] a low birth weight complications-reducing or -limiting
amount of at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof, or combination thereof, or
[0071] a therapeutically effective amount of at least one nucleic
acid molecule encoding a low birth weight complications-reducing or
-limiting amount of said at least one colony stimulating factor-1
protein (CSF-1), and/or at least one precursor, variant, analogue,
derivative thereof or combination thereof.
[0072] Certain embodiments disclosed provide pharmaceutical
compositions for treating, reducing or limiting complications
arising from or related to low birth weight in mammals, such as in
humans, pigs, horses, dogs or other livestock, such as in premature
infants, in low birth weight infants or in foetuses comprising
administering to said mammals:
[0073] a low birth weight complications-reducing or -limiting
amount of at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof or combination thereof, or
[0074] a therapeutically effective amount of at least one nucleic
acid molecule encoding a low birth weight complications-reducing or
-limiting amount of said at least one colony stimulating factor-1
protein (CSF-1), and/or at least one precursor, variant, analogue,
derivative thereof or combination thereof.
[0075] Certain embodiments disclose methods of promoting organ
development and/or maturation in mammals, such as in humans, pigs,
dogs, horses or other livestock, such as in premature infants, in
low birth weight infants or in foetuses are provided. In certain
aspects, methods of promoting organ development and/or maturation
in mammals are disclosed that comprise the step of administering to
the mammal such as the human, pig, horse or other livestock, such
as the premature infant, low birth weight infant or foetus:
[0076] colony stimulating factor-1 protein (CSF-1), and/or a
precursor, variant, analogue, derivative thereof, or combination
thereof or
[0077] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), and/or a precursor, variant, analogue,
derivative thereof or combination thereof.
[0078] In certain aspects, methods of promoting organ development
and/or maturation in mammals are disclosed that comprise the step
of administering to the mammal such as the human, pig, horse, dogs
or other livestock, such as the premature infant, low birth weight
infant or foetus:
[0079] at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof or combination thereof, or
[0080] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), and/or at least one a
precursor, variant, analogue, derivative thereof or combination
thereof.
[0081] In certain aspects, the methods of promoting organ
development in a premature infant, in a low birth weight infant or
in a foetus disclosed comprise the step of administering to the
premature infant, the low birth weight infants or the foetus:
[0082] a premature infant, a low birth weight infant or a foetus
organ development-enhancing amount of at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof, or
[0083] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus organ development-enhancing amount of said at
least one colony stimulating factor-1 protein (CSF-1), and/or at
least one precursor, variant, analogue, derivative thereof or
combination thereof.
[0084] Certain embodiments disclosed provide pharmaceutical
compositions for promoting organ development in a premature infant,
in a low birth weight infant or in a foetus that comprise
[0085] a premature infant, a low birth weight infant or a foetus
organ development-enhancing amount of at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof, or
[0086] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus organ development-enhancing amount of said at
least one colony stimulating factor-1 protein (CSF-1), and/or at
least one precursor, variant, analogue, derivative thereof or
combination thereof.
[0087] In certain aspects, methods are disclosed that promote
growth and/or enhance lung development and/or maturation in
mammals, such as in humans, pigs, horses, dogs or other livestock,
such as in premature infants, in low birth weight infants or in
fetuses. In certain aspects, the methods of promoting lung growth
and/or enhancing lung development and/or maturation in mammals,
such as in humans, pigs, horses, dogs or other livestock, such as
in premature infants, in low birth weight infants or in foetuses
comprising administering to said mammals:
[0088] colony stimulating factor-1 protein (CSF-1), and/or a
precursor, variant, analogue, derivative thereof or combination
thereof, or
[0089] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), and/or a precursor, variant, analogue,
derivative thereof or combination thereof.
[0090] In certain aspects, the methods of promoting lung growth
and/or enhancing lung development and/or maturation in a premature
infant, in a low birth weight infant or in a foetus disclosed
comprise the step of administering to the infant or foetus:
[0091] at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof, or combination thereof, or
[0092] at least one nucleic acid molecule encoding said at least
one colony stimulating factor-1 protein (CSF-1), and/or at least
one precursor, variant, analogue, derivative thereof or combination
thereof.
[0093] In certain aspects, the methods of promoting lung growth
and/or enhancing lung development and/or maturation in a premature
infant, in a low birth weight infant or in a foetus disclosed
comprise the step of administering to the infant or foetus:
[0094] a premature infant, a low birth weight infant or a foetus
lung growth promoting and/or lung development and/or
maturation-enhancing amount of at least one colony stimulating
factor-1 protein (CSF-1), and/or at least one precursor, variant,
analogue, derivative thereof or combination thereof, or
[0095] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus lung growth promoting and/or lung development
and/or maturation-enhancing amount of said at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0096] Certain embodiments disclosed provide pharmaceutical
compositions for promoting lung growth and/or enhancing lung
development and/or maturation in a premature infant, in a low birth
weight infant or in a foetus that comprise
[0097] a premature infant, a low birth weight infant or a foetus
lung growth promoting and/or lung development and/or
maturation-enhancing amount of at least one colony stimulating
factor-1 protein (CSF-1), and/or at least one precursor, variant,
analogue, derivative thereof or combination thereof, or
[0098] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus lung growth promoting and/or lung development
and/or maturation-enhancing amount of said at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0099] In certain embodiments, methods of promoting growth,
maturation and/or enhancing kidney development in mammals, such as
in humans, pigs, horses, dogs or other livestock, such as in
premature infants, in low birth weight infants or in foetuses. In
certain aspects the methods of promoting growth, maturation and/or
enhancing kidney development in mammals, such as in humans, pigs,
horses, dogs or other livestock, such as in premature infants, in
low birth weight infants or in foetuses comprising administering to
said mammals:
[0100] colony stimulating factor-1 protein (CSF-1), and/or a
precursor, variant, analogue, derivative thereof or combination
thereof, or
[0101] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), and/or a precursor, variant, analogue,
derivative thereof or combination thereof.
[0102] In certain aspects the methods of promoting growth,
maturation and/or enhancing kidney development in a premature
infant, in a low birth weight infant or in a foetus comprise the
step of administering to the infant or foetus;
[0103] at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof, or combination thereof, or
[0104] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0105] In certain aspects, the methods of growth, maturation and/or
enhancing kidney development in a premature infant, in a low birth
weight infant or in a foetus disclosed comprise the step of
administering to the infant or foetus:
[0106] a premature infant, a low birth weight infant or a foetus
kidney growth maturation and/or development-enhancing amount of at
least one colony stimulating factor-1 protein (CSF-1), and/or a
precursor, variant, analogue, derivative thereof or combination
thereof, or
[0107] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus kidney growth maturation and/or
development-enhancing amount of said at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0108] Certain embodiments disclosed provide pharmaceutical
compositions for promoting growth, maturation and/or enhancing
kidney development in a premature infant, in a low birth weight
infant or in a foetus that comprise
[0109] a premature infant, a low birth weight infant or a foetus
kidney growth maturation and/or development-enhancing amount of at
least one colony stimulating factor-1 protein (CSF-1), and/or at
least one precursor, variant, analogue, derivative thereof or
combination thereof, or
[0110] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus kidney growth maturation and/or
development-enhancing amount of at least one said colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0111] In certain aspects, methods are disclosed that promote
growth and/or enhance bone development and/or maturation in
mammals, such as in humans, pigs, horses, dogs or other livestock,
such as in premature infants, in low birth weight infants or in
foetuses. In certain aspects, the methods of promoting bone growth
and/or enhancing bone development and/or maturation in mammals,
such as in humans, pigs, horses, dogs or other livestock, such as
in premature infants, in low birth weight infants or in foetuses
comprising administering to said mammals:
[0112] colony stimulating factor-1 protein (CSF-1), and/or a
precursor, variant, analogue, derivative thereof or combination
thereof or
[0113] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), and/or a precursor, variant, analogue,
derivative thereof or combination.
[0114] In certain aspects, the methods of promoting bone growth
and/or enhancing bone development and/or maturation in a premature
infant, in a low birth weight infant or in a foetus disclosed
comprise the step of administering to the infant or foetus:
[0115] at least one colony stimulating factor-1 protein (CSF-1),
and/or at least one precursor, variant, analogue, derivative
thereof or combinations thereof or
[0116] at least one nucleic acid molecule encoding said at least
one colony stimulating factor-1 protein (CSF-1), at least one
precursor, at least one variant, at least one analogue, at least
one derivative thereof or combinations thereof.
[0117] In certain aspects, the methods of promoting bone growth
and/or enhancing bone development and/or maturation in a premature
infant, in a low birth weight infant or in a foetus disclosed
comprise the step of administering to the infant or foetus:
[0118] a premature infant, a low birth weight infant or a foetus
bone growth promoting and/or bone development and/or
maturation-enhancing amount of at least one colony stimulating
factor-1 protein (CSF-1), and/or at least one precursor, variant,
analogue, derivative thereof or combination thereof or
[0119] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus bone growth promoting and/or bone development
and/or maturation-enhancing amount of said at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
[0120] Certain embodiments disclosed provide pharmaceutical
compositions for promoting bone growth and/or enhancing bone
development and/or maturation in a premature infant, in a low birth
weight infant or in a foetus that comprise
[0121] a premature infant, a low birth weight infant or a foetus
bone growth promoting and/or bone development and/or
maturation-enhancing amount of at least one colony stimulating
factor-1 protein (CSF-1), and/or a precursor, variant, analogue,
derivative thereof or combination thereof, or
[0122] a therapeutically effective amount of at least one nucleic
acid molecule encoding a premature infant, a low birth weight
infant or a foetus bone growth promoting and/or bone development
and/or maturation-enhancing amount of said at least one colony
stimulating factor-1 protein (CSF-1), and/or at least one
precursor, variant, analogue, derivative thereof or combination
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0123] FIG. 1. Resident renal macrophages. Left panel: FACS profile
of GFP+ cells isolated from embryonic day 15.5 kidneys of
c-fms-EGFP transgenic mice represent 2.8% of total cells. Middle
panel: RNA section in situ hybridisation of macrophage-specific
outlier shows punctate expression within the interstitial
macrophages of the kidney. Right panel: Two colour confocal image
of a renal macrophage (arrowhead) between renal proximal
tubules.
[0124] FIG. 2. Metanephric explants cultured with 100U/.mu.L (1.25
ng/.mu.L) human recombinant CSF-1 for 3 days compared to control
explants. Immunofluorescence was performed to reveal the ureteric
tree (calbindin 28 KD) and the forming nephrons (WT1). 6 day
cultures with WT1, calbindin and DAPI (nuclei) merged show the
overall increase in size.
[0125] FIG. 3. Representative micrographs demonstrating the
histology of mice following 50 minutes of IR injury at 1 week after
injury (Panel A and B) compared to mice with IR injury following
delayed administered CSF-1 at the same time point (Panel C and D).
Numerous tubular casts (black arrows) were evident in the renal
medulla by 1 week after IR injury (Mag.times.200) (A). At higher
power (Mag.times.400) interstitial matrix expansion is shown with a
prominent inflammatory cell infiltrate (white arrows; B). In IR
mice following CSF-1 administration starting at 3 days post-renal
artery clamping the majority of the tubular epithelium showed
normal histology (black arrows) with very few tubular casts present
at 1 week white arrows; C Mag.times.200). At higher power (D;
Mag.times.400) there was widespread tubular epithelial cell
replacement (black arrows) with attenuated interstitial matrix
expansion.
[0126] FIG. 4. Measurement of urinary albumin levels (A) and the
albumin/creatinine ratio (B) in IR mice with or without the
administration of CSF-1 delivered day 3-5 after initiation of
injury. The administration of CSF-1 to IR mice was found to reduce
urinary protein levels and the albumin/creatinine ratio comparable
to control animals. **P<0.03; Data are means.+-.SD.
[0127] FIG. 5: Immunofluorescence microscopy of type IV collagen in
c-fms-GFV mice following IR receiving vehicle (A and B) or CSF-1
treatment (C and D). Panel A demonstrates increased numbers of
GFP-positive macrophages in the renal interstitium that was
associated with collagen type IV accumulation leading to
interstitial expansion (arrows; Mag.times.400). At higher power (B;
Mag.times.1,000) arrows show tubular cast formation (white arrows)
in the majority of proximal tubules as a result of loss of
epithelial cell integrity. Interstitial macrophages (large
arrowheads) can be seen associated with type IV collagen
accumulation in IR kidneys. Following CSF-1 treatment, IR kidneys
displayed decreased numbers of interstitial GFP-macrophages, and a
normal tubulointerstitium that contained a fine framework of
collagen type IV (arrows; C; Mag.times.400) comparable to normal
kidneys. At higher power (D; Mag.times.1,000) the CSF-1 treated IR
kidneys showed normal architecture with an intact proximal tubular
epithelial cell lining (arrows) that was surrounded by few
GFP-macrophages in the interstitium (large arrowheads) without
evidence of fibrosis.
