Emerging Therapeutic Targets - Volume Two, Number 1

Oncologic, Endocrine & Metabolic

Proposed target

Inhibition of Ca2+-calmodulin dependent phosphodiesterase (PDE1B1).

Proposed mechanism of action

Inhibition of specific PDE1 or PDE4 isoforms and the resultant elevation of

intracellular cAMP has been shown to induce apoptosis in human leukaemic
cells. PDE1B1 is chosen as the preferred initial target because its expression
appears restricted to the brain and transformed or proliferating
lymphocytes; whereas, the expression of PDE4 isoforms is more widespread.
However, our recent studies with PDE4 show expression of specific subtypes
of this gene family induced in leukaemic cells, suggesting that these PDE4
subtypes may also provide good potential targets.

Potential therapeutic
indications

Leukaemia.

Proposed therapeutic
intervention

Oligodeoxynucleotides (ODNs).

Research site

Department of Pharmacology, University of Connecticut Health Center, 263

Farmington Ave., Farmington, CT 06030-6125, USA

Telephone number

+1 860 679 2810

Fax number

+1 860 679 3693

Email address

Epstein@nso1.uchc.edu

Website address

www.uchc.edu

Author

Paul M Epstein

Current funding and associated

licensing restrictions

This work is currently supported by a research grant from the Robert Leet
and Clara Guthrie Patterson Trust, Fleet Bank, Trustee. There are no
associated licensing restrictions or exertion of intellectual rights by the
granting agency, over discoveries made through the support of this grant.

Relevant patent information

US patent filed. THE UNIVERSITY OF CONNECTICUT: US8940332 (30/9/97).

Permission for the development and use of these ODNs would need to be
obtained from the University.

Background

It is estimated that by the end of this year, 27,600 new cases of leukaemia
will be diagnosed, and 21,000 deaths will result this year from this disease.
While chemotherapy regimens are effective in inducing remission in many
cases, these are harsh treatments, and it has been found that 80% of adult
patients eventually relapse and die of the disease [1]. Hence, new and
effective treatments for leukaemia are therefore needed.

Inhibition ofEndocrine
Oncologic,
calcium-calmodulin
& Metabolic
dependent phosphodiesterase

[1]. BRAUN SE et al.: Gene therapy strategies for leukemia. Mol. Med. Today (1997)
3:39-46.

One novel approach that avoids harsh chemotherapeutic agents is the use
of antisense to block the expression of specific genes that would curtail the
growth or induce the death of the leukaemic cell. Antisense
oligodeoxynucleotides (AS ODNs) targeted to a number of oncogenes, such
as c-myc, c-myb, and c-raf, have all been shown to block proliferation and/or
induce apoptosis in leukaemic cells in vitro, or in severe combined
immunodeficient (SCID) mouse models of human leukaemia [1].
In our laboratory, we have cloned the cDNA for a form of Ca2+-calmodulin
dependent PDE1B1, from human leukaemic cells, which regulates levels of
the signalling molecule, cAMP [2]. We showed that PDE1B1 is expressed
only in activated and transformed lymphocytes, but not in normal, resting
lymphocytes. Moreover, PDE1B1 expression is restricted to proliferating
lymphocytes and to regions of the brain, and does not appear to be
109
1998 Ashley Publications Ltd. ISSN 1460-0412

110 Inhibition of calcium-calmodulin dependent phosphodiesterase

expressed anywhere else. In lymphoid cells, elevation of cAMP levels induces
apoptosis. Hence, inhibition of the expression of PDE1B1 by AS ODNs
should selectively elevate cAMP and induce apoptosis of actively
proliferating transformed lymphocytes, with no effect on normal, resting
lymphocytes, since PDE1B1 is not expressed there, and with little effect on
brain function, since ODNs penetrate the blood-brain barrier very poorly.
[2]. JIANG X et al.: Inhibition of calmodulin-dependent phosphodiesterase induces
apoptosis in human leukemic cells. Proc. Natl. Acad. Sci. USA (1996)
93:11236-11241.

Indeed, we synthesised an 18 nt phosphorothioate AS ODN targeted to the

translation initiation region of the PDE1B1 mRNA and showed that it
down-regulated the mRNA and protein for PDE1B1 in a sequence specific
manner, and induced apoptosis of human leukaemic cells [2], with no effect
on normal resting lymphocytes [3]. Hence, use of AS ODN to PDE1B1 could
provide a novel therapeutic strategy for treatment of leukaemia. Moreover,
we also found recently that specific subtypes of the cAMP-specific
phosphodiesterase gene family (PDE4) are also induced in activated and
transformed lymphocytes [4], suggesting that development of AS ODNs
targeted to these specific PDE4 subtypes [3] may also provide novel
therapeutic treatments for immunoproliferative disorders and immune
dysfunction [4].
[3]. EPSTEIN PM: Antisense inhibition of phosphodiesterase expression. In: Methods:
A Companion to Methods in Enzymology (1998) 14:21-33.
[4]. JIANG X et al.: Expression and regulation of mRNA for distinct isoforms of
cAMP-specific phosphodiesterase (PDE4) in mitogen-stimulated and leukemic
human lymphocytes. Cell Biochem. Biophys. (1998) 28:135-160.

