U.S. patent application number 10/060759 was filed with the patent office on 2003-01-23 for compositions and methods for the treatment of chronic lymphocytic leukemia.
This patent application is currently assigned to The Trustees of Boston University. Invention is credited to Lerner, Adam.
Application Number | 20030018014 10/060759 |
Document ID | / |
Family ID | 22286061 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030018014 |
Kind Code |
A1 |
Lerner, Adam |
January 23, 2003 |
Compositions and methods for the treatment of chronic lymphocytic
leukemia
Abstract
Methods for treating patients with CLL with pharmaceutical
agents are disclosed. The methods of the present invention can be
used in patients that have not responded to standard treatment. In
addition, the methods can be used to augment the impact of standard
chemotherapy.
Inventors: |
Lerner, Adam; (Newton
Highlands, MA) |
Correspondence
Address: |
NIXON PEABODY LLP
101 FEDERAL ST
BOSTON
MA
02110
US
|
Assignee: |
The Trustees of Boston
University
|
Family ID: |
22286061 |
Appl. No.: |
10/060759 |
Filed: |
January 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10060759 |
Jan 30, 2002 |
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09423349 |
May 1, 2000 |
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6399649 |
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09423349 |
May 1, 2000 |
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PCT/US99/21518 |
Sep 17, 1999 |
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60101721 |
Sep 24, 1998 |
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Current U.S.
Class: |
514/80 ;
514/263.1 |
Current CPC
Class: |
A61K 31/4015 20130101;
A61K 31/00 20130101 |
Class at
Publication: |
514/80 ;
514/263.1 |
International
Class: |
A61K 031/675; A61K
031/52 |
Claims
I claim,
1. A method, comprising: a) providing: i) a patient having symptoms
of chronic lymphocytic leukemia, and ii) a formulation comprising
an inhibitor that specifically inhbits Type 4 cyclic adenosine
monophosphate phosphodiesterases; and b) administering a
therapeutically effective dose of said formulation to said patient
under conditions such that said symptoms are reduced.
2. The method of claim 1, wherein said administering is enteral
administration.
3. The method of claim 2, wherein said enteral administration is
oral administration.
4. The method of claim 1, wherein said administering is parenteral
administration.
5. The method of claim 1, wherein said patient is a naive
patient.
6. The method of claim 1, wherein said patient is
immunocompromised.
7. The method of claim 1, wherein said patient is unresponsive to
chemotherapy with alkylating agents.
8. A method, comprising: a) providing: i) a patient having symptoms
of chronic lymphocytic leukemia, and ii) a formulation comprising
rolipram; and b) administering a therapeutically effective dose of
said formulation to said patient under conditions such that said
symptoms are reduced.
9. The method of claim 8, wherein said administering is enteral
administration.
10. The method of claim 9, wherein said enteral administration is
oral administration.
11. The method of claim 8, wherein said administering is parenteral
administration.
12. The method of claim 8, wherein said patient is a naive
patient.
13. The method of claim 8, wherein said patient is
immunocompromised.
14. The method of claim 8, wherein said patient is unresponsive to
chemotherapy with alkylating agents.
Description
FIELD OF THE INVENTION
[0001] The present invention pertains to the treatment of patients
with chronic lymphocytic leukemia (CLL) with pharmaceutical
compositions comprising Type 4 cyclic adenosine monophosphate
phosphodiesterase inhibitors, and more particularly, with an
inhibitor that specifically inhibits Type 4 cyclic adenosine
monophosphate phosphodiesterase.
BACKGROUND
[0002] Leukernias are malignant neoplasms of hematopoietic tissues.
These neoplasms are categorized into two predominant forms: chronic
and acute. While acute leukenias are characterized by
undifferentiated cell populations, chronic leukemias usually
present a more mature morphology. Notwithstanding these
classifications, however, the pathological impairment of normal
hematopoiesis is the hallmark of all leukemias,
[0003] Chronic lymphocytic leukemia (CLL) is a neoplasm in which a
clonal expansion of small lymphocytes accumulate in the marrow,
lymph nodes, blood, spleen liver, and sometime other organs. The
CLL cell is the neoplastic counterpart of an immunologically
immature, incompetent lymphocyte. In over 95 percent of the cases,
the clonal expansion is of a Bell lineage. See Cancer: Principles
& Practice of Oncology (3rd Edition) (1989) (pp. 1843-1847). In
less than 5 percent of cases the tumor cells have a T-cell
phenotype.
[0004] CLL, while accounting for only about 0.8 percent of all
cancers in the United States, is the most prevalent leukemia
afflicting adults in modem countries, accounting for 30 percent of
all leukemias. A majority of patients are over 60 years at the time
of disease and 90 percent are over age 50.
[0005] Most patients are diagnosed following a routine physical
examination or a blood count. The earliest and most frequent
symptoms are fatigue and malaise. Later symptoms include
lymphadenopathy and splenomegaly. Anemia and thrombocytopenia is
found in approximately 15 percent of patients.
[0006] The general goal of leukemia therapy is to arrest the
proliferation of abnormal morphologies and restore "normal"
hematopoiesis in the bone marrow. Treatment regimens include
chemotherapy. Unfortunately, chemotherapy is not always successful.
Indeed, while CLL patients may have initial clinical responses to
alkylating agents such as chlorambucil or adenosine analogs such as
fludarabine, many ultimately become refractory to therapy.
