Language selection

Search

Patent 2290598 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2290598
(54) English Title: GANGLIOSIDE GM3 INDUCED APOPTOSIS OF NEURAL CELLS
(54) French Title: APOPTOSE DE CELLULES NEURONALES INDUITE PAR UN GANGLIOSIDE GM3
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • A61K 31/70 (2006.01)
  • A61K 31/7032 (2006.01)
  • A61K 45/00 (2006.01)
  • A61P 25/00 (2006.01)
  • A61P 35/00 (2006.01)
  • A61P 43/00 (2006.01)
(72) Inventors :
  • BLACK, PETER MCLAREN (United States of America)
  • NAKASUJI, YUJI (United States of America)
  • NOLL, ELIZABETH (United States of America)
  • MILLER, ROBERT H. (United States of America)
(73) Owners :
  • BLACK, PETER MCLAREN (Not Available)
  • NAKASUJI, YUJI (Not Available)
  • NOLL, ELIZABETH (Not Available)
  • MILLER, ROBERT H. (Not Available)
(71) Applicants :
  • CASE WESTERN RESERVE UNIVERSITY (United States of America)
(74) Agent: SIM & MCBURNEY
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1998-05-22
(87) Open to Public Inspection: 1998-11-26
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US1998/010390
(87) International Publication Number: WO1998/052577
(85) National Entry: 1999-11-22

(30) Application Priority Data:
Application No. Country/Territory Date
60/047,430 United States of America 1997-05-22

Abstracts

English Abstract




The present invention relates to the ability of the ganglioside, GM3 to
inhibit proliferation and induce apoptosis in proliferating CNS cells. The
present invention further demonstrates the ability for GM3 to reduce cell
numbers in primary cultures of rapidly proliferating human glial tumors and
the 9L rat gliosarcoma cell line. In addition, GM3 is shown to have no effect
on quiescent cultures of normal human CNS cells. A single injection of GM3
three days after intracranial implantation of tumor cells in a murine
xenograft model system resulted in a significant increase in the symptom-free
survival period of host animals. Therefore, GM3 is useful as a
chemotherapeutic agent for human high grade gliomas.


French Abstract

La présente invention concerne l'aptitude du ganglioside, GM3, à inhiber la prolifération et à induire l'apoptose dans des cellules proliférantes CNS. L'invention concerne également l'aptitude de GM3 à réduire le nombre de cellules dans des cultures primaires de tumeurs gliales humaines proliférant rapidement et de la lignée cellulaire 9L du gliosarcome du rat. En outre, GM3 n'a aucun effet sur des cultures quiescentes de cellules CNS humaines. Une injection simple de GM3, trois jours après l'implantation intracrânienne de cellules tumorales dans un système murin constituant une hétérogreffe, provoque une augmentation significative de la période de survie sans symptôme chez des animaux hôtes. GM3 est, en outre, utilisé comme agent chimiothérapeutique destiné au traitement des gliomes très avancés chez l'homme.

Claims

Note: Claims are shown in the official language in which they were submitted.




-17-


Having thus described the invention, it is claimed:

1. A composition for induction of apoptosis of proliferating
neural cells comprising an apoptosis inducing effective amount of the
ganglioside
GM3.

2. The composition of claim 1 wherein the apoptosis inducing
effective amount is between about 5µM and about 50µM GM3.

3. The composition of claim 1 further including conventional
therapeutic adjuvants.

4. The composition of claim 3 wherein the conventional
therapeutic adjuvants include normal saline (sterile).

5. The composition of claim 1 further including other
chemotherapeutic agents.

6. A method for selectively inducing apoptosis of proliferating
neural cells comprising introducing an apoptosis inducing effective amount of
ganglioside GM3 to said proliferating neural cells.

7. The method of claim 6 wherein the apoptosis inducing
effective amount is between about 5µM and about 50µM.

8. The method of claim 6 wherein the neural cells are one or
more of glial tumors, glioblastoma multiformes (GBMs), astrocytomas, and
oligodendrocytomas.

9. A method for treating a patient having a brain tumor
comprising administering to said patient a therapeutically effective amount of
a
chemotherapeutic agent comprising ganglioside GM3.




-18-

10. The method of claim 9 wherein said brain tumor is a high or
low grade glioma or astrocytoma.

11. The method of claim 9 which comprises administering GM3
to said patient as a single application, as multiple applications or as a
polymeric
slow release suspension application, as required for inhibition of tumor
growth.

12. The method of claim 9 wherein said administration is via GM3
in combination with a slow release biopolymer which is placed in the tumor
bed,
through direct application of GM3 to the tumor, by intraarterially injecting
GM3,
or by intratumorally injecting GM3.

13. The method of claim 11 wherein said administration lasts for
a period of between about one day and about six months.

14. The method of claim 9 wherein the GM3 is selective towards
proliferating neural cells in said patient.

15. The method of claim 9, further comprising pre- or post-administration
of an adjuvant therapy in conjunction with the GM3 treatment of the
brain tumor.