[0128] FIG. 6 provides a graph demonstrating the average mouse body
weight (n=3/group) in mice receiving CSF-1 compared to litter mate
control treated mice.
[0129] FIG. 7 provides a graph showing the effect on kidney weight
of CSF-1 delivery to newborn mouse pups.
[0130] FIG. 8 provides a graph showing a stereological estimation
of glomerular number in the kidneys from mice receiving CSF-1 or
phosphate buffered saline (PBS).
[0131] FIG. 9 shows the histology of kidneys from control (A;
Mag.times.100) and CSF-1 treated mice (B; Mag.times.100) killed at
day 29 and stained with haematoxylin and eosin; and the histology
of lungs of control (C; Mag.times.200, E; Mag.times.400) and
CSF-1-treated (D; Mag.times.200, F; Mag.times.400) mice.
DETAILED DESCRIPTION
[0132] While CSF-1 has previously been observed to assist G-CSF in
preventing renal damage (i.e., reno-protection) and to modulate
recruitment of macrophages to the glomerulus of rats having
lipid-induced nephrotoxocity (although its precise role remains
controversial), the present invention has arisen, at least in part,
from the surprising observation that CSF-1 stimulates macrophages
to promote growth, regeneration and/or repair of the kidney. By
extension, the production of CSF-1 in renal disease forms part of a
protective/regenerative response that fails only when there is
ongoing tissue damage elicited by a separate causal agent. Thus, it
proposed that treatment with CSF-1 could provide a paradoxical and
unexpected approach to therapy for renal diseases and/or
conditions.
[0133] As used herein "CSF-1 protein" includes and encompasses any
CSF-1 protein (also known as macrophage colony stimulating factor
or M-CSF) of mammalian origin, including any biologically active
fragment of a CSF-1 protein
[0134] It will be appreciated that the invention also contemplates
use of any of a number of modified and/or fragmentary forms of
CSF-1.
[0135] For example, U.S. Pat. No. 6,322,779 describes an isolated
recombinant, dimeric CSF-1 is which is unglycosylated and which can
be produced essentially endotoxin and pyrogen-free.
[0136] In particular, several C-terminally truncated fragments of
CSF-1 have been described which retain biological activity.
[0137] By way of example, reference is made to U.S. Pat. No.
6,204,020 and U.S. Pat. No. 6,146,851 which describe various
carboxy-truncated forms of CSF-1 protein and their encoding nucleic
acids.
[0138] A biologically active CSF-1 dimer is described in U.S. Pat.
No. 5,861,150, wherein at least one of the CSF-1 monomers has one
or more amino acid substitutions together with a carboxy
truncation.
[0139] U.S. Pat. No. 5,672,343 sets forth a CSF-1 protein
consisting of amino acids 4-522 of the 536 amino acid CSF-1
sequence and fragments of CSF-1 comprising truncations at various
positions C-terminal of residue 149.
[0140] The invention also contemplates use of any other molecule
that has CSF-1 agonist activity, including but not limited to any
molecule capable of binding, dimerizing and/or activating the
cognate CSF-1 receptor (CSF-1R or c-fms).
[0141] CSF-1 protein may be in native form purified from a natural
source, including but not limited to human urine. An example of
such a product is Mirimostim.TM. from Mitsubishi Pharma.
[0142] CSF-1 may also be in recombinant or chemical synthetic
form.
[0143] For example, the present invention contemplates chemical
synthesis of CSF-1 protein, inclusive of solid phase and solution
phase synthesis. Such methods are well known in the art, although
reference is made to examples of chemical synthesis techniques as
provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson
(Blackwell Scientific Publications) and Chapter 15 of CURRENT
PROTOCOLS IN PROTEIN SCIENCE, Eds., Coligan, et al., (John Wiley
& Sons, Inc., NY, USA, 1995-2001).
[0144] The invention also contemplates recombinant DNA technology
as a means of producing recombinant CSF-1, including but not
limited to, standard protocols as for example described in
Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual, (Cold
Spring Harbor Press, 1989), in particular Sections 16 and 17;
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Eds., Ausubel, et al.,
(John Wiley & Sons, Inc., NY, USA, 1995-2001), in particular
Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE, Eds.,
Coligan, et al., (John Wiley & Sons, Inc., NY, USA, 1995-2001,
in particular Chapters 1, 5, and 6).
[0145] In one embodiment, the CSF-1 protein is
bacterially-expressed, non-glycosylated human recombinant
CSF-1.
[0146] However, use of glycosylated forms of CSF-1 (such as
produced by mammalian cell expression systems) are also suitable
for use according to the invention.
[0147] Preferably, the CSF-1 protein consists of a C-terminal 150
amino acid fragment of CSF-1 protein.
[0148] The invention also contemplates CSF-1 protein "derivatives",
which have been altered, for example by addition, conjugation or
complexing with other chemical moieties or by post-translational
modification techniques, as are well understood in the art.
[0149] By way of example only, the invention contemplates
derivatives of CSF-1 such as, but not limited to, chemical
modification of side chains (e.g., pegylation of nucleophilic
groups such as lysyl .epsilon.-amino groups or sulphydryl oxidation
by performic acid oxidation to cysteic acid), chemical modification
of the C-terminus (e.g., carbodiimide activation via O-acylisourea
formation followed by subsequent derivitization to a corresponding
amide), chemical modification of the N-terminus (e.g., acylation
with acetic or succinic anhydride), incorporation of non-natural
amino acids and/or their derivatives during protein synthesis and
the use of crosslinkers, labels (e.g., fluorochromes,
radionuclides, biotin) and other adducts.
[0150] Other CSF-1 derivatives may comprise additional amino acid
sequences such as fusion partner sequences. Fusion partner
sequences, by way of example, assist in protein purification and/or
identification. For instance, these include "epitope tags" such as
c-myc, FLAG and influenza haemagglutinin tags, polyhistidine (e.g.,
HIS6), maltose binding protein, green fluorescent protein (GFP),
immunoglobulin heavy chain Fc portion and glutathione S
-transferase (GST), although without limitation thereto.
[0151] For the purposes of fusion polypeptide purification by
affinity chromatography, relevant matrices for affinity
chromatography are antibody, protein A- or G-, glutathione-,
amylose-, and nickel- or cobalt-conjugated resins respectively.
Many such matrices are available in "kit" form, such as the
QIAexpress.TM. system (Qiagen) useful with (HIS6) fusion partners
and the Pharmacia GST purification system.
[0152] Isolated Nucleic Acids and Expression Constructs
[0153] It will be appreciated from the foregoing and also from
renal treatment methods and compositions to be described in more
detail hereinafter, that the invention also provides use of an
isolated nucleic acid encoding a CSF-1 protein.
[0154] The term "nucleic acid" as used herein designates single- or
double-stranded mRNA, RNA, cRNA, RNAi and DNA inclusive of cDNA and
genomic DNA and DNA-RNA hybrids. Nucleic acids may also be
conjugated with fluorochromes, enzymes and peptides as are well
known in the art.
[0155] The invention also contemplates variant CSF-1 nucleic acids
having one or more codon sequences altered by taking advantage of
codon sequence redundancy.
[0156] A particular example of a variant CSF-1 nucleic acid is
optimization of a nucleic acid sequence according to codon usage,
as is well known in the art. This can effectively "tailor" a
nucleic acid for optimal expression in a particular organism, or
cells thereof, where preferential codon usage has been
established.
[0157] In certain embodiments, said isolated CSF-1 nucleic acid may
be present in an expression construct, wherein the said isolated
nucleic acid is operably linked or connected to one or more
regulatory sequences in an expression vector.
[0158] In one particular embodiment, the expression construct is
suitable for bacterial expression of CSF-1 protein in bacteria such
as E. coli.
[0159] In another particular embodiment, the expression construct
is for expression in one or more mammalian cells, tissues or organs
in vitro or in vivo.
[0160] According to this embodiment, the mammalian cells, tissues
or organs include kidney cells, resident renal macrophages and/or
bone marrow-derived macrophages.
[0161] Accordingly, an "expression vector" may be either a
self-replicating extra-chromosomal vector such as a plasmid, or a
vector that integrates into a host genome, inclusive of vectors of
viral origin such as adenovirus, lentivirus, poxvirus and
flavivirus vectors as are well known in the art.
[0162] By "operably linked or connected" is meant that said
regulatory nucleotide sequence(s) is/are positioned relative to the
recombinant nucleic acid of the invention to initiate, control,
regulate or otherwise direct transcription and/or other processes
associated with expression of said nucleic acid.
[0163] Regulatory nucleotide sequences will generally be
appropriate for the host cell used for expression. Numerous types
of appropriate expression vectors and suitable regulatory sequences
are known in the art for a variety of host cells.
[0164] Typically, said one or more regulatory nucleotide sequences
may include, but are not limited to, promoter sequences, leader or
signal sequences, ribosomal binding sites, transcriptional start
and termination sequences, translational start and termination
sequences, splice donor/acceptor sequences and enhancer or
activator sequences.
[0165] Constitutive promoters (such as CMV, SV40 and human
elongation factor promoters) and inducible/repressible promoters
(such as tet-repressible promoters and IPTG-, alcohol-,
metallothionine- or ecdysone-inducible promoters) are well known in
the art and are contemplated by the invention, as are
tissue-specific promoters such as .alpha.-crystallin promoters. It
will also be appreciated that promoters may be hybrid promoters
that combine elements of more than one promoter (such as SRa
promoter).
[0166] The expression construct may also include a fusion partner
(typically provided by the expression vector) so that the
recombinant CSF-1 protein is expressed as a fusion polypeptide with
said fusion partner, as hereinbefore described.
[0167] Expression constructs may also include a selection marker
nucleic acid that confers transformed host cell resistance to a
selection agent. Selection markers useful for the purposes of
selection of transformed bacteria include bla, kanR and tetR while
transformed eukaryotic cells may be selected by markers such as
hygromycin, G418 and puromycin, although without limitation
thereto.
[0168] Expression constructs may be introduced into cells or
tissues by any of a number of well known methods typically referred
to as "transfection" "transduction", "transformation" and the like.
Non-limiting examples of such methods include transformation by
heat shock, electroporation, DEAE-Dextran transfection,
microinjection, liposome-mediated transfection (e.g. lipofectamine,
lipofectin), calcium phosphate precipitated transfection, viral
transformation, protoplast fusion, microparticle bombardment and
the like.
[0169] Pharmaceutical Compositions and Methods of Treatment
[0170] A variety of diseases and conditions can damage kidney
parenchyma, such as atheroembolic disease, renal vein thrombosis,
renal artery embolism, thrombosis, diabetic nephropathy,
glomerulonephritis of various etiology, toxic nephrosis, and
pyelonephritis. As a result of the damage, renal failure, whether
arising from an acute or chronic decline in renal function, is a
grave condition that can result in substantial or complete failure
of the filtration, reabsorption, endocrine, and homeostatic
functions of the kidney.
[0171] In one aspect, the invention therefore provides a method of
prophylactically or therapeutically treating a renal disease or
condition in an animal, such as by regenerating renal tissue in
vivo in the animal, by administering a CSF-1 protein, or an
expression construct encoding a CSF-1 protein, to the animal.
[0172] It will be appreciated that CSF-1 may be administered alone
or together with one or more other therapeutic agents that
facilitate or assist in treating the renal disease or
condition.
[0173] A non-limiting example of such a therapeutic agent includes
immunosuppressive agents {e.g. cyclosporine) and antibiotics (e.g.
amoxicillin, cephalosporins, levofloxacin and ciprofloxacin).
[0174] In one particular embodiment relating to prophylactic
treatment, said one or more other therapeutic agents is not
G-CSF.
[0175] It will also be appreciated that the invention contemplates
combination with other treatments such as dialysis, surgery and
transplantation.
[0176] In a preferred embodiment, the invention provides a method
of treating an existing renal disease or condition in an
animal.
[0177] The term "renal disease or condition" broadly includes and
encompasses both acute and chronic renal failure.
[0178] By "acute renal failure" is meant sudden loss of the ability
of the kidneys to excrete wastes, concentrate urine, and/or
conserve electrolytes.
[0179] Acute renal failure occurs relatively rapidly, such as in
the postoperative patient, due to nephrotoxic or ischaemic insult
during treatment for another condition.