Consequently, there is a pressing need for the identification of
novel approaches to this disease.
SUMMARY OF THE INVENTION
[0007] The present invention pertains to the treatment of patients
with chronic lymphocytic leukemia with pharmaceutical compositions
comprising Type 4 cyclic adenosine monophosphate phosphodiesterase
inhibitors, and more particularly, with an inhibitor that
specifically inhibits Type 4 cyclic adenosine monophosphate
phosphodiesterase. One embodiment of the present invention
contemplates a method comprising: a) providing: i) a patient having
symptoms of chronic lymphocytic leukemia, and ii) a formulation
comprising an inhibitor that specifically inhibits Type 4 cyclic
adenosine monophosphate phosphodiesterases; and b) administration
of a therapeutically effective dose of said formulation to said
patient under conditions such that said symptoms are reduced.
[0008] The present invention is not limited by the method of
administration. In one embodiment, the administration is enteral
administration. In another embodiment, said enteral administration
is oral administration. In still another embodiment, said
administration is parenteral administration. In these embodiments,
said parenteral administration can be topical administration or by
a transdermal patch. In another embodiment, said parenteral
administration is subcutaneous administration. While in still
another embodiment, said parenteral administration utilizes an
aerosol.
[0009] The present invention is not limited by the nature of the
patient. In one embodiment, said patient is a naive patient (e.g.,
has not undergone prior treatment for CLL), while in other
embodiments said patient is unresponsive or refractory to standard
chemotherapy (e.g., alkylating agents). In still another
embodiment, said patient is immunocompromised. In one embodiment,
said patient is over fifty years of age.
[0010] The present invention is also not limited by the method of
determining response to treatment. In one embodiment, said symptoms
comprise lymphadenopathy and splenomegaly. In a yet another
embodiment, said symptoms comprise the histology of a lymph node
that is consistent with CLL.
[0011] The present invention contemplates usage of a variety of
specific inhibitors. A preferred inhibitor is rolipram. While it is
not intended that the present invention be limited to a specific
mechanism by which the inhibitors of the present invention achieve
therapeutic success, it has been empirically found (as the data
herein shows) that the specific inhibitors of the present invention
(e.g., rolipram) augment apoptosis induced by commonly used drugs
(e.g., doxorubicin, chlorambucil and fludarabine). Consequently,
the present invention specifically contemplates the use of the
inhibitors in combination with other drugs, including but not
limited to cytotoxic drugs.
[0012] Definitions
[0013] As used herein, the term "enteral administration" means the
introduction of a composition to a patient such that it is absorbed
in the intestinal tract of the patient (e.g., pill, tablet, elixir,
etc.)
[0014] As used herein, the term "oral administration" means the
introduction of a composition to a patient through the oral cavity
(i.e., in the mouth).
[0015] As used herein, the term "parenteral administration" means
administration of a composition other than enteral (e.g.,
injection, transdermal, aerosol, etc.).
[0016] As used herein, the term "topical administration" means the
introduction of a composition to a patient by application to the
surface of the skin.
[0017] As used herein, the term, "subcutaneous administration"
means introduction of a composition to a patient under the surface
of the skin (e.g., injection with a hypodermic needle).
[0018] As used herein, the phrase "naive patient" refers to a
patient that has not undergone prior treatment for chronic
lymnphocytic leukemia.
[0019] As used herein, the phrase "an inhibitor that specifically
inhibits Type 4 cyclic adenosine monophosphate phosphodiesterase"
refers to a compound that inhibits Type 4 but not Type 1 or 3
phosphodiesterases. Of course, background level inhibition of Type
1 or 3 phosphodiesterases is permitted within the definition. Where
the inhibitor inhibits Type 4 as well as Type 1 and/or 3, but
inhibits Type 4 to a greater extent (the amounts being subject to
quantitative determination by assays described herein), the phrase
"preferentially inhibits Type 4 phosphodiesterases" is used herein
(as distinct from "Type 4 specific."
DESCRIPTION OF THE FIGURES
[0020] FIG. 1 is a gel showing the results of PCR on normal and CLL
cells using oligonucleotides specific for human
phosphodiesterases.
[0021] FIG. 2 is a Northern blot using the PCR products of FIG.
1.
[0022] FIG. 3 graphically shows the PDE activity of CLL cells (left
panel) and a murine B lymphoma cell line (right).
[0023] FIG. 4 graphically shows cAMP levels in CLL cells, WMC, and
resting B cells.
[0024] FIG. 5 shows the DNA fragmentation results on gel
electrophoresis using a 1.5% agarose gel wherein the bands are
visualized with ethidium bromide.
[0025] FIG. 6 shows the results of Hoechst 33342 flow
cytometry.
[0026] FIG. 7 graphically shows the increasing percent of apoptotic
cells as a function of time and rolipram dose.
[0027] FIG. 8 graphically shows the different sensitivity of
various cells to the specific inhibitors of the present
invention.
[0028] FIG. 9 graphically shows that rolipram blocks cAMP
catabolism in both sensitive and resistant lymphoid
populations.
[0029] FIG. 10 graphically shows that sensitivity to rolipram
mirrors the sensitivity to dbcAMP.
[0030] FIG. 11 graphically shows an increase in the percent
apoptotic cells with increasing doses of the inhibitor XX5.