16. The method of claim 15 wherein the adjuvant therapy is radiation
therapy.

17. The method of claim 15 wherein the brain tumor is one or more
of glioblastoma multiformes (GBMs), astrogliomas, oligodendrocytomas,
ependymomas, and mixed gliomas.

18. A chemotherapeutic regimen comprising administration of a
chemotherapeutically effective amount of GM3 to a patient in need thereof.



-19-

19. A method for inducing CdK inhibition by increasing the
presence of p27kip1 protein by administration of GM3 to astrocytes.

20. The method of claim 19 wherein hyperphosphorylated retino-blastoma
protein (pRb) is concomitantly reduced.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
GANGLIOSIDE GM3 INDUCED
APOPTOSIS OF NEURAL CELLS
This application claims priority to the filing date of U.S. Provisional
Application Serial No. 60/047,430 filed May 22, 1997.
Field of the Invention
The simple ganglioside GM3, a naturally occurring ligand, has been
found to selectively regulate cell proliferation and induce apoptosis of
astrocytes. In
addition, GM3 has been determined to be useful as a selective chemotherapeutic
agent for treating high grade gliomas in human patients.
Background of the Invention
During normal vertebrate development approximately twice as many
cells are born, as are finally incorporated into the mature nervous system.
This great
excess of cells requires that there is a mechanism to eliminate surplus cells,
thus
preventing an unnecessary nutritional burden to the animal. One mechanism for
selective pruning of excess cells is induction of programmed cell death, or
apoptosis.
Apoptosis plays an important role in the development of the central
nervous system (CNS). However, the factors which indure apoptosis in normal
CNS
development have not been clearly identified.
Gangliosides, surface components of all mammalian cells, are highly
expressed in the CNS compared to many other tissues ( Fishman, P.H. and R.O.
Brady, Biosynthesis and function of gangliosides, Science, 1976, 194: p.906-
915;
Ledeen, R.W. and R.K. Yu, Gangliosides: structure, isolation and analysis,
Methods Enzymol., 1982, 83: p. 1309-191). Specific ganglioside expression is
developmentally regulated (Irwin, L.N. and C.C. Irwin, Developmental changes
in
ganglioside composition of hippocampus, retina and optic tectum, Dev.
Neurosci.,
1979, 2: p. 129-138; Irwin, L.N., D.B. Michael, and C.C. Irwin, Ganglioside
patterns
of fetal rat and mouse brain, J. Neurochem., 1980, 34: p. 1527-1530; Hilbig,
R.,


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-2-
et al. , Developmental profiles of gangliosides in mouse and rate cerebral
cortex,
Roux's Arch., 1982, 191: p. 281-284; Rosner, H., Ganglioside changes in the
chicken optic lobes as biochemical indicators of brain development and
maturation,
Brain Res., 1982, 236: p. 49-61). During early development GD3 (Irwin, L.N.,
D.B. Michael, and C.C. Irwin, Ganglioside patterns of fetal rat and mouse
brain, J.
Neurochem., 1980, 34: p. 1527-1530; Hilbig, R., et al., Developmental profiles
of
gangliosides in mouse and rate cerebral cortex, Roux's Arch., 1982, 191: p.
281-284
and GM3 are (Heffer-Lauc, M., et al. , Anti-GM3 (113Neu5AGlactosylceramide)
ganglioside antibody labels human fetal purkinje neurons during the critical
stage
I 0 of cerebellar development, Neurosci. Lett., 1996, 213: p. 91-94) expressed
at high
levels in the CNS, which subsequently decrease (Goldman, J.E., et al., GD3
ganglioside is a glycolipid characteristic of immature neuroectodermal cells,
J.
Neuroimmunol., 1984, 7: p. 179-192). The highest levels of GD3 and GM3
expression correlate with periods of local cell proliferation suggesting that
gangliosides might regulate growth and differentiation of CNS neural cells
(Bremen
E.G., et al., Ganglioside - mediated modulation of cell growth, growth factor
binding and receptor phosphorylation, J. Biol. Chem., 1984, 258: p. 6818-6825;
Bremen E.G., J. Schlessinger, and S. Hakomori, Ganglioside-mediated modulation
of cell growth, Specific effects of GM3 on tyrosine phosphorylation of the
epidermal
gromth factor receptor, J. Biol. Chem., 1986, 261: p. 2434-2440; Cannella,
M.S.,
et al. , Comparison of epi-GM3 with GM3 and GMl as stimulators of neurite
outgrowth, Devel. Brain. Res., 1988, 39: p. 13'7-143; Durand, M., et al., ed.
Evidence for the effects of gangliosides on the development of neurons in
primary
cultures, Gangliosides and Neuronal Plasticity, ed. R. Tettamanti, et al.
1986,
Liviana: Padova, Italy. 295-307; Yim, S.H., et al., Differentiation of
oligodendrocytes cultured from developing rat brain is enhanced by exogenous
GM3
ganglioside, J. Neurosci. Res., 1994, 38: p. 268-281 ).
Gangliosides are present in the plasma membrane of most mammalian
cells and are enriched in the central nervous system (CNS) (P.H. Fishman, R.O.
Brady, Science 194: p. 906-15 (1976); R.W. Ledeen, R.K. Yu, Methods Enzymol
83:
p. 139-91 (1982)). The expression of the gangliosides is developmentally
regulated
I I T