[0180] A more comprehensive review and discussion of acute renal
failure can be found in Lameire, et al., 2006, JASN, 17, 923, and
Xue, et al., 2006, JASN, February 22.
[0181] By "chronic renal disease" is meant a gradual decline in
renal function which ultimately progresses to end stage renal
disease (ESRD) where the renal filtration rate falls below 10%.
[0182] In one embodiment, the chronic renal disease is not
lipid-induced nephrotoxocity.
[0183] In a preferred embodiment, the invention relates to
treatment of acute renal failure, such as where rapid renal repair
and/or regeneration is required.
[0184] However, it will be appreciated that immediate delivery of
CSF-1 in vivo may also be useful in ongoing treatment of chronic
renal disease.
[0185] In an alternative, less preferred embodiment, the invention
provides use of CSF-1 for prophylactic administration to an animal
to prevent, inhibit, suppress or otherwise protect against
subsequent renal damage and/or renal failure.
[0186] Suitably, according to such an embodiment CSF-1 is
administered to the animal in the absence of a therapeutically
effective amount of G-CSF.
[0187] As used herein, an example of a therapeutically effective
amount of G-CSF is an amount which is sufficient to protect against
subsequent renal failure.
[0188] An example of a therapeutically effective amount of G-CSF is
250 .mu.g/kg, such as described in Iwasaki, et al., 2005,
supra.
[0189] Preferably, CSF-1 is administered in the absence of
G-CSF.
[0190] Thus, a therapeutic agent administered according to the
invention may "consist of CSF-1 or "consist essentially of
CSF-1.
[0191] By "consist essentially of is meant that CSF-1 is the major,
therapeutically active agent administered to said animal.
[0192] For example, CSF-1 provides, accounts for, or constitutes at
least 60%, preferably at least 70%, more preferably at least 80%
and advantageously at least 85%, 90% or 95-99% of the therapeutic
activity administered to the animal.
[0193] In particular embodiments, CSF-1 is delivered as a
pharmaceutical composition that further comprises a
pharmaceutically acceptable carrier, diluent or excipient.
[0194] In general terms, by "pharmaceutically-acceptable carrier,
diluent or excipient is meant a solid or liquid filler, diluent or
encapsulating substance that may be safely used in systemic
administration. Depending upon the particular route of
administration, a variety of carriers, well known in the art may be
used. These carriers may be selected from a group including sugars,
starches, cellulose and its derivatives, malt, gelatine, talc,
calcium sulfate, vegetable oils, synthetic oils, polyols, alginic
acid, phosphate buffered solutions, emulsifiers, isotonic saline
and salts such as mineral acid salts including hydrochlorides,
bromides and sulfates, organic acids such as acetates, propionates
and malonates and pyrogen-free water.
[0195] A useful reference describing pharmaceutically acceptable
carriers, diluents and excipients is Remington's Pharmaceutical
Sciences (Mack Publishing Co., NJ, USA, 1991) which is incorporated
herein by reference.
[0196] Any safe route of administration may be employed for
providing a patient with the composition of the invention. For
example, oral, rectal, parenteral, sublingual, buccal, intravenous,
intra-articular, intra-muscular, intra-dermal, subcutaneous,
inhalational, intraocular, intraperitoneal,
intracerebroventricular, transdermal and the like may be employed.
Intra-muscular and subcutaneous injection is appropriate, for
example, for administration of proteinaceous and nucleic acid
molecules.
[0197] Dosage forms include tablets, dispersions, suspensions,
injections, solutions, syrups, troches, capsules, suppositories,
aerosols, transdermal patches and the like.
[0198] These dosage forms may also include injecting or implanting
controlled releasing devices designed specifically for this purpose
or other forms of implants modified to act additionally in this
fashion. Controlled, release of the therapeutic agent may be
effected by coating the same, for example, with hydrophobic
polymers including acrylic resins, waxes, higher aliphatic
alcohols, polylactic and polyglycolic acids and certain cellulose
derivatives such as hydroxypropylmethyl cellulose. In addition, the
controlled release may be effected by using other polymer matrices,
liposomes and/or microspheres.
[0199] Pharmaceutical compositions of the present invention
suitable for oral or parenteral administration may be presented as
discrete units such as capsules, sachets or tablets each containing
a pre-determined amount of CSF-1 protein or an expression construct
encoding same, as a powder or granules or as a solution or a
suspension in an aqueous liquid, a non-aqueous liquid, an
oil-in-water emulsion or a water-in-oil liquid emulsion. Such
compositions may be prepared by any of the methods of pharmacy but
all methods include the step of bringing into association one or
more agents as described above with the carrier which constitutes
one or more necessary ingredients. In general, the compositions are
prepared by uniformly and intimately admixing the agents of the
invention with liquid carriers or finely divided solid carriers or
both, and then, if necessary, shaping the product into the desired
presentation.
[0200] The above compositions may be administered in a manner
compatible with the dosage formulation, and in such amount as is
pharmaceutically-effective. The dose administered to a patient, in
the context of the present invention, should be sufficient to
effect a beneficial response in a patient over an appropriate
period of time. The quantity of agent(s) to be administered may
depend on the subject to be treated inclusive of the age, sex,
weight and general health condition thereof, factors that will
depend on the judgement of the practitioner.
[0201] Preferably, pharmaceutical compositions are deliverable
directly to the kidney in a manner which avoids or lessens the
likelihood of CSF-1-induced side effects that might result from
systemic delivery.
[0202] In this regard, a CSF-1 reservoir may be utilized which is
transplantable into an animal, preferably at a site in proximity to
the kidney, which delivers a controlled, metered dosage of CSF-1
over time.
[0203] In one embodiment, CSF-1 may be delivered by an implanted
osmotic pump that delivers CSF-1 to a location proximal to the
kidney and/or into the renal blood supply such as via the renal
artery.
[0204] In another embodiment, microsphere-based delivery may be
achieved by reconstituting CSF-1 in solvent including distilled
water and chitosan and combining this solution with
polylactic-co-glycolic acid in an aqueous solution so that an
emulsion is formed by ultrasonic treatment. The membrane is
permeable to CSF-1 and biocompatible and biodegradable with human
kidney tissue without forming toxic waste products. Release can be
controlled by the biodegradation kinetics of the chitosan used. For
example, recombinant human colony stimulating factor 1 (rhCSF-1)
was delivered in chitosan microcapsules that were injected locally
into injured mouse brain so that CSF-1 was constitutively released
for different lengths of time to enhance survival of neurons in
injured brain (Berezovskaya, et al., 1996, Acta Neuropathol., 92,
479-86).
[0205] Another particular, non-limiting example of CSF-1 protein
delivery technology is provided in United States Patent Application
20040191215.
[0206] In one embodiment, CSF-1 may be provided in a macroporous
reservoir comprising CSF-1 in a biologically and chemically inert
particle having interconnected pores. The pores are open to the
particle surface for communication between the exterior of the
particle and the internal pore spaces. Examples of particles for
formation of such macroporous reservoirs are described, for
example, in U.S. Pat. No. 5,135,740.
[0207] In another embodiment, CSF-1 reservoir may be provided by
way of a microcapsule and/or microparticle, having CSF-1 contained
or dispersed therein. Both microcapsules and microparticles are
well known in the pharmaceutical and drug delivery industries (see,
for example, Baker, R. W., CONTROLLED RELEASE OF BIOLOGICALLY
ACTIVE AGENTS, John Wiley & Sons, NY, 1987; Ranade V. and
Hollinger, M., DRUG DELIVERY SYSTEMS, CRC Press, 1996).
[0208] A microcapsule would typically comprise a reservoir or bolus
of CSF-1 contained within a polymer membrane shell.
[0209] A microparticle would typically be a monolithic system where
CSF-1 is dispersed throughout the particle.
[0210] Specific procedures for encapsulation of biologically active
agents which may be relevant to CSF-1 are disclosed in U.S. Pat.
No. 4,675,189 and U.S. patent application No. 20010033868.
[0211] In yet another embodiment, the invention contemplates a
polymer gel formulation comprising CSF-1. An example of a polymer
for use in such a gel formulation is a
polyoxyethylene-polyoxypropylene block copolymer (Pluronic.RTM.).
These copolymers exhibit reverse thermal gelation behavior, have
good drug release characteristics, and have a low toxicity. The
copolymers gel as a function of temperature and polymer
concentration, where an aqueous solution gels as the solution is
warmed. The gel has a low viscosity at room temperature, but at a
typical body temperature the viscosity increases.
[0212] Other suitable polymers for preparation of CSF-1 delivery
reservoirs include, but are not limited to collagen (Pieper et al.,
2000, Biomaterials, 21, 1689-1699); fibrin (Grassl, et. al., 2002,
J. Biomed. Mater. Res., 60, 607-612); yaupon gels (Ramamurthi, et
al., 2002, J. Biomed. Mater. Res., 60, 196-205); derivatized
dextrans (Letourneur, et al., 2002, J. Biomed. Mater. Res., 60,
94-100); heparin alginate (Laham, et al., 1999, Circulation, 100,
1865-1871); alginate (U.S. Pat. Nos. 6,238,705 & 6,096,344);
and chochleates (U.S. Pat. No. 6,403,056).
[0213] In yet another embodiment, CSF-1 may be delivered by way of
a liposome.
[0214] Liposomes are typically, although not exclusively, spherical
lipid vesicles, ranging in size from 0.01 to 10 microns, and
consist of one or more lipid bilayer encapsulating an aqueous
space. A variety of amphipathic lipids are used to form the
bilayer, such as phospholipids, as for example described in U.S.
Pat. No. 5,013,556. The lipid molecules are generally arranged with
their polar head groups toward the water phase and the hydrophobic
hydrocarbon tails adjacent to one another in the bilayer, thus
forming closed, concentric bimolecular lipid leaflets separating
aqueous compartment.
[0215] As previously described, the invention also contemplates
delivery of an expression construct that comprises an isolated
nucleic acid encoding CSF-1 protein.
[0216] For example, the invention contemplates intravenous
injection of a plasmid DNA expression construct comprising a CMV
promoter operably linked or connected to a CSF-1 nucleotide
sequence using a procedure such as described for delivery of
hepatocyte growth factor to renal glomeruli of mice (Dai, et al.,
2004, J Am Soc Nephrol., 15, 2637-47).
[0217] In another example, the invention contemplates transduction
of primary cultures of isolated macrophages with a CSF-1 expression
construct (for increased expression). Macrophages are then injected
systemically or into the renal artery and will localize to the
damaged kidney, as has been demonstrated with respect to IL-10 in
rats with nephritis (Wilson, et al., 2002, Mol Ther., 6,
710-7).
[0218] In yet another example, the invention contemplates
therapeutic delivery of a recombinant viral vector encoding CSF-1
injected intravenously into renal disease patients
[0219] Delivery of CSF-1 expression constructs may be facilitated
by use of appropriate delivery agents.
[0220] Biodegradable hydrogels may be formulated from cationized
gelatin prepared through aminization containing plasmid DNA
including a human CSF-1 nucleotide sequence operably linked or
connected to a promoter operable in a mammalian cell (e.g., a CMV
promoter).
[0221] Similar approaches using microspheres and hydrogels
(containing DNA constructs encoding recombinant matrix
metalloproteinases) have shown that injection into the renal
subcapsule of C57BL/6 mice which have had streptozotocin-induced
diabetes, showed promise as a prophylactic treatment of kidney
fibrolysis and dysfunction in the STZ-induced diabetic mouse model.
(Aoyama, et al., 2003, Tissue Eng., 9, 1289).
[0222] The invention also provides a method of regenerating,
repairing or otherwise treating renal tissue ex vivo for
transplantation into an animal, by administering a CSF-1 protein to
one or more kidney cells, tissues or organs in vitro prior to
transplantation.
[0223] As used herein, "transplantation" includes and encompasses
transplantation of autologous and heterologous cells, tissues and
organs, as understood in the art.
[0224] With improved surgical techniques and medical management of
rejection, renal transplantation has become the treatment of choice
for chronic and end-stage renal disease (ESRD).
[0225] The use of immunosuppressive agents such as cyclosporine,
OKT3, and FK506 has resulted in a 1-year survival rate for
mismatched renal grafts of 80%. A 90% 1-year graft survival rate
has been reported with non-identical grafts from living related
donors and a 95% 1-year success rate for grafts with identical
human lymphocyte antigen. The half-life of grafts from living
related donors varies from 13-24 years. Other medical managements
have further extended the functional life of renal transplants
while ensuring a better quality of life for the transplant
recipient.
[0226] Surgical techniques for transplantation were recently
advanced with the use of laparoscopic surgical techniques. The
frequency of left kidney harvesting via a laparoscopic approach has
resulted in more frequent transplantation of kidneys with multiple
renal arteries.