[0031] FIG. 12 graphically shows that rolipram augments
fludarabine-induced apoptosis in CLL cells.
[0032] FIG. 13 graphically shows that rolipram augments
chlorambucil-induced apoptosis in CLL cells.
[0033] FIG. 14 graphically shows that rolipram augments
doxorubicin-induced apoptosis in CLL cells.
DESCRIPTION OF THE INVENTION
[0034] The present invention pertains to the treatment of patients
with chronic lymphocytic leukemia with pharmaceutical compositions
comprising Type 4 cyclic adenosine monophosphate phosphodiesterase
inhibitors, and more particularly, with an inhibitor that
specifically inhibits Type 4 cyclic adenosine monophosphate
phosphodiesterase. The description of the invention discusses 1)
apoptosis generally, 2) phosphodiesterases as a target for CLL
therapy, and 3) treating CLL patients.
[0035] A. Apoptosis And Intracellular cAMP Levels
[0036] The first observation that some lymphoid cells die following
exposure to agents that raise intracellular cAMP levels was made by
Daniel et al who found that the murine lymphoma cell line S49.1
underwent cytolysis following 48 hours of exposure to the
combination of theophylline and a cell permeable 3': 5'cyclic AMP
analog, dibutyryl cAMP (dbcAMP). V. Daniel et al., "Induction of
cytolysis of cultured lymphoma cells by adenosine 3':5'-cyclic
monophosphate and isolation of resistant variants," Proc. Natl.
Acad. Sci. USA 70:76 (1973). When mutant S49.1 clones resistant to
the cytolytic effects of dbcAMP were isolated in soft agar, they
were shown to be defective in the regulatory subunit of protein
kinase A, confirming that cytolysis occurred as a direct result of
PKA-mediated phosphorylation of unknown lymphoid target proteins.
Subsequent work has demonstrated that cAMP-induced cytolysis occurs
by apoptosis. D. J. McConkey et al, "Agents that elevate cAMP
stimulate DNA fragmentation in thymocytes," J Immunol. 145:1227
(1990). Certain normal T and B lymphoid subsets express the same
marked sensitivity to cAMP-induced toxicity as tumor cell lines.
Within the T lineage, CD4+CD8+thymocytes appear to be more
sensitive to the induction of apoptosis by cAMP than mature T
cells. Apoptosis in resting human B lymphocytes, which occurs
spontaneously at a high rate in culture, can be augmented by the
addition of stimuli which elevate intracellular cAMP levels, such
as the diterpene adenylate cyclase activator forskolin J. Lomo et
al., "TGF-b1 and cyclic AMP promote apoptosis in resting human B
lymphocytes,"J. Immunol 154:1634 (1995).
[0037] D B. Phosphodiesterases As A Target For Therapy Of CLL
[0038] Cyclic AMP is catabolized within cells to 5'-AMP by
3':5'cAMP phosphodiesterases (PDE), a diverse group of enzymes
encompassing 15 gene products and 7 classes of enzymes which have
proven to be the target of successful pharmaceutical agents for
neurologic, cardiovascular and inflammatory disorders. Despite this
large array of cyclic nucleotide PDEs, only a subset of these
enzymes have been reported in human lymphoid cells. Among them, the
most commonly reported enzymes in human T cells are types 1, 3 and
4. Calcium-calmodulin dependent type 1 PDE activity has been
detected in phytohemagglutinin-stimulated but not resting
peripheral blood lymphocytes. One isoform from this family, PDE1B1,
was recently detected in acute lymphocytic leukemia cells;
inhibition of this enzyme was reported to induce apoptosis. PDE1
enzymes, which can catalyze the degradation of both cAMP and cGMP,
are specifically inhibited by vinpocetine (IC50=21 mMol/L). Two
groups have reported both type 3 and type 4 PDE in human T
lymphocytes; lectin-mediated proliferation was completely
suppressed only by treating cells with specific inhibitors of both
classes of enzymes. While four human PDE4 genes have been cloned,
only three of the isoforms (PDE4A, B and D) have been identified in
lymphocytes. Type 4 enzymes are specifically inhibited by rolipram
[4-(4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone] (IC50=1
mMol/L) and the structurally related compound XX5 (IC50=2 mMol/L).
U. Schwabe et al.,
"4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711): a
potent inhibitor of adenosine cyclic 3',5'-monophosphate
phosphodiesterases in homogenates and tissue slices from rat
brain," Molecular Pharmacology 12:900 (1976). H. Sheppard et al., "
Structure-activity relationships for inhibitors of
phosphodiesterase from erythrocytes and other tissues," Adv Cyclic
Nucl Res 1:103 (1972).
[0039] Theophylline induces apoptosis in CLL cells, but it is not a
specific Type 4 inhibitor. Moreover, a clinical application of
theophylline for CLL is complicated by its activity as an adenosine
receptor antagonist. As an alternate approach, the present
invention contemplates specific inhibitors.
[0040] C. Treating Patients
[0041] While the present invention is not limited by the nature of
the prior treatment of the subject, it is contemplated that, in one
embodiment, the present invention be utilized in patients who have
not undergone prior treatment for their condition (ie., naive
patients), as well as patients who have not responded to (or are
refractory to) standard chemotherapeutic agents (e.g., alkylating
agents). Thus, the present invention specifically contemplates
treating patients who have failed standard therapy.