CA 02290598 1999-11-22
_ WO 98/52577 PCT/US98/10390
-3-
in the brain, and the simplest ganglioside GM3 is expressed predominantly in
rat
brain until embryonic day 16 (E16), and at lower levels during further
development
(R.K. Yu, L.J. Macala, T. Taki, H.M. Weinfield, F.S. Yu, J. Neurochem 50: p.
1825-9 ( 1988)), with low levels of expression persisting throughout
adulthood. GM3
is expressed in the ventricular zone of early human brain development (M.
Stojiljkovic, et al., Int. J. Dev. Neurosci 14: p.35-44 (1996)), and in adult
rat brain,
GM3 is intensely expressed in the white matter and layer VI of cerebrum (M.
Kotani,
et al., Glycobiology, 4: p. 855-65 (1994)). In vitro, exogenously added
gangliosides
are rapidly incorporated into the plasma membranes of cells (T.W. Keenan, E.
Schmid, W. W. Franke, H. Wiegandt, Exp. Cell Res. 92: p. 259-70 ( 1975); R.A.
Laine, S. Hakomori, Biochem. Biophys. Res. Commun. 54: p. 1039-45 ( 1973)) and
cause numerous biological effects. One common effect is the modulation of the
cell
growth. Proliferation of EGF driven 3T3 fibrobEast and A431, KB epidermoid
carcinoma cell lines (E.G. Bremer, S. Hakomori, P.D. Bowen, E. Raines, R.
Ross,
J. Btol. Chem. 259: p. 6818-25 (1984); E.G. Bremer, J. Schlessinger, S.
Hakomori,
J. Biol. Chem. 261: p. 2434-40 (1986)), PDGF driven 3T3 fibroblast and SH-SYSY
neurobIastoma cell lines (E.G. Bremen S. Hakomori, P.D. Bowen, E. Raines, R.
Ross, J. Biol. Chern. 259: p. 6818-25 (1984); E.G. Bremen J. Schlessinger, S.
Hakomori, J. Biol. Chem. 261: p. 2434-40 (1986); D.L. Hynds, M. Summers, B.J.
Van, M.S. O'Dorisio, A.J. Yates, J. Neurochem. 65: p. 2251-8 (1990), FGF
driven
BHK fibroblast cell line (E.G. Bremen S. Hakomori, Biochem. Biophys. Res.
Commun. 106: p. 711-8 (1982)), and insulin driven HL-60 leukemia cell line (N.
Nojiri, M. Stroud, S. Hakomori, J. Biol. Chem. 266: p. 4531-7 (1991)), are
inhibited by exogenously added GM3. The phosphorylation of the appropriate
receptors, EGF-receptor (R), PDGF-R, and Insulin-R, are inhibited by GM3 (E.G.
Bremer, S. Hakomori, P.D. Bowen, E. Raines, R. Ross, J. Biol. Chem. 259: p.
6818
25 (1984); E.G. Bremen J. Schlessinger, S. Hakomori, J. Biol. Chem. 261: p.
2434
40 (1986); N. Nojiri, M. Stroud, S. Hakomori, J. Biol. Chem. 266: p. 4531-7
( 1991 ); Q. Zhou, S. Hakomori, K. Kitamura, Y. Igarashi, J. Biol. Chem. 269:
p.
1959-65 ( 1994)).

i i
CA 02290598 1999-11-22
_ WO 98/52577 PCT/US98/1039U
-4-
Several studies suggest the another ganglioside, GM 1, has supportive
effects on CNS neurons (C.L. Schengrund, Brain Res. Bull. 24: p. 131-41
(1990)).
Less is known about the supportive effects of GM3. GM3 also inhibits cell
growth
and modulates cell differentiation of the human leukemia cell lines HL-60 and
U937,
and induces monocytic differentiation (N. Nojiri, F. Takaku, Y. Terui, Y.
Miura, M.
Saito, Proc. Natl. Acad. Sci. USA. 83: p. 782-6 ( 1986)), while anti-GM3-
antibody
mediates differentiation of neuro-2a neuroblastoma cells (D. Chatterjee, M.
Chakraborty, G.M. Anderson, Brain Res. 583: p: 31-44 (1992); M. Chakraborty,
G.M. Anderson, A. Chakraboriy, D. Chatterjee, Brain Res. 625: p. 197-202 ( I
993)).
The neuritegenesis of neuro-2a and PC-12 cells are increased by GM3 (M.S.
Cannella, F.J. Roisen, T. Ogawa, M. Sugimoto, R. W. Ledeen, Brain Res. 467: p.
137-43 1988)), and the number of thickness of processes of cultured
oligodendrocytes are also increased (S.H. Yim, E. Yavin, J.A. I-Iammer. R.H.
Quarles, J. Neurochem. 57: p. 2144-7 (1991); S.H. Yim, R.G. Farrer, J.A.
Hammer,
E. Yavin, R.l I. Quarles, J. Neurosci Res. 38: p. 268-81 ( 1994)).
Although gangliosides are abundant in brain, little is known about
their function on CNS cells especially during development.
Gangliosides have been proposed to regulate cellular differentiation
in neurons (Cannella, M.S., et al., Comparison of epi-GM3 with GM3 and GMl as
stimulators of neurite outgrowth, Devel. Brain. Res., 1988, 39: p. 137-143;
Dimpfel, W., W. Moller, and U. Mengs, ed. Ganglioside-induced neurite
forrrcation
in cultures neuroblastoma cells, Gangliosides in Neurological function, ed.
M.M.
Rapport and A. Gorio. 1981, Raven: New York. I19-124; Ferrari, G., M. Fabris,
and A. Gorio, Gangliosides enhance neurite outgrowth in PC12 cells, Dev. Brain
Res., 1983, 8: p. 2i5-221; Katoh-semba, R. S.D. Skaper, and S. Varon,
Interaction
of GMl ganglioside with PC12 phenochromocytoma cells: serumand NGF-
dependent effects on neurtitic growth and proliferation, J. Neurosci. Res.,
1984, 12:
p. 299-310; Leskawa, K.C. and E.L. Hogan, Quantitiation of the in vitro
neuroblastoma response to exogenous purified gangliosides, J. Neurosci. Res.,
1985,
13: p. X39-550; Matta, S.C., G. Yorke, and F.J. Roisen, Neuritic and metabolic
effects of individual gangliosides and their interaction with nerve growth
factor in