[0227] The invention therefore contemplates treatment of whole
kidney or isolated kidney tissue in vitro, such as by soaking or
perfusing with CSF-1, to thereby facilitate the effectiveness of
transplantation to a recipient. The CSF-1 treatment may further
comprise other agents such as immunosuppressants (e.g., OKT3,
cyclosporine or FK506), growth factors and/or cytokines other than
CSF-1 that suppress rejection and/or assist renal regeneration
and/or repair (e.g., Ccl and Cxcl).
[0228] In a particular embodiment, the invention contemplates
enhancing growth of renal progenitor or stem cells (once committed
to a renal fate) by addition of CSF-1 to culture before injection
into the renal capsule.
[0229] It will also be appreciated that resident, renal macrophages
may be used therapeutically.
[0230] Although not wishing to be bound by any particular theory,
it is possible that CSF-1 acts to induce renal macrophages to
produce soluble factors that promote renal cell growth and
development.
[0231] Examples of such factors include the chemokines within the
Ccl and Cxcl families. The expression of receptors for Ccl and Cxcl
chemokines on renal cells, including podocytes and collecting duct
cells (Huber, et al., 2002, J. Immunol., 168, 6244-52.) suggests
that these ligands can signal to the kidney itself rather than
simply playing a role in monocyte attraction
[0232] Therefore, the invention contemplates delivery of isolated
renal macrophages to renal tissue to thereby promote CSF-1-mediated
repair and regeneration of renal tissue.
[0233] Resident renal macrophages may be readily isolated by way of
surface markers such as c-fins, class II MHC, CD83, CD 14 and/or
CD86 by cell isolation methods well known in the art (e.g., by FACS
sorting or by magnetic bead enrichment).
[0234] While in preferred forms the invention provides methods of
treatment of renal diseases or conditions in humans, the invention
also contemplates veterinary treatments of non-human animals such
as poultry, livestock (e.g., cattle, horses, goats and sheep),
performance animals (e.g., racehorses including sires and
broodmares) and domestic animals, although without limitation
thereto.
[0235] So that preferred forms of the invention may be better
understood and put into practical effect, reference is made to the
following non-limiting examples.
[0236] In general, because of the strong similarities between all
placental mammals in terms of organo genesis, the mouse provides an
excellent predictive model for organogenesis in humans, pigs,
horses, dogs and other placental mammals.
[0237] Growth factor known as colony stimulating factor-1 protein
(CSF-1) (also known as macrophage colony stimulating factor
(M-CSF)) controls the survival, proliferation and differentiation
of cells of the monocyte/macrophage lineage, and acts by binding to
the CSF-1 receptor (CSF-1R), a cell-surface tyrosine kinase
receptor encoded by the c-fms proto-oncogene. Previous studies have
shown that c-frns mRNA is found in the placenta, localised to cells
of a macrophage specific lineage (Hume, Monkley et al., 1995). The
present embodiments relate to and elucidate the role(s) that CSF-1
has in embryonic development. The applicants have found,
surprisingly, that in newborn mice, CSF-1 was able to treat
complications arising from or related to low birth weight, to
promote organ development, and more particularly, increased growth
and/or enhanced lung maturation and nephrogenesis in the kidney and
increased bone growth and/or enhanced bone maturation in
co-occurrence with an overall increase in size and body weight.
Lung and kidney development is incomplete in newborn mice, newborn
mice, therefore, provide a useful model for lung and kidney
development in the human foetus and premature infants. In addition,
newborn mice undergo bone and cartilage remodelling and growth
postnatally. Thus, as disclosed herein the administration of CSF-1
to premature infants and pregnant mothers at risk of premature
birth (or for whom premature birth is desirable) may permit
treatment and/or prevention of diseases and conditions associated
with underdeveloped organs such as the lungs and kidneys and bone
formation.
[0238] In certain embodiments, based on studies conducted to
elucidate what role(s) CSF-1 might have in embryonic development,
the present applicants surprisingly found that in certain
embodiments in newborn mice, CSF-1 was able to promote organ
development (as reflected in, for some organs, an increase in organ
weight), and more particularly, increased growth and/or enhanced
nephrogenesis and lung maturation.
[0239] In certain embodiments, methods of promoting organ
development and/or maturation in a premature infant or foetus, the
methods comprising the step of administering to the infant or
foetus;
[0240] colony stimulating factor-1 protein (CSF-1), or a precursor,
variant, analogue or derivative thereof, or
[0241] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), or a precursor, variant, analogue or
derivative thereof.
[0242] The methods disclosed herein may be used to treat
complications arising from or related to low birth weight, to
promote the development of one or more organs such as, but not
limited to, the lung, kidney, brain, eye and organs of the
gastrointestinal (G.I.) tract, in certain aspects in particular the
small intestine, and may be used to promote bone growth and
development in mammals, such as humans, pigs, horses, dogs and
other livestock, such as in premature infants or in foetuses. The
organ development that may be achieved by the method disclosed
herein can result in cell growth and cell differentiation so as to
cause organ maturation (e.g. in terms of organ structure and
function) towards that of infants born following a normal,
full-term pregnancy and foetal development. The premature infant or
foetus treated in accordance with certain embodiments may thereby
avoid or defer, for example, developing hypertension and/or reduced
renal function following injury later in life, respiratory distress
syndrome (RDS) and bronchopulmonary dysplasia, intraventricular
hemmorhage and neural development disorders that can lead to
learning problems, behavioural problems and cerebral palsy,
retinopathy due to abnormal growth of blood vessels, and hearing
loss.
[0243] In certain embodiments, the methods involve the
administration of CSF-1, at least one nucleic acid encoding CSF-1,
or combinations thereof. In certain preferred embodiments, the
methods disclosed involve the administration of human CSF-1, a
least one nucleic acid encoding human CSF-1, or combinations
thereof. However, it is also suitable in certain embodiments to
administer a precursor, variant, analogue or derivative of CSF-1,
at least one nucleic acid encoding same, or combinations thereof.
In certain aspects it is preferred to administer, at least one
precursor, at least one variant, at least one analogue, at least
one derivative of human CSF-1, at least one nucleic acid encoding
same, or combinations thereof.
[0244] The term "precursor" is to be understood to refer to any
molecule that is converted or metabolised within the body to CSF-1.
Thus, one example of a suitable CSF-1 precursor is an immature
CSF-1 comprising its native, or a heterologous, secretory signal,
which can be processed by proteolytic cleavage to produce CSF-1
(i.e., mature CSF-1).
[0245] The term "variant" is to be understood to refer to an
isoform of CSF-1 encoded by, for example, an allelic variant.
[0246] The term "analogue" is to be understood to refer to any
molecule that differs from CSF-1 but retains similarity, or
substantial similarity, in biological function of CSF-1, in
particular the ability to promote organ development. In certain
aspects, an analogue may have substantial overall structural
similarity with CSF-1 or only structural similarity with one or
more regions or domains of CSF-1 responsible for its biological
function. Typically, an analogue of CSF-1 will be provided by, or
be the result of, the addition of one or more amino acids to the
amino acid sequence of CSF-1, deletion of one or more amino acids
from the amino acid sequence of CSF-1, and/or substitution of one
or more amino acids of the amino acid sequence of CSF-1, and/or
combinations thereof. In certain aspects, inversion of amino acids
and other mutational changes that result in the alteration of the
amino acid sequence are also encompassed. Such an analogue may be
prepared by introducing nucleotide changes into a nucleic acid
molecule such that the desired amino acid changes are achieved upon
expression of the mutagenised nucleic acid molecule, or by
otherwise synthesising an amino acid sequence incorporating the
desired amino acid changes. The substitution of an amino acid may
involve conservative or non-conservative amino acid substitution.
By conservative amino acid substitution, it is meant that an amino
acid residue is replaced with another amino acid having similar, or
substantially similar, characteristics and which does not
substantially alter the desired biological function of the protein.
Exemplary conservative amino acid substitutions are provided in
Table 1 below. In certain aspects, particular conservative
substitutions envisaged are: G, A, V, I, L, M; D, E, N, Q; S, C, T;
K, R, H; and P, N-.alpha.-alkylamino acids. In certain aspects,
conservative amino acid substitutions may be selected on the basis
that they do not have any substantial effect on (a) the structure
of the peptide backbone in the region of the substitution, (b) the
charge or hydrophobicity of the protein at the site of
substitution, (c) the bulk of the side chain at the site of
substitution, and/or combinations thereof.
TABLE-US-00001 TABLE 1 Exemplary conservative amino acid
substitutions Conservative Substitutions Ala Val*, Leu, Ile Arg
Lys*, GIn, Asn Asn Gln*, His, Lys, Arg, Asp Asp Glu*, Asn Cys Ser
Gln Asn*, His, Lys, Glu Asp*, .gamma.-carboxyglutamic acid (Gla)
Gly Pro His Asn, Gln, Lys, Arg* Ile Leu*, Val, Met, Ala, Phe,
norleucine (Nle) Leu Nle, Ile*, Val, Met, Ala, Phe Lys Arg*, Gln,
Asn, ornithine (Orn) Met Leu*, Ile, Phe, Nle Phe Leu*, Val, Ile,
Ala Pro GIy*, hydroxyproline (Hyp), Ser, Thr Ser Thr Thr Ser Trp
Tyr Tyr Trp, Phe*, Thr, Ser Val Ile, Leu*, Met, Phe, Ala, Nle
*indicates preferred conservative substitutions
[0247] In certain aspects, where an analogue is prepared by
synthesis, the analogue may also include an amino acid or amino
acids not encoded by the genetic code, such as
.gamma.-carboxyglutamic acid and hydroxyproline. For example,
D-amino acids rather than L-amino acids may be included. A list of
amino acids not encoded by the genetic code is provided in Table 2.
In a certain preferred embodiments, the analogue is a mimetic of
CSF-1 such as a peptido-mimetic. However, it is not always
necessary that an analogue of CSF-1 have amino acid sequence
identity and/or similarity. In certain aspects an analogue may not
be proteinaceous at all. In certain embodiments an analogue may
have at least 75%, such as at least 80%, at least 85%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
homology with CSF-1.
TABLE-US-00002 TABLE 2 List of amino acids not encoded by the
genetic code .alpha.-aminobutyric acid D-.alpha.-methylhistidine
L-N-methyl-t-butylglycine .alpha.-amino-.alpha.-methylbutyrate
D-.alpha.-methylisoleucine L-norleucine Aminocyclopropane-
D-.alpha.-methylleucine L-norvaline carboxylate Aminoisobutyric
acid D-.alpha.-methyllysine .alpha.-methyl-aminoisobutyrate
Aminonorbornyl- D-.alpha.-methylmethionine
.alpha.-methyl-.alpha.-aminobutyrate carboxylate Cyclohexylalanine
D-.alpha.-methylornithine .alpha.-methylcyclohexyl alanine
Cyclopentylalanine D-.alpha.-methylphenylalanine
.alpha.-methylcyclopentyl alanine L-N-methylisoleucine
D-.alpha.-methylproline .alpha.-methyl-.alpha.-napthyl alanine
D-alanine D-.alpha.-methylserine .alpha.-methylpenicillamine
D-arginine D-.alpha.-methylthreonine N-(4-aminobutyl)glycine
D-aspartic acid D-.alpha.-methyltryptophan N-(2-aminoethyl)glycine
D-cysteine L-N-methylalanine N-(3-aminopropyl)glycine D-glutamate
L-N-methylarginine N-amino-.alpha.-methyl butyrate D-glutamic acid
L-N-methylasparagine .alpha.-napthylalanine D-histidine
L-N-methylaspartic acid N-benzylglycine D-isoleucine
L-N-methylcysteine N-(2-carbamylediyl) glycine D-leucine
L-N-methylglutamine N-(carbamylmethyl) glycine D-lysine
L-N-methylglutamic acid N-(2-carboxyethyl)glycine D-methionine
L-N-methylhistidine N-(carboxymethyl)glycine D-ornithine
L-N-methylleucine N-cyclobutylglycine D-phenylalanine
L-N-methyllysine N-(N-(3,3-diphenylpropyl carbamylmethyl)glycine
D-proline L-N-methylmethionine N-(N-(2,2-diphenylethyl
carbamylmethyl)glycine D-serine L-N-methylnorleucine
1-carboxy-1-(2,2-diphenyl- ethylamino)cyclopropane D-threonine
L-N-methylnorvaline L-.alpha.-methyltryptophan D-tryptophan
L-N-methylornithine N-cycloheptylglycine D-tyrosine
L-N-methylphenylalanine N-cyclohexylglycine D-valine
L-N-methylproline N-cyclodecylglycine D-.alpha.-methylalanine
L-N-medlylserine L-.alpha.-methylnorleucine
D-.alpha.-methylarginine L-N-methylthreonine
L-.alpha.-methylornithine D-.alpha.-methylasparagine
L-N-methyltrytophan L-.alpha.-methylproline
D-.alpha.-methylaspartate L-N-methyltyrosine
L-.alpha.-methylthreonine D-.alpha.-methylcysteine L-N-methylvaline
L .alpha.-methyltyrosine D-.alpha.-methylglutamine
L-N-methylethylglycine L-N-methylhomo- phenylalanine
D-.alpha.-methyltyrosine L-.alpha.-methylleucine
L-.alpha.-methylserine L-.alpha.-methylmethionine
L-.alpha.-methyllysine L-.alpha.-methylphenylalanine
L-.alpha.-methylnorvaline L-.alpha.-methylvaline
[0248] The term "derivative" is to be understood to refer to any
molecule that is derived (substantially derived) or obtained
(substantially obtained) from CSF-1, but retains similarity, or
substantial similarity, in biological function of CSF-1. In certain
aspects, the biological function is the ability to promote organ
development. A derivative may, for instance, be provided as a
result of cleavage of CSF-1 to produce biologically-active
fragments, cyclisation, bioconjugation and/or coupling with one or
more additional moieties that improve, for example, solubility,
stability or biological half-life, or which act as a label for
subsequent detection or the like. A derivative may also result from
post-translational or post-synthesis modification such as the
attachment of carbohydrate moieties, or chemical reaction(s)
resulting in structural modification(s) such as alkylation or
acetylation of an amino acid(s) or other changes involving the
formation of chemical bonds. In a particularly preferred embodiment
of a derivative suitable for use in the present invention, the
derivative is the mature domain of CSF-1. In another preferred
embodiment of a derivative suitable for use in the methods
disclosed herein, the derivative is a biologically active,
C-terminal fragment of CSF-1 (e.g. a CSF-1 fragment comprising the
C-terminal amino acids 1 to 150 of the 536 amino acid protein).