[0042] On the other hand, the present invention specifically
contemplates the use of the inhibitors in combination with other
drugs, including but not limited to cytotoxic drugs. This
combination therapy is based on findings (described herein) that
specific inhibitors can augment apoptosis when used with such
drugs.
[0043] It is contemplated that the methods of the present invention
be administered alone or can be administered with a pharmaceutical
carrier selected on the basis of the chosen route of administration
and standard pharmaceutical practice. In one preferred embodiment,
the specific inhibitor is administered orally in solid dosage
forms, such as capsules, tablets, or powders, or in liquid dosage
forms, such as elixirs, syrups, and suspensions; however, it can
also be administered parenterally, in sterile liquid dosage forms,
or rectally in the form of suppositories.
[0044] One skilled in the art will be capable of adjusting the
administered dose depending upon known factors such as the mode and
route of administration; age, health, and weight of the recipient;
nature and extent of symptoms, kind of concurrent treatment,
frequency of treatment, and the effect desired. In one embodiment,
the dosage is increased to overcome a non-responsive condition.
[0045] Additionally, the specific (and preferential) inhibitors of
the present invention can be employed in admixture with
conventional excipients, i.e., pharmaceutically acceptable organic
or inorganic carrier substances suitable for parenteral (e.g.
topical application) or enteral (e.g., oral) which do not
deleteriously react with the active compounds.
[0046] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions, alcohols, gum arabic,
vegetable oils, benzyl alcohols, polyethylene glycols, gelatine,
carbohydrates such as lactose, amylose, or starch, magnesium
stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, pentaerythritol fatty acid
esters, hydroxy methylcellulose, polyvinyl pyrrolidone, merely to
name a few. The pharmaceutical preparations can be sterilized and
if desired mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifier, salts for
influencing osmotic pressure, buffers, coloring, flavoring, and/or
aromatic substances and the like which do no deleteriously react
with the active compounds. They can also be combined where desired
with other agents, e.g., vitamins and/or antibiotics.
[0047] For enteral application, particularly suitable are tablets,
liquids, drops, suppositories, or capsules. A syrup, elixir, or the
like can be used wherein a sweetened vehicle is employed. Sustained
or directed release compositions can be formulated, e.g., liposomes
or those wherein the active compound is protected with
differentially degradable coating, e.g., by microencapsulation,
multiple coatings, etc.
[0048] In this manner, the present invention may be introduced into
a subject in polymeric microspheres for the controlled release of
the compound. Methods of producing microspheres from polymer can be
found in U.S. Pat. No. 5,601,844 to Kagayama, et al and U.S. Pat.
Nos. 5,529,914 and 5,573,934 to Hubbel, et al., herein incorporated
by reference.
[0049] Other medicaments can be produced in a known manner, whereby
the known and customary pharmaceutical adjuvants as well as other
customary carrier and diluting agents can be used. Examples
include, but are not limited to, gelatins, natural sugars such as
sucrose or lactose, lecithin, pectin, starch (for example
cornstarch), alginic acid, tylose, talc, lycopodium, silica (for
example colloidal silica), glucose, cellulose, cellulose
derivatives for example, cellulose ethers in which the cellulose
hydroxyl group are partially etherified with lower aliphatic
alcohols and/or lower saturated oxyalchohols, for example, methyl
hydroxypropyl cellulose, methyl cellulose, cellulose phthalate,
stearates, e.g., methylstearate and glyeeryl stearate, magnesium
and calcium salts of fatty acids with 12 to 22 carbon atoms,
especially saturated acids (for example, calcium stearate, calcium
laurate, magnesium oleate, calcium palmitate, calcium behenate and
magnesium stearate), emulsifiers, oils and fats, especially of
plant origin (for example, peanut oil, castor oil, olive oil,
sesame oil, cottonseed oil, corn oil, wheat germ oil, sunflower
seed oil, cod-liver oil), mono, di, and triglycerides of saturated
fatty acids (C.sub.12 H.sub.24 O.sub.2 to C.sub.18 H.sub.36 O.sub.2
and their mitues), e.g., glyceryl monostearate, glyceryl
distearate, glyceryl tristearate, glyceryl trilaurate),
pharmaceutically compatible mono- or polyvalent alcohols and
polyglycols such as glycerine, mannitol, sorbitol, pentaerythritol,
ethyl alcohol, diethylene glycol, triethylene glycol, ethylene
glycol, propylene glycol, dipropylene glycol, polyethylene glycol
400, and other polyethylene glycols, as well as derivatives of such
alcohols and polyglycols, esters of saturated and unsaturated fatty
acids (2 to 22 carbon atoms, especially 10 to 18 carbon atoms),
with monohydricaliphatic alcohols (1 to 20 carbon atom alkanols),
or polyhydric alcohols such as glycols, glycerine, diethylene
glycol, pentaerythritol, sorbitol, mannitol, ethyl alcohol, butyl
alcohol, octadecyl alcohol, etc., e.g., glyceryl stearate, glyceryl
palmitate, glycol distearate, glycol dilaurate, glycol diacetate,
monoacetin, triacetin, glyceryl oleate, ethylene glycol stearate;
such esters of polyvalent alcohols can in a given case be
etherified, benzyl benzoate, dioxolane, glycerine formal,
tetrahydrofurfuryl alcohol, polyglycol ethers with 1 to 12 carbon
atom alcohols, dimethyl acetamide, lactamide, lactates, e.g. ethyl
lactate, ethyl carbonate, silicones (especially middle viscosity
dimethyl polysiloxane).