CA 02290598 1999-11-22
_WO 98/52577 PCT/US98/10390
-5-
cultures of neuroblastoma and phenochromocytoma, Dev. Brain Res., 1986, 27: p.
243-252), while a variety of gangliosides are expressed in normal and
transformed
glia (Irwin, L.N., D.B. Michael, and C.C. Irwin, Ganglioside patterns of fetal
rat and
mouse brain, J. Neurochem., 1980, 34: p. 1527-1530; Goldman, J.E., S.S. Geier,
and
M. Hirano, Differentiation of astrocytes and oligodendrocytes from germinal
matrix
cells in primary culture, J. Neurosci, 1986, 6(1): p. 52-60; Sung, C.C., et
al.,
Glycolipids and myelin proteins in human oligodendrogliomas, Glycoconjugate
J.,
1996, 13: p. 433-443; Kim, S.U., G. Moretto, and R.K. Yu, Neuroimmunology of
gangliosides in human neurons and glial cells in culture, J. Neurochem., 1986,
15:
p. 303-321; Shinoura, N., et al., Ganglioside composition and its relation to
clinical
data in brain tumors, Neurosurg., 1992, 31: p. 541-549; Stojiljkovic, M., et
al.,
Gangliosides GMl and GM3 in early human brain development: an
immunocyltochemical study, Int. J. Devel. Neurosci., 1996, 14: p. 35-44). The
majority of human brain tumors are of glial origin. In children gliomas
comprise
67% of CNS tumors, and are the second most frequently diagnosed childhood
malignancy (Parkin, D.M., et al., International incidence of childhood cancer,
1988,
IARC Scientific Publication, International Agency for Research on Cancer). In
adults brain tumors represent between 40% and 67% of primary CNS tumors
{Bohnen, N.I., et al., ed. Descriptive and analytic epidemiology of brain
tumors,
Cancer of the Nervous System, ed. P.M. Black and J.S. Loeffler, 1997,
Blackwell
Science: Cambridge Mass. 3-24). CNS tumors account for approximately 2% of all
adult malignancies (Giles, G.G., B.K. Armstrong, and L.N. Smith, Cancer in
Australia, 1987), but are responsible for disproportionately high number of
years of
life lost (Hoffinan, R.M., ed. Fertile seed and rich soil; The development of
clinically
relevant models of human cancer by surgical orthotopic implantation of intact
tissue,
Anticancer drug development guide: Preclinical screening, clinical trials and
approval., ed. B.A. Teicher, 1997, Humana Press: Totowa, N.J. 127-144). Of
primary CNS malignancies, glioblastoma multiforme (GBM) has the poorest
prognosis, with median survival between 29 to 36 weeks (Ammirati, M., et al.,
Effects of the extent of surgical resection on survival and quality of life in
patients
with supratentorial glioblastomas and anaplastic astrocytomas, Neurosurgery,
1987,