Further embodiments of a derivative of CSF-1 include CSF-1
comprising chemically modified side chains (e.g. pegylation of
lysyl e-amino groups), C- and/or N-termini (e.g. acylation of the
N-terminal with acetic anhydride), or linked to various carriers
(e.g. human serum albumin or histidine (His.sub.6) tag).
[0249] CSF-1 produced from synthetic protein synthesis and chemical
ligation may be used as a source for delivery of large amounts of
protein to animals or infants. These synthesized protein analogues
may have improved potency or pharmacokinetic properties in
comparison to natural CSF-1. CSF-1 protein may be made by first
making individual peptide segments of the protein using solid-phase
peptide synthesis (SPPS) and then after purification, joining the
segments chemically, or via ligatation, in solution to form the
full-length polypeptide. To facilitate the ligation of individual
peptide segments an N-terminal cysteine residue (generally
occurring naturally in the protein sequence) and a C-terminal
thioester (prepared on-resin) may be needed. After synthesis, the
unfolded full-length CSF-1 polypeptide may be folded into its
biologically active conformation.
[0250] Preferably, certain embodiments disclosed involve the
administration of at least one recombinant human CSF-1 (rhCSF-1),
in particular, bacterially-expressed, non-glycosylated recombinant
human CSF-1.
[0251] CSF-1, or a precursor, variant, analogue or derivative
thereof, may be administered to the premature infant or foetus by
any effective method, some of which are known. For example, for the
premature infant, the route of administration can be selected from,
for example, intramuscular (i.m.), intravenous (i.v.), topical,
such as inhalational administration, intratracheal, subcutaneous
(s.c.) administration and/or combinations thereof. On the other
hand, for the foetus, the route of administration may be selected
from i.m., i.v., s.c, intrauterine (i.u.), oral, inhalational
administration to the pregnant mother, and/or combinations
thereof
[0252] In some embodiments, rather then systemic administration, it
may be desirable for the route of administration to be localized to
one or more specific organs or portions of the body, such as by
direct application of the therapeutic to the target treatment area
or areas. In some embodiments, the composition may be an immediate
release dosage form. In other embodiments the composition may be a
time release dosage form, including an implantable controlled
release form. In some embodiments, the dosage forms may include
tablets, dispersions, suspensions, solutions, injections, syrups,
troches, capsules suppositories, aerosols, transdermal patches and
the like.
[0253] Certain embodiments may be administered to ventilated
premature infants using aerosol delivery. "Preterm" or "premature"
birth can be defined as delivery before approximately the
thirty-seventh week of pregnancy. Preterm deliveries can be further
delineated as either "very preterm" (before approximately the
thirty-third week) or "moderately preterm" (between the
approximately thirty-third and approximately the thirty-sixth
weeks). Mechanical ventilation that is heated or non-heated; and
humidified or non-humidified may be performed in infants. Because
of the small tidal volumes and high respiratory rates required for
an infant, ventilation may be time or pressure cycled, with a
continuous flow of gas circulating through the ventilator
circuit.
[0254] Certain embodiments of the composition and methods disclosed
may be include delivery of CSF-1 with ventilation by aerosol
delivery using either a vibrating mesh nebulizer, a jet nebuliser,
a metered dose inhaler (MDI), an ultrasonic nebulizer, or an
electric pump nebuliser. For example, CSF-1 may be administered in
the nebulizer as a bolus dose before the initiation of positive
pressure ventilation. In another example CSF-1 may be delivered
using continuous feed through an infusion set into a nebulizer.
Such a method will allow dosing of aerosol at different rates by
adjusting the flow of the drug/unit of time into the nebulizer.
Nebulization of certain embodiments disclosed herein may ensure
more effective drug delivery to, for example, the lung alveoli of
the premature infants.
[0255] In certain embodiments, nebulization treatment may be
delivered as soon as possible after birth and delivered either
intermittently or with continuous aerosol therapy. Continuous
nebulization therapy may involve the delivery of prescribed dose
of, for example, CSF-1 in diluents or sterile saline or phosphate
buffered-saline over 8 hour periods. CSF-1 may be delivered with a
small volume-limited or large-volume nebuliser with infusion pump.
The volume of CSF-1 to be delivered to infants in the nebulizer may
be in the range of 5-15 ml. The CSF-1 may be added to the pediatric
nebulizer unit in the inspiratory limb of the ventilator circuit
about 10-30 cm away from the patient wye.
[0256] The nebulisers may be placed in the ventilator manifold and
set to deliver a CSF-1 to an infant at a dose ranging from
0.01-1000 .mu.g/hour of CSF-1 continuously over 8 hours for 1 day,
2 days, 3 days, 4 days, 5 days, 6 days or 7 consecutive days.
Typically, such an amount may be, in the case of administration to
the premature infant, in the range of about 0.1 to 500 .mu.g/h,
about 0.05 to 500 .mu.g/h, about 0.2 to 400 .mu.g/h, 0.1 to 1000
.mu.g/h, about 0.05 to 1000 .mu.g/h, about 0.1 to 1000 .mu.g/h,
about 0.5 to 300 .mu.g/h, about 0.75 to 200 .mu.g/h about 1 to 100
.mu.g/h, about 1 to 100 .mu.g/h about 1.25 to 30 .mu.g/h, or about
0.5 to 50 .mu.g/h and, in certain aspects about 0.5 to 200 .mu.g/h,
about 0.05 to 100 .mu.g/h, about 0.25 to 150 .mu.g/h, about 0.5 to
50 .mu.g/h, about 1 to 100 .mu.g/h, about 0.75 to 200 .mu.g/h,
about 0.5 to 30 .mu.g/h, or about 0.1 to 75 .mu.g/h.
[0257] Additionally, in some embodiments, the nebulisers may
deliver CSF-1 to infants at a dose ranging from 0.01-1000 mg/hour
of CSF-1. CSF-1 to the infants at a dose ranging from 0.01-1000
mg/hour of CSF-1 continuously over 8 hours for 1 day, 2 days, 3
days, 4 days, 5 days, 6 days or 7 consecutive days. Typically, such
an amount may be, in the case of administration to the premature
infant, in the range of about 0.1 to 500 mg/h, about 0.05 to 500
mg/h, about 0.2 to 400 mg/h, 0.1 to 1000 mg/h, about 0.05 to 1000
mg/h, about 0.1 to 1000 mg/h, about 0.2 to 400 mg/h, about 0.5 to
300 mg/h, about 0.75 to 200 mg/h about 1 to 100 mg/h, about 1 to
100 mg/h about 1.25 to 30 nig/h, or about 0.5 to 50 mg/h and, in
certain aspects about 0.5 to 200 mg/h, about 0.05 to 100 mg/h,
about 0.25 to 150 mg/h, about 0.5 to 50 mg/h, about 1 to 100 mg/h,
about 0.75 to 200 mg/h, about 0.5 to 30 mg/h, or about 0.1 to 75
mg/h.
[0258] Alternatively, in some embodiments, CSF-1 and/or a
precursor, variant, analogue, derivative thereof or combination
thereof, may be administered to a premature infant directly into
the bloodstream by intramuscular (i.m.), intravenous (i.v.),
subcutaneous (s.c.) administration and/or combinations thereof. The
most familiar type of vascular access is a peripheral intravenous
line (PIV) attached to an i.v. pump. In newborns, PIVs often may be
placed in veins of the hand, foot, or scalp that may enable
delivery of CSF-1 in combination with fluids, nutrients or other
pharmaceutical agents. The PIV may enable the continuous infusion
or pulse infusion of CSF-1 for hours to days. CSF-1 may be
delivered by i.v infusion to the infants at a dose ranging from
0.01-1000 .mu.g/hour of CSF-1 continuously over 8 hours for 1 day,
2 days, 3 days, 4 days, 5 days, 6 days or 7 consecutive days.
Typically, such an amount may be, in the case of administration to
the premature infant, in the range of about 0.1 to 500 .mu.g/h,
about 0.05 to 500 .mu.g/h, about 0.2 to 400 .mu.g/h, 0.1 to 1000
.mu.g/h, about 0.05 to 1000 .mu.g/h, about 0.1 to 1000 .mu.g/h,
about 0.5 to 300 .mu.g/h, about 0.75 to 200 .mu.g/h about 1 to 100
.mu.g/h, about 1 to 100 .mu.g/h about 1.25 to 30 .mu.g/h, or about
0.5 to 50 .mu.g/h and, in certain aspects about 0.5 to 200 ng/h,
about 0.05 to 100 .mu.g/h, about 0.25 to 150 .mu.g/h, about 0.5 to
50 .mu.g/h, about 1 to 100 .mu.g/h, about 0.75 to 200 .mu.g/h,
about 0.5 to 30 .mu.g/h, or about 0.1 to 75 .mu.g/h.
[0259] CSF-1 may be delivered to infants by i.v infusion at a dose
ranging from 0.01-1000 mg/hour of CSF-1 at a dose ranging from
0.01-1000 mg/hour of CSF-1 continuously over 8 hours for 1 day, 2
days, 3 days, 4 days, 5 days, 6 days or 7 consecutive days. CSF-1
may be delivered in the range of about 0.1 to 500 mg/h, about 0.05
to 500 mg/h, about 0.2 to 400 mg/h, 0.1 to 1000 mg/h, about 0.05 to
1000 mg/h, about 0.1 to 1000 mg/h, about 0.2 to 400 mg/h, about 0.5
to 300 mg/h, about 0.75 to 200 mg/h about 1 to 100 mg/h, about 1 to
100 mg/h about 1.25 to 30 mg/h, or about 0.5 to 50 mg/h and, in
certain aspects about 0.5 to 200 mg/h, about 0.05 to 100 mg/h,
about 0.25 to 150 mg/h, about 0.5 to 50 mg/h, about 1 to 100 mg/h,
about 0.75 to 200 mg/h, about 0.5 to 30 mg/h, or about 0.1 to 75
mg/h.
[0260] In some embodiments, the CSF-1, at least one precursor, at
least one variant, at least one analogue, at least one derivative,
or combinations thereof, may be administered in the form of a
composition comprising a carrier (e.g. a pharmaceutically
acceptable vehicle or diluent such as saline). Infants that are
intubated may receive CSF-1 as an aerosol with nebulizer treatment
before or in combination with another pharmaceutical agent
including but not limited to surfactants such as artificial or
natural surfactants, such as EXOSURF, PUMACTANT, KL-4, VENTICUTE.