[0050] For injectable solutions or suspensions, non-toxic
parenterally compatible diluting agents or solvents can be used,
for example: Water, 1,3 butane diol, ethanol, 1,2-propylene glycol,
polyglycols in a mixture with water, Ringer's solution, isotonic
solution of sodium chloride or also hardened oils including
synthetic mono or diglycerides or fatty acids like oleic acid.
[0051] Known and customary solution assistants or emulsifiers can
be used in the production of the preparations. The following are
examples of solution assistants and emulsifiers which can be used:
Polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbian
trioleate, phosphatides such as lecithin, acacia, tragacath,
polyoxethylated sorbitan monooleate and other ethoxyated fatty acid
esters of sorbitan, polyoxyethylated fats, polyoxyethylated
oleotriglycerides, linolized oleotriglycerides, polyethylene oxide
condensation products of fatty alcohols, alkyl phenolene or fatty
acids or also 1-methyl-3-(2-hydroxyethyl)imidazolidone2). The term
polyoxyethylated means in this context that the substances in
question contain polyoxyethylene chains whose polymerization is
generally between 2 to 40 and especially between 10 to 20.
[0052] Furthermore, there can be added preservatives stabilizers,
buffers, for example, calcium hydrogen phosphate, colloidal
aluminum hydroxide, taste correctives, antioxidants and complex
formers (for example, ethylene diamine tetraacetic acid) and the
like. In a given case for stabilization of the active molecule, the
pH is adjusted to about 3 to 7 with physiologically compatible
acids or buffers. Generally, there is preferred as neutral as
possible to weak acid (to pH 5) pH value.
[0053] As antioxidants, there can be used, for example, sodiummeta
bisulfite, ascorbic acid, gallic acid, alkyl gallates, e.g., methyl
gallate and ethyl gallate, butyl hydroxyanisole,
nordihydroguararetic acid, tocopherols as well as tocopherol and
synergists (materials which bind heavy metals by complex formation,
for example, lecithin, ascorbic acid, phosphoric acid). The
addition of synergists increases considerably the antioxidant
activity of tocopherol. As preservatives, there can be used, for
example, sorbic acid, p-hydroxybenzoic acid esters (for example,
lower alkyl esters such as the methyl ester and the ethyl ester)
benzoic acid, sodium benzoate, trichloroisobutyl alcohol, phenol,
cresol, benzethonium chloride, and formalin derivatives.
[0054] Experimental
[0055] The following example serves to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
[0056] A. Reagents
[0057] The following reagents were obtained from commercial
sources: alkaline phosphatase, cAMP, dibutyryl cAMP, calmodulin,
forskolin (Sigma Chemical Co., St. Louis, Mo.); vinpocetine (Alexis
Biochemicals, San Diego, Calif.); recombinant human IL2 (Genzyne,
Boston, Mass.); F(ab')2 fragment goat anti-human IgG and IgM
(Jackson Immunoresearch Laboratories, West Grove Pa.); Hoechst
33342 (Molecular Probes, Eugene, Oreg.). Rolipram (racemate of
4-[3'-cyclopentyloxy-4'-methoxyphenyl]-2-py- rrolidone) was a gift
from Dr. Ronald Wohl, Berlex Laboratories (Wayne, N.J.).
[0058] B. Patient Selection
[0059] After IRB-approved informed consent, blood was drawn in
heparinized tubes from patients with flow cytometry-verified CLL
who were either untreated or at least one month post-chemotherapy.
Patients with active infections or other serious medical conditions
were not included in this study. Charts were reviewed to establish
patients' sensitivity to chemotherapy and the stage of CLL.
Resistance to a chemotherapeutic agent was defined as a rise in
peripheral leukemic cell count or progression of adenopathy or
splenomegaly prior to the initiation of the next scheduled cycle of
chemotherapy.
[0060] C. Cell Purification And Culture
[0061] Mononuclear cells were obtained by density gradient
centrifugation over Histopaque 1077 (Sigma Chemical Company, St.
Louis, Mo.). As flow cytometry demonstrated that CLL cells made up
more than 95% of the mononuclear cells so purified, both apoptosis
and cAMP assays were performed with these patient cell
preparations. For PCR experiments on CLL cells or for all
experiments on normal circulating B cells, the whole mon'onuclear
cells were further purified by incubation with Dynal anti-CD19
magnetic beads at a 1:1 bead:cell ratio, extensive washing with a
magnetic particle concentrater and elution with CD19 Detachabead
reagent Dynal, Lake Success, N.Y.). Leukemic cells from two CLL
patients showed sensitivity to rolipram-mediated apoptosis whether
or not they were further purified by anti-CD19 magnetic beads.
Cells were cultured in RPMI 1640 media (Biowhittaker, Walkersville,
Md.) supplemented with 10% fetal calf serum 50 uMol/L
2-mercaptoethanol, 2 mMol/L L-glutamine, 10 mMol/L Hepes pH 7.4,
100 U/mL penicillin, and 100 U/mL streptomycin (Sigma Chemical
Company, St. Louis, Mo.) at 37.degree. C. and 5% CO.sub.2 in
air.