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-6_
21: p. 201-206; Harsch, G.R., et al., Reoperating for recurrent glioblastoma
and
anaplastic astrocytoma, Neurosurgery., 1987, 21: p.615-621 ). Even with
aggressive
surgical resection followed by adjuvent therapy (radiotherapy. immunotherapy,
chemotherapy), the vast majority of GBM patients succumb to their disease
(Wen,
P.Y. and D. Schiff, ed. Clinical evaluation of patients with astrocytomas,
Contemporary issues in Neurological surgery - Astrocytomas: Diagnosis,
Treatment
and Biology., ed. P.M. Black, W.C. Schoene, and L.A. Lampson, 1993, Blackwell
Scientific: Boston, Mass. 26-36). Treatment of CNS malignancies is complicated
due to the sensitivity of collateral brain tissue and the profound
consequences of
damage to cells adjacent to the tumor.
Summary of the Invention
The present invention relates to the ability of the ganglioside, GM3
to inhibit proliferation and induce apoptosis in proliferating CNS cells. The
present
invention further demonstrates the ability for GM3 to reduce cell numbers in
primary
cultures of rapidly proliferating human filial tumors and the 9L rat
gliosarcoma cell
line. In addition, GM3 is shown to have no effect on quiescent cultures of
normal
human CNS cells. A single injection of GM3 three days after intracranial
implantation of tumor cells in a murine xenograft model system resulted in a
significant increase in the symptom-free survival period of host animals.
Therefore,
GM3 is useful as a chemotherapeutic agent for human high grade gliomas.
As such, a first aspect of the invention includes a novel
chemotherapeutic agent for inducing apoptosis in proliferating neural cells
comprising
GM3.
A second aspect of the invention relates to selective induction of
apoptosis in proliferating neural cells using a GM3 chemotherapeutic agent.
A further aspect of the invention relates to treatment of a patient
having a brain tumor to inhibit proliferation and induce apoptosis of
proliferating
neural cells in said patient.
An additional aspect of the invention relates to a chemotherapeutic
regimen to treat patients in need thereof.
,r i r


CA 02290598 1999-11-22
_ WO 98/52577 PCTNS98/10390
Therefore, the present invention has the advantage of being able to
selectively treat brain tumors including high grade gliomas in patients
thereby
enhancing the prognosis of the patients by effectively increasing the median
survival
time for such patients.
Another advantage relates to the use of a chemical agent normally
produced in the body in a chemotherapeutically effective composition thereby
reducing the risk of adverse side effects typically related to such
treatments.
Still other advantages and benefits of the invention will become
apparent to those skilled in the art upon reading and understanding the
following
detailed description of the preferred embodiments.
Brief Description of the Drawings
FIGURE 1 shows TUNEL labeling showing DNA fragmentation for
proliferating astrocyte cells treated with SOpM GM3.
FIGURE 2A and 2B show proliferative astrocyte morphology without
GM3 treatment and with GM3 treatment.
FIGURE 3A and 3B show the effect of GM3 on non-proliferative
astrocytes.
FIGURE 4 shows TUNEL labeling showing DNA fragmentation for
proliferating neurons and glial precursor cells treated with GM3.
FIGURE 5 shows the effect of GM3 treatment on human glial tumor
growth and on rat 9L cell line in vitro.
FIGURE 6A-6E show morphological changes in control cell cultures
(6A, 6C) and GM3 treated cell cultures (6B, 6D, 6E).
FIGURE 7 shows the effect of increasing the concentration of GM3
on human glioblastoma multiforms (GBM's) in vitro.
FIGURE 8 shows the effects of GM3 on normal human central
nervous system (CNS) tissue cultures.
FIGURE 9 shows the effect of GM3 treatment on survival time of
animals implanted with 9L rat tumor cells.


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
_g_
Detailed Description of the Preferred Embodiment
Exposure of proliferating rat CNS (central nervous system) neural cells
to the ganglioside GM3 inhibits cell proliferation and induces apoptosis. The
present
invention demonstrates that GM3 treatment of human CNS tumor cells inhibits
their
growth in vitro and results in prolonged symptom-free post-implant intervals
in a
xenograft brain tumor model.
The mechanism by which GM3 inhibits the expansion of human GBM
in vitro is unclear. Without intending to be bound by any particular theory,
it seems
likely that GM3 may act to both inhibit tumor cells proliferation, as well as
to induce
apoptosis in actively proliferating cells. Consistent with this hypothesis,
GM3
treatment resulted in a significant inhibition of proliferation of rat neural
cell
precursors and a rapid induction of apoptosis which is correlated with an up
regulation of p27'''~'-' expression and a reduction of pRb
(hyperphosphorylated
retinoblastoma protein) expression. Two lines of evidence suggest that human
tumor
cells also undergo apoptosis in response to GM3 exposure. First, the number of
cells
in GM3 treated cultures was reduced compared to both the starting population
and
to control cultures suggesting cell death. Second, the rounded morphology and
nuclear fragmentation seen following GM3 exposure is characteristic of
apoptosis.
The degree of response of cultures to GM3 exposure appears to be cell
or tumor specific. While all tumors studied in the present invention were
characterized as glioblastoma multiforme (GBM) and where grown at similar
densities under identical conditions, their response to GM3 varied. In some
cultures
the reduction in cell number was greater than 80%, while in parallel
experiments
using cultures of a different tumor, the reduction in cell number was only
35%. This
difference in response to GM3 treatment is unlikely to be due to culture
passage
number since the seven primary cultures were derived from the original
plating, or
early passages of the resected tumor.
One factor which might regulate responsiveness of cells from different
tumors to GM3 treatment is the rate of proliferation. Non-proliferative rodent
CNS
cells do not undergo apoptosis in response to GM3 treatment in vitro, but
rather
become increasingly differentiated. Similarly, intraventricular injection of
GM3 into
i i o