ALVEOFACT, CUROSURF, INFASURF or SURVANTA, anti-inflammatory agents
or corticosteroids, fluids for hydration, heparin, albuterol,
antibiotics, ibuprofen, nutritional supplements, vitamin
supplements, mineral supplements, sildenafil, other colony
stimulating factors such as G-CSF or GM-CSF, and/or IGF-I, IGF-II,
HGF, EGF, or mixtures thereof.
[0261] In some embodiments, CSF-1 may be delivered to the foetus
via the pregnant woman by i.v bolus injection typically at a
concentration range of 0.1-1 g/kg body weight. The exact amount may
vary depending upon a variety of factors including the relative
activity, metabolic stability and length of action of the CSF-1,
precursor, variant, analogue or derivative thereof, the route and
time of administration, the degree, or likely degree, of organ
underdevelopment, and, in the case of the foetus, the general
health of the pregnant mother.
[0262] Alternatively, in some embodiments, CSF-1 may be delivered
by aerosol nebulisation with or without ventilation. CSF-1 may be
administered in the nebulizer as a bolus dose before the initiation
of positive pressure ventilation. Alternatively, CSF-1 may be
delivered by continuous feed through an infusion set into a
nebulizer. The doses and timing for CSF-1 delivery may be similar
to infants described above.
[0263] In some embodiments, in pregnant warm blooded animals, such
as mares, pigs, catties, dogs and other livestock, CSF-1 may be
delivered to the foetus by bolus injection into the bloodstream of
the mother using intramuscular, intravenous, or subcutaneous
administration or combinations of the above at a concentration
range typically from 0.1-1 g/kg of body weight. Alternatively, in
other embodiments, CSF-1 may be delivered to such animals or to
infant animals by aerosol nebulisation with or without ventilation
by administration of a bolus in the nebuliser before initiation of
positive pressure or by using a continuous feed through an infusion
set into a nebuliser. The doses and timing for CSF-1 delivery may
be similar to human infants described above.
[0264] In some embodiments, the CSF-1, at least one precursor, at
least one variant, at least one analogue, at least one derivative,
or combinations thereof, may be administered in the form of a
composition comprising a carrier (e.g. a pharmaceutically
acceptable excipient, vehicle or diluent). Such compositions may
further comprise other therapeutic agents (e.g. IGF-I, IGF-II, HGF,
EGF, or mixtures thereof) and may be formulated by, for example,
employing conventional solid or liquid excipients, vehicles,
diluents or combinations thereof, as well as pharmaceutical
additives of a type appropriate to the mode of desired
administration (for example, excipients, binders, preservatives,
stabilisers, flavours, colorants, buffers etc.). Non-limiting
examples of suitable excipients, vehicles and diluents may be found
in Gennaro, Alfonso, Remington's Pharmaceutical Sciences, 18.sup.th
edition. Mack Publishing Co. (1990), in Gennaro, Alfonso,
Remington: The Science and Practice of Pharmacy, 19.sup.th edition
(1995) and, 20.sup.th edition (2003) and/or in University of the
Sciences in Philadelphia (editor) Remington: The Science and
Practice of Pharmacy and 21.sup.St edition (2005), the entire
contents of each of which is hereby incorporated by reference.
Examples of some excipients include sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil, saline solutions, aqueous dextrose solutions, aqueous
glycerol solutions, buffers, proteins such as serum albumin, amino
acids such as aspartic acid, glutamic acid, lysine, arginine,
glycine, histidine, peptides, carbohydrates such as saccharides,
polymeric additives, antimicrobial agents, sweeteners,
antioxidants, antistatic agents, surfactants (e.g., polysorbates
such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids,
fatty acids), steroids (e.g., cholesterol), and chelating agents,
or combinations thereof. Examples of some buffers that may be used
include salts prepared from an inorganic acid such as mineral acid
salts, such as hydrochlorides, bromides, and sulfates and salts
prepared from an organic acid or base, such as salts of citric
acid, propionic acid, malonic acid, ascorbic acid, gluconic acid,
carbonic acid, tartaric acid, succinic acid, acetic acid, or
phthalic acid or Tris, tromethamine hydrochloride, phosphate
buffers, or combinations thereof.
[0265] The CSF-1, at least one precursor, at least one variant, at
least one analogue, at least one derivative, or combinations
thereof, may be administered in any amount that is effective in
treating complications arising from low birth weight in mammals,
such as humans, pigs, horses, dogs or other livestock, such as in
premature infants or foetuses, in promoting organ development in
the premature infant or foetus and/or in promoting bone growth
and/or enhancing bone development and/or maturation in mammals,
such as humans, pigs, horses, dogs or other livestock, such as in
premature infants or foetuses. Typically, such an amount will be,
in the case of administration to the premature infant, in the range
of about 0.1 to 500 .mu.g/kg/day, about 0.05 to 500 .mu.g/kg/day,
about 0.1 to 500 .mu.g/kg/day, about 0.2 to 400 .mu.g/kg/day, about
0.5 to 300 .mu.g/kg/day, about 0.75 to 200 .mu.g/kg/day about 1 to
100 .mu.g/kg/day, about 1 to 100 .mu.g/kg/day about 1.25 to 30
.mu.g/kg/day, or about 0.5 to 50 .mu.g/kg/day and, in certain
aspects more preferably, about 0.5 to 200 .mu.g/kg/day, about 0.05
to 100 .mu.g/kg/day, about 0.25 to 150 .mu.g/kg/day, about 0.5 to
50 .mu.g/kg/day, about 1 to 100 .mu.g/kg/day, about 0.75 to 200
.mu.g/kg/day, about 0.5 to 30 .mu.g/kg/day, or about 0.1 to 75
.mu.g/kg/day and for the foetus (where administration is via the
pregnant mother). However, the exact amount may substantially vary
depending upon a variety of factors including, but not limited to,
the relative activity, metabolic stability and length of action of
the CSF-1, at least one precursor, at least one variant, at least
one analogue, at least one derivative, or combinations thereof, the
route and time of administration, the degree, or likely degree, of
organ underdevelopment, the type and severity of complications
arising from the low birth weight, the age of the foetus or
premature infant and, in the case of the foetus, the general health
of the pregnant mother.
[0266] Administration of the CSF-1, at least one precursor, at
least one variant, at least one analogue, at least one derivative,
or combinations thereof, to the premature infant may commence upon
birth and continue until a desired level of organ development is
observed. Thus, for the lung, a desired level of lung development
may be achieved when the saccular phase has been completed and/or
the infant no longer requires a mechanical ventilator. For the
kidney, a desired level or organ development may be achieved when
renal function has been improved and/or the numbers of nephrons has
been increased since birth. For the foetus, administration of the
CSF-1, at least one precursor, at least one variant, at least one
analogue, at least one derivative thereof, or combinations thereof,
may commence from about gestational week 4, about gestational week
5, about gestational week 7, about gestational week 10, about
gestational week 12, about gestational week 14, or about
gestational week 19, but in certain aspects preferably, commences
after about week 20, about week 22, about week 24, or about week
26.
[0267] Certain embodiments disclosed also encompasses the
administration of at least one nucleic acid molecule encoding
CSF-1, at least one precursor, at least one variant, at least one
analogue, at least one derivative thereof, or combinations thereof,
such that the CSF-1, at least one precursor, at least one variant,
at least one analogue, at least one derivative thereof, or
combinations thereof, are expressed from the nucleic acid molecule
by the premature infant or foetus (and/or pregnant mother).
[0268] In certain embodiments, suitable nucleic acid molecules may
be single or double stranded, such as mRNA, ssRNA, dsRNA, ssDNA and
dsDNA. However, in certain preferred aspects, the nucleic acid
molecule will be dsDNA.
[0269] The nucleic acid molecule may be incorporated into an
expression construct or vector in accordance with any effective
method, some of which are known. Typically, in certain aspects the
nucleic acid molecule will be introduced into such an expression
construct or vector such that transcription of the nucleic acid
molecule is driven by a promoter sequence provided by the
expression construct or vector. In certain aspects it is preferred
that the expression construct or vector is adapted for expression
in mammalian cells, tissues or organs such as lung and/or kidney
cells.
[0270] In certain aspects it is preferred that the nucleic acid
molecule is incorporated into at least one viral vector such as an
adenovirus, lentivirus or poxvirus vector.
[0271] The nucleic acid molecule may be administered to the
premature infant or foetus (via the pregnant mother) by any
effective method, some of which are well know (e.g.
liposome-mediated transfection, or for viral vectors, viral
transformation).
[0272] Administration of the at least one nucleic acid molecule
encoding the CSF-1, at least one precursor, at least one variant,
at least one analogue, at least one derivative thereof, or
combinations thereof, to the premature infant may occur upon birth,
whereas for the foetus, administration of the at least one nucleic
acid molecule encoding the CSF-1, at least one precursor, at least
one variant, at least one analogue, at least one derivative
thereof, or combinations thereof, may occur from about gestational
week 4, about gestational week 5, about gestational week 7, about
gestational week 10, about gestational week 12, about gestational
week 14, or about gestational week 19, but in certain aspects
preferably, commences after about week 20, about week 22, about
week 24, or about week 26.
[0273] Certain methods disclosed are particularly suitable for
increasing growth and/or enhancing lung maturation and
nephrogenesis in the kidney.
[0274] In certain aspects disclosed, the methods of promoting
growth and/or enhanced lung development and/or maturation in a
premature infant or foetus, comprise the step of administering to
the infant or foetus;
[0275] at least one colony stimulating factor-1 protein (CSF-1), or
at least one precursor, at least one variant, at least one
analogue, at least one derivative thereof, or combinations thereof,
or
[0276] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), at least one precursor, at
least one variant, at least one analogue, at least one derivative
thereof, or combinations thereof.
[0277] In certain aspects disclosed, the methods of promoting
growth and/or enhanced lung development and/or maturation in a
premature infant or foetus, comprise the step of administering to
the infant or foetus;
[0278] colony stimulating factor-1 protein (CSF-1), or precursor,
variant, analogue, or derivative thereof or
[0279] nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), precursor, variant, analogue, or
derivative thereof.
[0280] And, in certain aspects disclosed a method of promoting
growth, maturation and/or enhanced kidney development in a
premature infant or foetus is provided, said method comprising the
step of administering to the infant or foetus;
[0281] colony stimulating factor-1 protein (CSF-1), or a precursor,
variant, analogue or derivative thereof, or
[0282] a nucleic acid molecule encoding said colony stimulating
factor-1 protein (CSF-1), or a precursor, variant, analogue or
derivative thereof.
[0283] In certain aspects disclosed, methods are provided for
promoting growth, maturation and/or enhanced kidney development in
a premature infant or foetus, comprising the step of administering
to the infant or foetus;
[0284] at least one colony stimulating factor-1 protein (CSF-1), at
least one precursor, at least one variant, at least one analogue,
at least one derivative thereof or combinations thereof, or
[0285] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), at least one precursor, at
least one variant, at least one analogue, at least one derivative
thereof, or combinations thereof.
[0286] It is anticipated that the certain methods disclosed may be
equally applicable to newborn non-human animals and non-human
foetuses. In particular, it is anticipated that the methods of the
invention might be used in relation to warm blooded animal, for
example. But not limited to, thoroughbred horses, stud animals and
companion animals such as dogs and cats.
[0287] In order that the nature of the present inventions may be
more clearly understood, preferred forms thereof will now be
described with reference to the following non-limiting
examples.
[0288] In some embodiments, the methods of treating complications
arising from low birth weight in mammals, such as humans, pigs,
horses, dogs or other livestock, such as in premature infants or
foetuses, in promoting organ development in the premature infant or
foetus and/or in promoting bone growth and/or enhancing bone
development and/or maturation in mammals, such as humans, pigs,
horses, dogs or other livestock, such as in premature infants or
foetuses may include co-treatment with other therapeutic
modalities.
[0289] For example, in some embodiments, prior to birth the methods
may include treatment of the mother by administering to the
mother
[0290] at least one colony stimulating factor-1 protein (CSF-1), at
least one precursor, at least one variant, at least one analogue,
at least one derivative thereof or combinations thereof, or
[0291] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), at least one precursor, at
least one variant, at least one analogue, at least one derivative
thereof, or combinations thereof;
[0292] in combination with treatment with one or more drugs or
other substances to treat an underlying cause of low birth weight
or complications resulting from low birth weight, such as drugs for
hypertension, infections or diabetes, coticosteroids, tocolytics,
nutritional supplements, vitamin supplements, mineral supplements,
albuterol, antibiotics, heparin, other colony stimulating factors
such as G-CSF or GM-CSF, surfactants such as artificial or natural
surfactants, such as EXOSURF, PUMACTANT, KL-4, VENTICUTE.
ALVEOFACT, CUROSURF, INFASURF or SURVANTA, IGF-I, IGF-II, HGF, EGF,
sildenafil, ibuprofen or in combination with surgical treatment of
the mother and/or fetus.