[0062] D. RT PCR And Northern Analysis
[0063] RNA was isolated from CLL or whole mononuclear cells using
Ultraspec reagent (Biotecx, Houston, Tex.). cDNA was synthesized
from 10 ug of total RNA using oligo d(T) primers and Maloney murine
leukemia virus reverse transcriptase in a final volume of 40 uL
(Stratagene, La Jolla, Calif.). One mL of the first strand cDNA
product was then used as template for PCR amplification with
AmpliTaq DNA polymerase (Roche Molecular Systems, Branchburg, N.J.)
by 40 thermocycles of 94.degree. C. for 1 minute, 60.degree. C. for
1 minute and 72.degree. C. for 1 minute. The PDE PCR assay products
were as follows with oligonucleotide sequences given 5'->3':
Human PDE1BI (Genbank accession # U56976) was 430 bp (1st base
1660; sense=GTC TTC ATT GAG TCC AAA GTG , antisense=GAC CTG CCA GCT
AAG ATC TGG). Human PDE3A (cGIP1, HSPDE3B) (X95520) was 340 bp (1st
base 2999, sense=GTA ACT CCI ATG ATG CTG CTG G, antisense=CTA TTC
CTC TTC ATC TGC CTC). Of note, these PDE3 PCR oligonucleotides are
selective for the human cGIP1 PDE, homologous to rat PDE3A, as the
amplified sequence has only 50% nucleotide homology to the
cardiac/platelet form of human PDE3 (cGIP2). Human PDE4A (M37744)
was 461 bp (1st bp 1819, sense=GGA GGA AGA AAT ATC AAT GGC CC,
antisense=GAT GTG TCC TCC CCA AAT GTC). Human PDE4B (L20966) was
479 bp (1st bp 2213, sense=ATT CTG AAG GAC CTG AGA AGG,
antisense=CAG TGA GTT CAG TCA CTG TCG). For hybridization to
Northern blots, these PCR products were subdloned into a plasmid
vector (pCRII, Invitrogen, Carlsbad, Calif.) and subsequently
utilized for PCR-based amplification of a32P dATP-labelled
probes.
[0064] E. cAMP Assay
[0065] One million cells in 1 mL were treated for two hours with or
without drugs. 0.8 mL of cells were pelleted by spinning at 4,000
rpm (RCF=1,310) in microcentrifuige tube. After discarding 0.7 mL,
400 uL of ethanol was added, vortexed and left on ice for five
minutes. Particulate cell debris was removed by centrifuigation at
14,000 rpm (RCF=16,000). The supematant was stored at -20.degree.
C. until the day of assay, at which time it was dried in a
Speedivac (Savant, Farmingdale, N.Y.) to a volume of 50 uL. After
ten-fold dilution in 10 mMol/L Tris pH 8.0, 1 mMol/L EDTA, the cAP
sample was analyzed for cAMP concentration using a cAMP RIA kit
(NEN, Boston, Mass.) according to the manufacturer's instructions
using the nonacetylated protocol.
[0066] F. cAMP PDE Assay
[0067] The technique of Robicsek et al., itself adapted from an
assay described by Thompson and Appleman, was used in modified
form. S. A. Robicsek et al., "Multiple high-affinity
cAMP-phosphodiesterases in human T-lymphocytes," Biochem.
Pharmacol. 42:869 (1991). W. J. Thompson and M. M. Appleman,
"Multiple cyclic nucleotide phosphodiesterase activities from rat
brain," Biochemistry 10:311 (1991). 150 million purified CLL cells
were pelleted and sonicated (Branson 350 Sonifier with microtip
probe, output=2, 50% duty cycle) on ice in 1.0 nL of a buffer which
contained 20 mMol/L Tris (pH 6.8), 1 mMol/L EDTA, aprotinin (50
u/mL), pepstatin (1 mg/mL), PMSF (1 mMol/L) and 3.75 mMol/L b-ME.
Assay buffer contained 100 mMol/L Tris (pH 8.0), 20 mMol/1
MgCl.sub.2, 0.2% BSA and 7.5 mMol/L b-ME. [.sup.3H]-cAMP (NEN,
Boston, Mass.) was incubated with PDE for 10 minutes at 30.degree.
C. in 20 uL volumes (10 uL of sonication buffer and 10 uL of assay
buffer) which contained 0.22 units of alkaline phosphatase. 10
uMol/L rolipram or 0.2 mMol/L calcium/20 nMol/L calmodulin were
added to the assay buffer as appropriate. Reactions were halted by
the addition of 0.5 mL of a 1:3 slurry w/v slurry of AG1-X8 anion
exchange resin and a mixture of equal volumes of water and
isopropanol. The resin bound the unreacted nucleotide but not the
dephosphorylated nucleoside. Microcentrifuge tubes were spun at
3000 rpm RCF=735) for 15 minutes. The radiolabelled nucleosides in
the supernatant were counted using Ecoscint scintillation fluid
(National Diagnostics, Atlanta, Ga.). Three to five enzyme
dilutions were assayed to determine each velocity. Linearity of
velocity with respect to enzyme concentration and time were
verified.