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-9-
developing rats results in cell death only in ventricular and subventricular
proliferating cell populations. As seen in the present invention, GM3 did not
reduce
cell number in quiescent cultures of normal human brain cells and there were
no
changes in neurological behavior associated with injection of GM3 in rats.
Thus,
S cells in cultures of different tumors may have different cell cycle times
and the
response to GM3 may be directly correlated with cell cycle time (i.e. faster
cycle
times result in increased cell death).
The present invention demonstrates that GM3 treatment significantly
decreased the growth of primary cultures of human and rodent tumor cells,
while not
significantly altering cell number in quiescent cultures of normal human
brain. In
addition, a single treatment with GM3 significantly extended the symptom-free,
post
implant period in nude mice with intracranially implanted rat 9L brain tumor
cells.
Taken together, these data demonstrate that GM3 provides an effective
therapeutic
treatment for human high grade gliomas.
Below are several examples showing both in vitro and in vivo results
upon application of GM3 to both tumorous and normal cell lines. The GM3 used
in the examples was obtained from Sigma and was 98% pure from bovine brain.
Normal saline {sterile) was used as a carrier.
Example 1
In this example, GM3 is shown to induce apoptosis of proliferating
astrocytes. Astrocyte cells were prepared from cerebral cortices of newborn
(PO)
Sprague-Dawley rats as described in Smith et al., Dev.Biol. 138, 377-390
(1990), and
maintained in Delbecco's modified Eagles medium (DMEM) supplemented with 10%
fetal bovine serum (FBS).
To examine the mechanisms of GM3 induced astrocyte cell death, the
cells were double labeled with anti-GFAP antibody and the terminal
deoxynucleotidyl
transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) method.
Morphological changes of astrocytes were apparent after 18 hours of incubation
with
50 ~,M of GM3. By 72 hours, most of the cells were highly shrunk and their
nuclei
condensed. compatible with the induction of apoptosis. Consistent with these


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
- 10-
morphological changes, the proportion of T LJNEL positive cells increased
substantially. By 72 hours of incubation, some of the dead cells detached from
cover
slips, so the proportion of ~fLJNEL positive cells in these cultures may be an
understatement of the extent of cell death. In all the cultures, a few GFAP
negative
cells, presumably meningeal or endothelial cells, appeared normal even after
72 hours
of incubation with GM3.
Since any random DNA cleavage caused by necrosis can potentially
be labeled by TUNEL, apoptotic internucleosomal DNA fragmentation was assayed
(Figure 1 ). Characteristic DNA fragmentation was first seen after 48 hours
and was
clearly apparent after 72 hours of incubation with GM3, consistent with the
TUNEL
staining data. Ultrastructurally, characteristic features of apoptotic cell
death such
as cell shrinkage, condensation of chromatin, loss of the integrity of the
nuclear and
plasma membranes were observed (Figure 2).
Example 2
To determine if the response to GM3 was correlated with the
proliferative state of astrocytes, GM3 was added to contact-inhibited non-
proliferating
astrocyte cultures. GM3 did not induce changes in cell morphology or cell
death in
these contact-inhibited non-proliferating astrocytes (Figure 3).
Upon analyzing the results from Examples 1 and 2, it is seen that
GM3 selectively induces apoptosis in immature, proliferating astrocytes.
Further, the
results also demonstrate that active proliferation of neural cells also
contributes to the
susceptibility of those cells to GM3 treatment.
Example 3
In this example, GM3 is shown to induce apoptosis of neurons and
glial precursors. Mixed cell cultures (post-mitotic neurons, neuronal and
glial
precursors) were prepared from E15 brain according to Smith et al., Dev.
Biol., 138,
377-390 (1990), and maintained in DMEM + 10% FBS or in DMEM + 1% FBS +
1 % N2 supplement.
,r i ?


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
To evaluate the effect of GM3 on neurons and glial precursors, the
mixed cultures were treated with GM3, and cell death was assessed by double
labeling with anti-(3-tubulin-positive and A2B5-positive cells were TUNEL-
positive
and the characteristic DNA fragmentation became apparent (Figure 4). By 72
hours
of incubation, approximately 80 % of the cells were dead. The remaining cells
appeared intact and the majority of these cells were (3-tubulin-positive
neurons,
consistent with the process-outgrowth promoting effect of GM3 on mature
neurons.
When these remaining cells were incubated with BrdU during the last 24 hours
of
GM3 treatment and double labeled with anti-~3-tubulin and anti-BrdU
antibodies, the
majority (>99%) of remaining viable cells were ~3-tubulin+/BrdU-, suggesting
that
post-mitotic neurons, like non-proliferative astrocytes were less sensitive to
GM3
treatment. Together, these observations indicate that exposure to GM3 induces
apoptosis in all classes of proliferating but not non-proliferating neural
cells.
In the following Examples, all tumor specimens were diagnosed as
glioblastoma multiforme (GBM), corresponding to W.H.O. grade 1.1.3 and/or St.
Anne-Mayo grade IV. Non-neoplastic cells were obtained from a 3 yr. old male
that
underwent a left hemispherectomy for Rasmussen's disorder.
Cultures were generated using standard protocols. Briefly, tumor
samples were dissociated enzymatically, plated at densities between 1X105-6 on
l2mm poly-L-lysine coated cover slips and maintained at 37°C with 5%
CO,.
Cultures were grown for at least 24 hours prior to addition of 100 ~.M GM3
suspended in media to experimental cultures. Parallel control cultures
received media
alone at the commencement of the experiment. The majority of assays were
performed from cells derived from the original plating or first passage of a
tumor
sample. Cultures were grown in DMEM media with 10% FBS and N2 supplement
containing insulin, transferrin, selenium, progesterone, putrescine, 3,3',5
triiodo-L-
thyronine, thyroxine and fraction V BSA.
Following exposure to GM3 for seven days, cultures were fixed in 4%
paraformaldehyde, stained with the DNA stain Dapi, ( 1:10,000; Molecular
Probes)
for 5 min. and examined on a Zeiss Axiophot microscope. To quantify remaining
cells, the number of cells in 10 consecutive 40X randomly selected fields was