[0293] In some embodiments, prior to birth the treatment of the
mother may include treatment in a dosage form that enhances
transplacental drug delivery, such as using a liposomal form of the
administered treatment. Such liposomal forms may be created having
a variety of sizes, charges and lipid compositions. In some
embodiments, such liposomal forms may be anionic small unilamellar
liposomes.
[0294] In some embodiments after birth, the methods herein may
include administering to the premature or low birth weight
infant
[0295] at least one colony stimulating factor-1 protein (CSF-1), at
least one precursor, at least one variant, at least one analogue,
at least one derivative thereof or combinations thereof, or
[0296] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), at least one precursor, at
least one variant, at least one analogue, at least one derivative
thereof, or combinations thereof;
[0297] in combination with treatment with one or more drugs or
other substances to treat an underlying cause or complication of
low birth weight, such as coadministration of surfactant therapy,
such as administration of surfactant, such as artificial or natural
surfactants, such as EXOSURF, PUMACTANT, KL-4, VENTICUTE.
ALVEOFACT, CUROSURF, INFASURF or SURVANTA, albuterol, heparin,
sildenafil, ibuprofen, nutritional supplementation, vitamin and/or
mineral supplementation, surgery, IGF-I, IGF-II, HGF, EGF,
antibiotic therapy, other colony stimulating factors such as G-CSF
or GM-CSF, other drug therapy to treat any complications of low
birth weight.
[0298] In some embodiments, the CSF-1 may be provided as a kit,
such as at least one colony stimulating factor-1 protein (CSF-1),
at least one precursor, at least one variant, at least one
analogue, at least one derivative thereof or combinations thereof,
or
[0299] at least one nucleic acid molecule encoding said colony
stimulating factor-1 protein (CSF-1), at least one precursor, at
least one variant, at least one analogue, at least one derivative
thereof, or combinations thereof in conjunction with instructions
for administration, including dosing instructions, adverse event
information and adverse interaction information, other dosing
equipment or coadministration therapy-related drugs, equipment
and/or instructions.
[0300] For example, in some embodiments of the kits, at least one
colony stimulating factor-1 protein (CSF-1), at least one
precursor, at least one variant, at least one analogue, at least
one derivative thereof or combinations thereof, may be provided as
an ampule that may be added to a nebulisation unit or as a
pre-dosed nebulisation system that attaches to a ventilator system
for premature babies. In some embodiments of the kits, at least one
colony stimulating factor-1 protein (CSF-1), at least one
precursor, at least one variant, at least one analogue, at least
one derivative thereof or combinations thereof, may be provided in
pre-dosed syringes, with infant-specific i.v. lines and
infant-sized needles or other administration devices. In some
embodiments, CSF-1 kits may be provided as a mixture of at least
one colony stimulating factor-1 protein (CSF-1), at least one
precursor, at least one variant, at least one analogue, at least
one derivative thereof or combinations thereof with saline,
buffered saline or other diluent and/or as a mixture with other
colony stimulating factors, such as G-CSF and/or CM-CSF, drugs such
as albuterol, antibiotics, or IGF-I, IGF-II, HGF, EGF, or a
surfactant or mixtures thereof.
EXAMPLES
Example 1
[0301] Characterizing the Location and Arrival of Resident Renal
Macrophages
[0302] Materials and Methods
[0303] CSF-1
[0304] All experiments described herein used human recombinant
CSF-1.
[0305] Isolation of Renal Embryonic Tissue and Macrophages
[0306] Outbred CD1 female mice were mated and sacrificed by
cervical dislocation for the collection of control kidney tissue at
E15.5. Embryonic kidneys were dissected in ice-cold PBS and stored
at -70.degree. C. in preparation for RNA extraction.
[0307] Male c-fms transgenic mice (Sasmono, et al., 2003, supra)
were mated to CD1 outbred females. Pregnant females were sacrificed
at E15.5 and transgenic offspring were determined by visualization
of c-fins EGFP expression in the placenta of the embryos. The
kidneys of both transgenic and non-transgenic embryos were
dissected separately in ice-cold PBS. 10-20 kidneys were incubated
in 1 ml Dissociation Media (1 mg/ml Collagenase B. 1.2 U/ml
Dispase, 5 U/ml DNase II, in HANKS media) at 37.degree. C. for 20
min. Kidneys were then dissociated with a P-1000 and incubated for
a further 5 min at 37.degree. C. This step was repeated before the
kidneys were dissociated with a 23-gauge syringe and passed through
a 40 uM cell strainer (BD Bioscience). An equal volume of ice-cold
PBS was washed through the strainer and the cells were centrifuged
at 3000.times.rpm for 5 min. The supernatant was discarded and the
cells resuspended in 2-3 ml of ice-cold PBS. The cells were passed
again through a 40 uM cell strainer, checked under a microscope to
ensure they were a single-cell suspension, and stored on ice ready
for fluorescence activated cell sorting (FACS). Isolation of EGFP
positive macrophages was carried out on a FACS Vantage SE DiVa flow
cytometer (BD Biosciences). Approximately 200 transgenic kidneys
were subjected to FACS analysis with non-transgenic littermate
kidneys used as a reference. All animal experimentation was covered
by Animal Ethics Committee number IMB/479/03/NIH.
[0308] RNA In Situ Hybridisation
[0309] Section RNA in situ hybridisation was performed as
previously described (Roche DIG Application Manual) with minor
modifications (Holmes G P, et al., Mech. Dev., 79:57-72, 1998).
Sections were dehydrated through an ethanol series prior to
hybridization overnight at 65.degree. C. Posthybridisation washes
consisted of 6.times.SSC (5 min, 65.degree. C.), 2.times.SSC/50%
formamide/10 mM EDTA (30 min, 65.degree. C.), 2.times.SSC
(2.times.30 min, 65.degree. C.) and 0.2.times.SSC (2.times.30 min,
65.degree. C.).
[0310] Isolation and Preparation of Tissue for Confocal
Analysis
[0311] Male c-fms transgenic mice (Sasmono, et al., 2003, supra)
were mated to CD1 outbred females. Pregnant females were sacrificed
at E11.5, E 12.5, E15 and newborn and transgenic offspring were
determined by visualization of c-fms EGFP expression in the
placenta of the embryos. The kidneys of transgenic embryos were
dissected separately in ice-cold PBS. c-fms kidneys were fixed in
4% paraformaldehyde for 3 hrs at room temperature. The kidneys were
subsequently equilibrated in 30% sucrose overnight at 4.degree. C.
before being mounted in Tissue-Tek OCT medium in isopentane cooled
over dry ice.
[0312] Results
[0313] To facilitate the isolation and characterisation of tissue
macrophages, we have generated transgenic mice in which the control
elements of the c-fms gene direct expression of a green fluorescent
protein (EGFP) reporter. In the so-called MacGreen mice, all tissue
macrophages, including interstitial macrophages in the kidney and
phagocytes in the embryo from the earliest appearance in the yolk
sac, express high levels of EGFP fluorescence (Sasmono, et al.,
2003, supra).
[0314] As shown in FIG. 1 (left), GFP+ cells isolated from
embryonic day 15.5 kidneys of c-fms-EGFP transgenic mice
represented 2.8% of total cells.
[0315] Confocal analysis of kidneys from these mice revealed that
these cells appear within the kidney from 12 days post coitum (dpc)
and are spread throughout the renal interstitium. As the tubules of
the developing nephrons arise and the interstitial space contracts,
the renal macrophages become intimately associated with the
basement membranes of the adjacent proximal and distal tubules.
Their cellular processes wrap around adjacent tubules (FIG. 1,
right) facilitating an intimate relationship with the cells of
these tubules.
Example 2
[0316] Human Recombinant CSF-1 has a Growth-Promoting Effect on the
Developing Kidney.
Materials and Methods
[0317] Metanephric Organ Culture
[0318] Metanephric organ culture was used to test the effect of
recombinant CSF-1 on the developing metanephros. Metanephroi from
E11.5 mice were grown for 1-6 days on Poretics 13 mm polycarbonate
inserts (Osmonics Inc) with a membrane pore size of 1.0 .mu.m at
37.degree. C. with 5% CO2 in 300 .mu.l of DMEM/Hams F12 media
(Invitrogen) supplemented with 50n/ml transferrin and 20 mM
glutamine. Metanephroi were either grown in media alone or media
supplemented with CSF1 to a final concentration of 100U/.mu.L (1.25
ng/.mu.L).
[0319] Immunofluorescence
[0320] Co-immunofluorescence for calbindin-D28K and WT1 was
performed at the end of the culture period to visualise growth and
differentiation of the ureteric epithelium and formation of early
nephron structures in explanted metanephroi as previously described
(Piper, et al., 2002, Int. J. Dev. Biol., 46, 545). Metanephroi
were fixed in 100% methanol at -20.degree. C. for 20 minutes.
Monoclonal anti-calbindin-D28K (Sigma Chemical Company) was used at
a dilution of 1:100 and C-terminal WT1 polyclonal antibody C19
(Santa Cruz, S.C.-192) was used at a dilution of 1:100. Secondary
antibodies used were Cy3-conjugated anti-rabbit IgG (Sigma) at a
dilution of 1:500, and Alexa Fluor 488 conjugated goat anti-mouse
(Molecular Probes) at a dilution of 1 :200. Explants were also
treated with DAPI for visualisation of individual nuclei. Digital
images were captured using a Dage "MTI" peltier cooled charge
coupled device digital camera attached to an AX70 Olympus
microscope, and artificially coloured and overlayed using Adobe
Photoshop 7 software.
[0321] Statistical Analysis
[0322] To semi-quantitatively assess the effects of CSF-1
conditioned media on in vitro metanephric development, branch tips,
branch points and WT1-positive bodies (forming nephrons) present in
each explanted metanephros were counted. A one-way ANOVA followed
by a Tukey's post-hoc test was used to determine if there was a
significant difference in the number of ureteric tip, branch and/or
WT1 positive bodies in CSF-1-treated metanephroi in comparison to
untreated metanephroi.
[0323] Results
[0324] At 11.5 dpc, mouse embryonic kidneys (metanephroi) are
comprised of a T-shaped UB surrounded by MM. These can be isolated
via microdissection and cultured as explants for up to 6 days.
During culture, the ureteric epithelium undergoes branching
morphogenesis and a mesenchyme-to-epithelial transition occurs,
generating immature nephrons. Metanephric explant culture is an
excellent model system for examining kidney development.
[0325] We have established kidney explant culture to screen
secreted factors for their ability to perturb or promote kidney
development. One of the proteins that we have added to explant
cultures is human recombinant CSF-1. The C-terminal 150 amino acids
of this protein is bioactive and contains 4 helix bundles similar
to those of other members of this cytokine family (G-CSF, GM-CSF).
It can be produced in bacteria and correctly fold to form a
bioactive protein.
[0326] Addition of recombinant CSF1 to kidney explants resulted in
a dramatic and statistically significant enhancement of renal
development (FIG. 2). Explants grew with the same morphology as
normal, but at a much greater rate and to a greater overall size.
This was evident after only 24 hours and detectable at doses as low
as 100U/.mu.L. This implies that renal CSF1 signalling via c-fms
(CSFR1) on resident macrophages plays a positive role in kidney
development.
Example 3
[0327] Human Recombinant CSF-1 has a Growth-Promoting Effect on the
Damaged Adult Kidney
[0328] Materials and Methods
[0329] Mouse Surgery
[0330] Male c-fms transgenic mice (20-25 g, Monash University
Animal House, Australia), carrying a green fluorescent protein
(GFP) driven by the c-fms (CSF-1 R) promoter, were divided into 3
groups. The first group (n=4) were anaesthetized with 2% inhaled
isofluorane (Abbott Australasia Pty Ltd, Kurnell, Australia) and
ischemia/reperfusion (IR) injury was induced via 50 minutes of left
renal artery clamping. A vascular clamp (0.4-1.0 mm; S&T Fine
Science Tools, CA) was used for this procedure via a flank
incision. Each mouse in this group received three intraperitoneal
injections of CSF-1 (20 .mu.G/timepoint) at day 3, 4, and 5 after
initiation of IR injury. The right contralateral kidney served as a
control for CSF-1 treatment.
[0331] The second group of mice (n=4) underwent 50 minutes of left
renal artery clamping and vehicle injections (phosphate buffered
saline; PBS) were administered at days 3, 4, and 5. The third group
(n=5) of mice served as a sham-operated control group where the
animals were anaesthetized and a flank incision was performed
without renal artery clamping. All experiments were approved by a
Monash University Animal Ethics Committee which adheres to the
"Australian Code of Practice for the Care and Use of Animals for
Scientific Purposes".