[0068] G. DNA Ladder Gel Assay
[0069] 10 million purified CLL cells were harvested by
centrifugation following exposure to drugs during a 72 hour tissue
culture incubation. Cells were lysed in 0.5 mL of 20 mMol/L Tris
(pH 7.4), 0.4 mMol/L EDTA, 0.25% Triton X. After 15 minutes of
incubation at room temperature, nuclei were removed by
centnfugation at 14,000 rpm (RCF=16,000). The supernatant was
transferred to a new tube and soluble DNA precipitated overnight at
-20.degree. C. following the addition of 55 mL of 5 Mol/L NaCl and
550 mL of isopropanol. After centrifugation at 14000 rpm for 10
minutes, followed by a 70% ethanol wash, the pellet was resuspended
in 20 uL of 10 mMol/L Tris (pH 8.0), 1 mMol/L EDTA, 0.1 mg/mL RNase
and incubated at 37.degree. C. for 30 minutes prior to
electrophoresis on 1.6% TBE agarose gels. DNA fragments were
visualized by UV light after staining the gels with ethidium
bromide.
[0070] H. Hoechst 33342 Apoptosis Assay
[0071] Hoechst 33342 was dissolved in water and frozen at 33 mg/mL
at -20.degree. C. One million cells/well were incubated in
duplicate or triplicate in 48 well tissue culture plates (Costar,
Cambridge, Mass.) with or without drug treatment for 48 hours in 1
mL of culture media. Cells were transferred to 12.times.75 mm
polystyrene Falcons.RTM.2054 FACS tubes (Becton Dickinson Labware,
Lincoln Park, N.J.) and incubated for ten minutes at 37.degree. C.
with Hoechst 33342 at a final concentration of 0.25 mg/mL.23 Cells
were stored on ice until analysis on a FACS Vantage flow cytometer
(Becton Dickinson, San Jose, Calif.). Hoechst 33342 dye
fluorescence was excited with a UV laser and detected using a 450
banddpass filter. Data was analyzed using Cellquest software
(Becton Dickinson, San Jose, Calif.).
EXAMPLE 1
[0072] This example describes the identification of PDE targets in
CLL cells. As an initial approach to identify potential PDE targets
in CLL, PCR was performed on cDNA derived from unpurified
mononuclear cells of a CLL patient with qAFX oligonucleotides
specific for human PDEI-LB, PDE3A, PDEAA and PDE4B, all of which
have been previously identified in various normal and malignant
primary lymphoid cells. Appropriate sized PCR products were
detected from all four transcripts using this template. In order to
reduce the likelihood of amplifying non-leukemic cell transcripts,
we purified CDl9-positive cells from this and two other CLL
patients prior to synthesizing cDNA. PDEl-LB, PDE-4A and PDE-4B
were still detected in these three templates, as they were in cDNA
derived from normal whole mononuclear cells FIG. 1 shows that CLL
cells contain transcripts for PDE 1B1, PDE4A and PDE4B. The cDNA
utilized in the PCR in the bottom 3 panels was derived from
leukemic cells from three different patients purified by positive
selection for CD19 expression. The lowest band in the MWM lane on
the left in the upper panel is 603 bp. Expected PCR product sizes
for PDE1, 3A, 4A and 4B are 430, 340, 461 and 470 bp
respectively.
[0073] Using the same four PCR products as probes on Northern
blots, only PDE-4B transcript was detectable in 10 ug of loaded
RNA. FIG. 2 shows that PDE4B levels fall in CLL cells following
culture but are partially maintained by treatment with 10 uMol/L
rolipram. RNA was isolated from 20 million CLL cells immediately
after cell purification (CT), or after 6 hours culture in media
alone (6 Hr) or with addition of 10 uMol/L rolipram (Roli). Equal
loading and transfer of RNA was confirmed by hybridization with an
actin probe as shown. These results are representative of Northern
analysis performed on leukemic cells from two patients. PDE-4B
transcript levels were significantly higher in freshly isolated CLL
cells than in CLL cells which had been cultured for six hours.
Addition of 10 uMol/L rolipram, a type 4 PDE-specific
phosphodiesterase inhibitor, significantly reduced this fall in
PDE-4B transcript levels during the six hour culture period.
EXAMPLE 2
[0074] In this example, it was determined whether the
above-described PDE transcripts were translated into protein with
constitutive activity. PDE enzyme assays were performed on CLL cell
lysates. FIG. 3 shows Lineweaver-Burk analysis of PDE enzymatic
activity in lysates of CLL cells and Bal-17 cells (the CLL data are
representative of enzymatic assays on three patients). Clearly,
Type 1 and 4 PDE activity differs between a murine B lymphoma cell
line (Bal-17) and CLL cells. We found no evidence of type 1 PDE
activity in CLL cells as the basal PDE activity was not augmented
by the addition of calcium and calmodulin (FIG. 3, left). PDE
activity in CLL cells was inhibited by 10 uMol/L rolipram (rolipram
altered both K.sub.m and the V.sub.max, consistent with its known
activity as a noncompetitive antagonist) but was not augmented by
the addition of 0.2 mMol/L calcium and 20 Mol/L calmodulin. On the
other hand, Bal-17 PDE activity is augmented by the addition of
calcium and calmodulin (FIG. 3, right). That is to say, it was
possible to identify substantial constitutive type 1 PDE activity
in the murine B lymphoma cell line Bal-17, as demonstrated by a
rise in PDE activity with the addition of calcium and
calmodulin.