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-12-
counted and the mean determined. For each tumor at least 3 independent
cultures
were assayed and the data pooled. To determine the effect of increasing GM3
concentrations, these studies were repeated at concentrations of 100 ~,M, and
400 ~.M
in parallel.
Example 4
In the following example, GM3 is applied to human high grade
gliomas in vitro to determine whether GM3 treatment inhibited expansion of
primary
human tumor cells. Tumor samples used in this example were high grade lesions
from a variety of regions of the tumor. Seven independent tumor samples were
assayed.
In each tumor sample, 100 ~M GM3 was applied in a single
application. This resulted in a significant reduction in the number of cells
in these
experimental cultures over a period of seven days (Figure 5).
The effect of GM3 treatment on cell number in the tumor cultures,
relative to matching untreated control cultures, was tumor specific. For
example,
while cell number was reduced by approximately 57% in cultures of GBM-1, cell
number was reduced by greater than 85% in cultures of GBM-7. The effect of GM3
was not limited to primary tumor cultures. Cell number in cultures of rat 9L
cell
line, an established brain tumor model for in vivo studies, was reduced by
approximately 65%, close to the average reduction in the human primary
cultures
(Figure 5).
The morphology of cells in GM3 treated cultures was different from
the control cultures (Figure 6A-6E). In control cultures, cell density
increased during
7 days in vitro and the majority of cells were flattened and well adhered. By
contrast in GM3 treated cultures, cell density decreased over the same period
and the
residual cells were rounded and highly refractile. Many cells in GM3 treated
cultures, but not in control cultures, had chromosomal fragmentation revealed
by the
DNA stain Dapi (Figures 6A-6E) and GM3 cultures contained a significant amount
of cellular debris. The effects of GM 3 treatment on cell number were
increased at
higher concentrations. In parallel cultures of tumor GBM-5 exposed to 100 p,M
or
~r i r


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-13-
400 ~,M GM3, there were significantly fewer cells in cultures treated with 400
~M
GM3 than in those treated with 100 ~M GM3 (Figure 7).
Example 5
To determine whether GM3 treatment reduced cell number in
quiescent cultures of normal human CNS, exogenous GM3 (100 ~.M) was added to
cultures derived from non-neoplastic CNS tissue. The density of cells was not
significantly altered curing 7 days in vitro. Furthermore, there was no
significant
difference in the number of cells in GM3 treated cultures compared to controls
(Figure 8), suggesting GM3 was not toxic to normal human CNS cells, consistent
with all previous studies.
Example 6
To assess the effects of GM3 on tumor growth in vivo a 9L rat
gliosarcoma cells were transplanted intracranially into mice. Between 1-
1.5X106
cells were stereotactically injected into the brain parenchyma of 18 adult
Swiss nude
mice, in two separate experiments. In each experiment, the animals were
randomly
assigned to either control or experimental groups immediately following the
implantation procedure. Seventy two hours after cell implantation control
animals
received an intracranial injection of 5 ml of 0.9% sodium chloride while
experimental animals received 0.3 mg GM3 in 5 mol of 0.9% sodium chloride. All
animals were evaluated daily for signs of neurological impairment (seizures,
hemiparesis, ataxia, dyskinesia, gait difficulties, etc.) and, when detected,
the animal
was sacrificed. Survival rates from both experiments were pooled, the means +/-
S.D.
[standard deviation] determined and the statistical significance assessed by a
2 tailed
Students t test. Of the animals in group 1, one of the animals demonstrated
symptoms of a rapidly progressing infection and had to be sacrificed prior to
exhibiting any symptoms of tumor growth. This animal was excluded from the
data
set.
Injection of GM3 significantly prolonged the symptom-free period in
host animals (Figure 9). Among control animals (N=9) the mean post implant