[0332] Preparation of Tissue for Microscopy
[0333] At 1 week after IR injury, mice were perfusion-fixed with 4%
paraformaldehyde (PFA) under 2% inhaled isofluorane anesthesia. A
midline incision was made to expose both the heart and the inferior
vena cava. A 27'' gauge needle was injected into the left ventricle
of mice and flushed for 3 minutes with PBS containing heparin and
NaNitropruside. At the same time, the inferior vena cava was cut to
provide an outlet for the perfusate. Mice were perfusion-fixed with
preheated 4% PFA at 100 mmHg for 10 minutes. Mid-coronal kidney
sections were immersion fixed in 4% PFA (Sigma-Aldrich), embedded
in paraffin wax and cut at 4 .mu.m. Sections were stained with
haematoxylin and eosin and Periodic Acid Schiff (PAS) for
histolopathological analysis.
[0334] For fluorescence visualization of c-fms-EGFP-macrophages,
following perfusion-fixation kidney tissue was fixed in 4% PFA for
8 hours, transferred to PBS containing 30% sucrose for overnight
incubation at 4.degree. C., embedded in O.C.T. (TissueTek.RTM.
Japan) and stored at -80.degree. C. Frozen sections were cut (5
.mu.m) using a cryostat (Leica, Germany) and visualized under an
Olympus Provis AX70 fluorescent microscope.
[0335] For determination of collagen type IV localisation, sections
were incubated with 1% bovine serum albumin (BSA). A goat
anti-human collagen type IV primary antibody (Southern Biotech,
Birmingham, Ala.; 1:100 dilution) was added for 1 hour followed by
a chicken anti-goat Alexa Fluor 647 conjugate (1:1000; Molecular
Probes). Sections were mounted with Fluorescent Mounting Media
(DakoCytomation) before visualization under an Olympus Provis AX70
fluorescent microscope.
[0336] Measurement of Proteinuria and Urine Creatinine
[0337] Mice were housed in metabolic cages, with free access to
food and water on the days of urine collection. Albumin and
creatinine levels, and the albumin/creatinine ratio were measured
in 24 hour urine samples using an Albuwell murine microalbuminuria
ELISA assay and creatinine companion kit (both from Exocell Inc.),
respectively.
[0338] Results
[0339] Promotion of Renal Repair in IR Kidneys with CSF-1
Treatment
[0340] At 1 week widespread damage was evident in IR kidneys of
mice receiving vehicle treatment. Characteristic of the renal
damage was extensive loss of tubular epithelium and tubular cast
formation particularly in the outer medullary region where numerous
tubular casts were observed (FIGS. 3A & B). In these IR
kidneys, interstitial matrix expansion was associated with the
accumulation of extracellular matrix proteins resulting in the
development of interstitial fibrosis (FIGS. 3 A & B). 50
minutes of IR injury led to a severe inflammatory response and the
extensive loss of the tubular epithelium, without the necrotic
insult demonstrated with longer durations of renal artery
clamping.
[0341] In comparison, mice with IR injury receiving CSF-1 treatment
starting at 3 days after initiation of injury, showed normal renal
histology in the cortical and medullary regions (FIG. 3C). In the
outer medullary region the tubules appeared intact with complete
re-epithelialisation evident (FIG. 3D). There were very few tubular
casts apparent in these kidneys (FIGS. 3 C & D). Furthermore,
in IR kidneys with CSF-1 treatment there was a marked attenuation
of interstitial matrix expansion as a result of diminished
interstitial fibrosis.
[0342] Functional Recovery of IR Mice with CSF-1 Treatment
[0343] Urine protein levels were measured in 24 hour urine samples
obtained from sham-operated control mice and IR mice with/without
CSF-1 delayed administration (FIG. 4). There was a significant
reduction in the urine protein levels in IR kidneys with CSF-1
treatment compared to IR kidneys without CSF-1 administration
(324.4+250.1 vs. 23.84+15.7; P<0.03). Although creatinine was
not found to be significantly different between the mice with IR
injury compared to control or IR+CSF-1 treatment, the
albumin/creatinine ratio was found to be significantly reduced
following CSF-1 treatment (42.24.+-.25.60 vs. 604.22+496.20;
P<0.03). The fact that urinary creatinine levels were not
significantly different between groups is probably reflective of
right kidney compensation following the left unilateral renal
artery clamping. However, the albumin/creatinine ratio is a good
indicator of kidney function.
[0344] CSF-1 Reduces Type IV Collagen Accumulation and the Number
of Interstitial Macrophages in the IR Kidney
[0345] In c-fms-GEF IR mice, increased numbers of GFP-positive
macrophages in the renal interstitium were associated with collagen
type IV accumulation and interstitial expansion (FIGS. 5A & B).
Tubular cast formation in the majority of proximal tubules was
observed (FIG. 5B) as a result of loss of epithelial cell
integrity. Following CSF-1 treatment, IR mouse kidneys displayed
decreased numbers of interstitial GFP-macrophages, and a normal
tubulointerstitium that contained a fine framework of collagen type
IV comparable to normal kidneys (FIGS. 5C & D). Furthermore,
the CSF-1 treated IR kidneys showed normal architecture with an
intact proximal tubular epithelial cell lining that was surrounded
by few GFP -macrophages in the interstitium without evidence of
fibrosis (FIG. 5D).
CONCLUSIONS
[0346] CSF-1 administration to mice with IR injury resulted in the
promotion of renal repair by accelerated tubular epithelial cell
replacement and attenuation of interstitial fibrosis. IR is a model
of acute tubular necrosis that is characterized pathologically by
tubule cell damage resulting from prolonged renal ischemia. The
accumulation of macrophages was distinctly observed in the
tubulointerstitium of IR kidneys at 1 week after the initiation of
injury. This was associated with numerous tubular casts formed as a
result of the complete loss of the loss of functioning tubular
epithelial cells leading to diffuse effacement and loss of the
proximal tubule cell brush border. In IR kidneys large numbers of
macrophages were also evident in the interstitium due to
inflammation induced from hypoxic insult. Elevated urine protein
levels were also observed in IR mice subsequent to the loss of
renal function.
[0347] CSF-1 was found to promote both a structural and functional
recovery of the kidneys from IR mice. Importantly, CSF-1 treatment
was initiated at 3 days after IR injury, a time when renal damage
and inflammation is already evident. CSF-1 treatment of IR mice
resulted in a restoration of the tubular epithelium, attenuation of
interstitial matrix expansion and recovery of renal function,
comparable to control kidneys.
[0348] Markedly reduced numbers of interstitial macrophages were
observed in the in CSF-1-treated IR kidneys, compared to IR kidneys
without treatment. The population of macrophages observed in the IR
mice with CSF-1-treatment appeared to surround re-epithelialised
renal tubules and were present without evidence of extracellular
matrix accumulation.
Example 4
[0349] Materials and Methods
[0350] Newborn Mouse Analysis
[0351] C57/B16 mice were time-mated and the mouse pups given three
intraperitoneal (i.p.) injections of recombinant human CSF-1
(Chiron Corporation, Emeryville, Calif., USA) at days 1, 2 and 3
after birth. The CSF-1 was administered at a dose of 1 .mu.g/g body
weight at a concentration of 1 .mu.g/ml where the final volume did
not exceed 50 .mu.l per injection. Litter mate aged-matched control
mice received vehicle (phosphate buffered saline) control
injections of the same volume. The CSF-1-treated mouse pups were
toe and tail clipped for identification and returned to their
mothers. The CSF-1-treated mouse pups and the control-treated pups
were killed at day 29 for light microscopy of kidney and lung
histology and estimation of glomerular number. Histology
[0352] Kidney and lung tissue was taken from CSF-1-treated and
litter mate control-treated mice, immersion fixed in 4%
paraformaldehyde and processed on short cycle before embedding into
paraffin for histological analysis.
[0353] The paraffin-embedded kidneys were each sectioned at 4
microns using a microtome (Leitz Wetzlar, Germany), and the
sections were then placed on poly-L-lysine slides and left to
adhere for 3 hours at 70.degree. C. The slides were dewaxed in
xylene, rehydrated through graded alcohols to water before staining
with haematoxylin and eosin by standard methods.
[0354] Stereological Assessment of Glomerular Number and Kidney
Volume
[0355] At day 29, mice with/without delivery of CSF-1 were killed
and their kidneys removed and processed for methacrylate embedding
and subsequent stereological counting. The processing involved
placing the kidneys into 10% formalin for 48 hours, 70% ethanol
overnight, and then three one hour washes with 100% ethanol
followed by butanol overnight and 72 hours in infiltration solution
(Technovik 7100, Electron Microscopy Sciences, QLD). Kidneys were
then embedded in methacrylate resin (Technovik 7100) and left to
set for three days. Once set, the backs of blocks were made using
Technovik 3040 solution (Technovik, Electron Microscopy Sciences,
QLD) and allowed to set for one hour. Sections were then serial
sectioned at 20 .mu.m using a microtome, and every 10th and 11th
section was collected beginning at a random number.
[0356] Sections were stained using periodic acid-Schiff (PAS)
staining, however the time in reagents was extended compared to a
standard paraffin protocol, due to the slow rate of penetration
through the resin.
[0357] Stereological counting was performed firstly on a micro
fische reader to determine kidney volume using the Cavalieri
Principal (Kett, M M et al., 1996). Complete sections were used for
nephron number estimation using a physical dissector/fractionator
combination (Bertram, J F, 1995)--whereby pairs of sections are
projected onto an unbiased 2.times.2 cm counting grid. Grid points
that lay upon kidney tissue, glomeruli and renal corpuscles were
tabulated, as were glomeruli disappearing events across each slide.
Using a formula, the nephron number was estimated.
[0358] Results and Discussion
[0359] The administration of CSF-1 (1 .mu.g/g body weight) was
given to mouse pups at Day 1, 2 and 3 after birth. In CSF-1-treated
mice, there was an overall increase in body weight (FIG. 6) and
individual kidney weights (FIG. 7). Mice killed at day 29 we
observed to have a 37% increase in overall body weight compared to
age-matched litter mates. CSF-1-treated mice had a 27% and 33%
increase in left and right kidney weights, respectively, at the
same timepoint. The mice with CSF-1 treatment were also found to
have a 29% increase in the number of kidney glomeruli compared to
age-matched litter mate control mice treated with phosphate
buffered saline (FIG. 8). This demonstrates that CSF-1 can promote
increased nephro genesis that is associated with increased kidney
growth.
[0360] FIG. 9 demonstrates the histology of the kidneys and lungs
from CSF-1 (FIG. 9B, D, F) and control-treated (FIG. 9A, C, E) mice
at day 29. There were no obvious structural abnormalities observed
in the kidneys from mice following CSF-1 injection compared to
control animals. On the other hand, the lungs from CSF-1-treated
mice appeared more developed; in particular, the alveolar wall
appeared to be thinner and less cellular, and there was less
connective tissue compared to litter mate control animals. This
corresponds to a greater degree of alveolarisation in the
CSF-1-treated animals.
[0361] In conclusion, CSF-1 was observed to have a growth promoting
effect on total and individual kidney weights when administered
systemically to newborn mice. This increase in kidney weight in the
CSF-1 treated mice was associated with increased, nephrogenesis. In
addition, the lungs of the CSF-1 treated mice appeared more
differentiated compared to control litter mate animals. Therefore,
it is considered that CSF-1 shows considerable promise for the
treatment of pregnant mothers at risk of premature delivery, as
well as in the treatment of premature babies with the objective to
promote growth and maturation of the lungs and kidneys to prevent
associated complications and disorders.
[0362] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0363] The invention described herein has been supported by a
research grant from the National Institutes of Health (USA).
[0364] Throughout this specification, the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
Various changes and modifications may be made to the embodiments
described and illustrated herein without departing from the broad
spirit and scope of the invention.
[0365] All computer programs, algorithms, patent and scientific
literature referred to in this specification are incorporated
herein by reference in their entirety.
[0366] All publications mentioned in this specification are herein
incorporated by reference. Any discussion of documents, acts,
materials, devices, articles or the like which has been included in
the present specification is solely for the purpose of providing a
context to the disclosed embodiments. It is not to be taken as an
admission that any or all of these matters form part of the prior
art base or were common general knowledge in the field relevant to
the present inventions as it existed in Australia or elsewhere
before the priority date of each claim of this application.
[0367] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
disclosed embodiments as shown herein without departing from the
spirit or scope of the inventions as disclosed. The present
embodiments are, therefore, to be considered in all respects as
illustrative and not restrictive.
* * * * *
References