Potential advantages of the

approach

The potential advantages and reduced toxicity of an antisense approach to

the treatment of leukaemia have prompted the development of AS ODNs
and experimental trials with them for treatment of leukaemia. However, the
genes targeted so far are oncogenes that are expressed in normal cells as
well, and thus use of these AS ODNs could interfere with normal cellular
functions. Our approach of targeting the PDE1B1 gene has the potential to
be more selective than other developed AS ODNs, since PDE1B1 is not
expressed in normal, resting lymphocytes, nor in other normal tissues where
the AS ODNs would be likely to act.
More than 10,000 autologous bone marrow transplants are performed
each year, and, of these procedures, 15% are for leukaemia, and the
number is increasing at an annual rate of 20% [1]. However, many of these
autologous bone marrow transplant patients relapse, and in an effort to
prevent this, the cells are often treated first, ex vivo, with genotoxic agents,
such as mafosfamide. Unfortunately, it has been found, recently, that
treatment with these genotoxic agents frequently results, some years later,
in the formation of secondary myelodysplastic syndrome and acute
myelogenous leukaemias [5]. Hence, AS ODN to PDE1B1 might be highly
useful to replace these genotoxic agents as an ex vivo bone marrow purging
agent for autologous bone marrow transplants.
[5]. SMITH MA et al.: The secondary leukemias: challenges and research directions. J.
Natl. Cancer Inst. (1996) 88:407-418.

Potential disadvantages of the

approach

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Although the use of AS ODNs shows great promise experimentally and in

early clinical trials for the treatment of a wide variety of diseases, no AS ODN
has yet been approved for clinical use, and hence, as with all new drugs on
the horizon, a lot of unknowns and uncertainties still remain regarding the
widespread clinical use of these agents.

Emerging Therapeutic Targets (1998) 2(1)

Oncologic, Endocrine & Metabolic 111

Current research goals

Develop additional AS ODNs that should be more effective than those we

have already developed in inducing apoptosis of leukaemic cells by
synthesising newer chemical derivatives, such as N3P5phosphoramidates, and chimeric backbone phosphorothioate AS ODNs,
and by targeting other regions of the mRNA for PDE1B1, such as areas of the
3-untranslated region rich in secondary RNA structure.
Detect and clone variant forms of PDE1B and PDE4, present in leukaemic
cells, in order to provide additional, and possibly more selective, targets for
the development of more AS ODNs.
Test (through collaboration) the AS ODNs that are the most effective in vitro
against cells in culture, for their ability to prevent or delay the onset of
leukaemia and increase the survival rate in in vivo models of leukaemia, in
which immunodeficient (SCID) mice are transplanted with human
leukaemic cells.

Biologicals in house

Cloned cDNA for human PDE1B1 and AS ODNs targeted to PDE1B1.

Tools available

Cultured human leukaemic cell lines, cloned cDNA for human PDE1B1, AS
ODNs targeted to PDE1B1, reverse transcription polymerase chain reaction
(RT-PCR) strategies to determine the expression of the mRNA for subtypes of
PDE1 and PDE4, assays for the measurement and detection of PDE gene
families.

Specific in-house expertise

Twenty five years experience in studying the biochemistry and molecular

biology of PDEs. Extensive experience in the development of PDE enzyme
assays, in biochemical purification of PDE isoforms, in cloning of PDE
isoforms, and in performing RT-PCR for examination of the expression of
individual PDE isoforms. Experience also in studying the biological
regulation of PDE isoforms, in development of AS ODNs to block the
expression of PDE, and in tissue culture, for maintenance of cell lines in
which PDE is being studied.

Collaborative research
envisaged

Funding for a team of 4 post-doctoral scientists, two already on staff, and

two to be newly appointed, to clone additional forms of PDE1 and PDE4
from human leukaemic cells, to develop newer and better AS ODNs to block
the expression of these PDEs, and to test them for their ability to induce
apoptosis of human leukaemic cells in culture. And in collaboration, the
partner to provide in vivo models of efficacy for these AS ODNs in which they
are given to immunodeficient (SCID) mice transplanted with human
leukaemic cells to generate and optimise lead AS ODNs as potential
antileukaemic agents.

Preferred level of interaction

Indicate order of priority

Low level of interaction: hands

off - 3 to 6 monthly meetings
and reports

Medium level of interaction:

regular meetings, exchange of
ideas

Fully interactive: week-to-week

contact & exchange of samples
& personnel, as and when
appropriate

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Emerging Therapeutic Targets (1998) 2(1)

112 Inhibition of calcium-calmodulin dependent phosphodiesterase

Preferred type of partner

Indicate order of priority

Multinational pharmaceutical
company

Pharmaceutical company with

an interest in Gene Therapy/
Biopharmaceuticals

Small to medium Biotechnology

company

Other (explain below)

Other

Pharmaceutical or biotechnology company dedicated to development of

therapeutic AS ODN.
A major pharmaceutical company with sufficient human and financial
resources to fund the above proposal and provide the developmental
machinery required to examine lead AS ODN candidates in in vivo mouse
models of leukaemia, and in Phase I and long-term clinical trials. The partner
would be expected to agree to suitable remuneration (against
predetermined decision criteria) to recompense the intellectual property
exchanged at the outset of the project.

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Emerging Therapeutic Targets (1998) 2(1)