CA 02290598 1999-11-22
_ WO 98/52577 PCT/US98/10390
-14-
survival period was 18 days (+/-3). At 13 days approximately 20% (2/9) animals
demonstrated neurological impairment and no animals survived without
noticeable
neurological impairment beyond 23 days post implantation. By contrast, among
experimental animals that received a single injection of GM3 (N=8), the mean
post
implant survival period was 23 days (P-value compared to control of 0.03).
Although one animal developed neurological impairment at day 17, two animals
were
symptom-free until 30 days post implant; that is 7 days after all control
animals were
sacrificed.
The above Example demonstrates that a single treatment of GM3
increased the mean symptom free survival time of host animals following
implantation of 9I. cells. Based on the in vitro assays, this increased
survival time
most likely reflects reduction in the rate of growth of implanted tumor cells
following GM3 treatment. Since the median survival for a patient diagnosed
with
a GBM is estimated between 29-36 weeks, any increase in median survival would
I S be beneficial, provided there was little inherent treatment toxicity. GM3
is a
naturally occurring molecule that enhances maturation rather than induce cell
death
in non-proliferative neural cells. Thus GM3 treatment is unlikely to severely
compromise the quality of remaining life.
Several additional approaches may enhance the symptom-free survival
period of GM3 treated host animals. First, in vitro studies suggest the
efficacy of
GM3 induced cell death is enhanced at higher concentrations. While any effect
on
non-proliferative neural cells may be more pronounced at higher
concentrations,
increasing GM3 concentrations may result in extended survival periods. Second,
the
current studies used a single injection of GM3, 3 days after tumor
implantation.
Multiple injections of GM3 should result in a greater limitation of tumor
growth and
concomitant extension of symptom-free survival periods. Recent in vitro
studies
indicate that sustained application of GM3 over a period of 1 week resulted in
greater cell loss in the majority of tumor cultures approaching I00%.
Taken together these studies identify GM3 as a treatment agent for
patients diagnosed with GBM. Treatment of proliferating CNS cells with GM3
inhibits cell proliferation and induces apoptosis. Likewise, growth of human
primary
,i i T


CA 02290598 1999-11-22
WO 98/52577 PCT/US98/10390
-15-
GBM tumor cells is inhibited by GM3 treatment. By contrast, normal, non-
proliferative CNS cells are not induced to undergo apoptosis following GM3
treatment. This strong proliferation dependence of GM3 responsiveness
indicates that
GM3 is a powerful agent in the treatment of highly proliferative human CNS
S malignancies such as GBM.
Induction of apoptosis of various types of neural cells may be
accomplished in practicing the present invention. Glioblastoma multiformes
(GBMs),
astrocytomas (astrogliomas}, oligodendrocytomas, ependymomas, glial tumors and
other various mixed gliomas are within the scope of cells to be treated
according to
the present invention.
Various treatment regimens may be utilized in accordance with the
present invention. For example, the administration of GM3 may be in a single
application, multiple applications, or as a slow release suspension polymer
formulation. The amount to be administered is dictated by the amount needed to
inhibit tumor growth. Typical ranges of effective amounts of GM3 to be
administered to a patient range from about SpM to ~OpM (or about lmg to 16g.).
The treatment regimen should last at least until tumor growth has been
completely
inhibited. Generally, this may be from about one day to about six months (or
longer,
if necessary).
Various conventional means for delivering chemotherapeutic agents
may be used in accordance with practicing the present invention. Such means
include direct application of the GM3 to the tumor (either alone or in
combination
with a slow release polymer), intraarterial injection, and stereotactic
injection
intratumorally.
In addition, other adjuvant therapies and chemotherapeutic treatment
methods may be used in conjunction with the GM3 treatment. For example, pre-
or
post- administration of radiation therapy is an effective adjuvant therapy
which may
be used in conjunction with the GM3 treatment.
The invention has been described with reference to the preferred
embodiments. Obviously, modifications and alterations will occur to others
upon
reading and understanding the preceding detailed description. It is intended
that the


CA 02290598 1999-11-22
_ WO 98/52577 PCT/US98/10390
-16-
invention be construed as including all such modifications and alterations
insofar as
they come within the scope of the appended claims or the equivalents thereof.
'f I T

Representative Drawing

Sorry, the representative drawing for patent document number 2290598 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1998-05-22
(87) PCT Publication Date 1998-11-26
(85) National Entry 1999-11-22
Dead Application 2003-02-24

Abandonment History

Abandonment Date Reason Reinstatement Date
2002-02-25 FAILURE TO RESPOND TO OFFICE LETTER

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $150.00 1999-11-22
Maintenance Fee - Application - New Act 2 2000-05-23 $50.00 1999-11-22
Extension of Time $200.00 2001-02-12
Maintenance Fee - Application - New Act 3 2001-05-22 $50.00 2001-05-03
Maintenance Fee - Application - New Act 4 2002-05-22 $50.00 2002-05-08
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
BLACK, PETER MCLAREN
NAKASUJI, YUJI
NOLL, ELIZABETH
MILLER, ROBERT H.
Past Owners on Record
None
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 1999-11-22 1 55
Description 1999-11-22 16 792
Claims 1999-11-22 3 74
Drawings 1999-11-22 9 633
Cover Page 2000-01-14 1 45
Correspondence 1999-12-22 1 2
Assignment 1999-11-22 4 135
PCT 1999-11-22 6 253
Correspondence 2001-02-12 1 42
Correspondence 2001-02-26 1 14
Fees 2001-05-03 1 46
Fees 2002-05-08 1 50