EXAMPLE 3
[0075] In this example, it was determined what role type 1 and type
4 PDE activity might play in the catabolism of cyclic nucleotides
in CLL cells. cAMP levels were measured after culturing leukemic
cells with varying concentrations of PDE-isoform specific
inhibitors, either alone or in conjunction with an activator of
adenylate cyclase, the diterpene forskolin. Specifically, one
million freshly isolated cells were incubated with the indicated
drugs for two hours prior to analysis of cAMP in cell lysates using
a radioimmunoassay. The results are shown in FIG. 4 and the data
are the mean of three individually assayed culture wells. The
experimental conditions indicated with an asterisk had a greater
[cAMP] than, as appropriate, the untreated or forskolin-treated
control cells (t test: one-tailed significance level<0.05).
[0076] In panel D, vinpocetine was added at 30 uMol/L. When
incubated with forskolin and the PDE-1 inhibitor vinpocetine, CLL
cell cAMP was not augmented above levels induced by forskolin alone
(FIG. 4, panel D). Incubation of Bal-17 cells with vinpocetine
reduced both basal and forskolin-stimulated cAMP levels, a result
in keeping with the reported primary effect of this drug on cGMP
rather than cAMP levels (data not shown). In contrast, addition of
the type 4 PDE inhibitor rolipram augmented CLL cAMP levels, both
when used alone and more dramatically when combined with forskolin
(FIG. 4, Panels A-D). CLL cells were not unique with respect to
their response to these isoform-specific inhibitors. cAMP levels in
both a predominantly T cell population (IL-2 supplemented normal
whole mononuclear cells; >90% CD3+T cells by flow cytometry) and
magnetic-bead purified CD19+B cells rose following inhibition of
type 4 but not type 1 PDE activity (FIG. 4, Panels E and F and data
not shown).
EXAMPLE 4
[0077] This example demonstrates that Type 4 PDE inhibition induces
CLL apoptosis The above-described results identified the type 4
cAMP phosphodiesterase family as an important regulator of cAMP
levels in CLL cells. Consequently, a study of the activity of type
4-specific POE inhibitors as inducers of cAMP-mediated apoptosis in
leukemic cells from CLL patients was carried out. CLL cells were
incubated for 72 hours either alone of with 10 uMol/L rolipram, 40
uMol/L forskolin or both agents. We tested whether rolipram induces
intemucleosomal DNA fragmentation characteristic of apoptosis by
isolating soluble DNA from the leukemic cells with detergent
treatment, then removing DNA from intact non-apoptotic nuclei by
centrifugation. Specifically, soluble DNA was isolated from ten
million CLL cells cultured for 72 hours in media (CT), 10 uMol/L
rolipram (Roli), 40 uMol/L forskolin (Fsk) or a combination of the
latter two agents (Ro/Fs). DNA fragments were resolved by
electrophoresis on a 1.5% agarose gel and visulized with ethidium
bromide. FIG. 5 shows the results. These data are representative of
the four leukemic cell samples tested. As shown in FIG. 5, while
culture of CLL cells in media alone resulted in only a faint DNA
"ladder", treatment with rolipram and/or forskolin resulted in
pronounced intemucleosomal DNA fragmentation.
[0078] As a more quantitative analysis of CLL apoptosis, we
utilized a flow cytometry method in which apoptotic cells are
distinguished both by their reduced size (FSC) and their increased
uptake of the lipophilic UV fluorescent dye Hoechst 33342 (FL-4)
when the intact, heterogeneous cell population is incubated with a
low concentration of the dye (0.25 ug/mL) for 10 minutes at
37.degree. C. Specifically, cells were cultured for 72 hours in
media (1), 1 uMol/L rolipram (2), 40 uMol/L forskolin (3) or a
combination of the two drugs (FIG. 6). The abcissa reflects forward
light scatter and the ordinate Hoechst 33342 fluorescence.
Apoptotic cells are characterized by reduced forward light scatter
and increased Hoechst 33342 fluorescence. Previous reports of cAMP
induced lymphoid apoptosis have noted that this form of programmed
cell death may take 48 to 72 hours to develop maximally. Using the
Hoechst 33342 assay in a time course experiment, we found that the
combination of 10 uMol/L rolipram and 40 uMol/L forskolin induced
significant CLL apoptosis which plateaued 48 to 72 hours after the
addition of these drugs (FIG. 7, left panel). CLL cells were
cultured for 72 hours in one mL of media with the indicated
concentration of rolipram with (black bars) or without (stippled
bars) the addition of 40 uMol/L forskolin. Using the 72 hour
culture period, we found a dose dependent increase in CLL cell
apoptosis when leukemic cells were incubated with rolipram (FIG. 7,
right panel). Treatment of CLL cells with forskolin alone induced
moderate apoptosis, but combination of forskolin with even low
doses of rolipram resulted in a supra-additive effect on induction
of CLL apoptosis (FIG. 7, right panel). Similar results were
obtained with a structurally distinct PDE4 inhibitor, XX5, or when
isoproterenol or prostaglandin E2 were utilized to activate CLL
adenylate cyclase rather than forskolin (data not shown).
[0079] When CLL cells were incubated with the type 1 PDE inhibitor,
vinpocetine, they underwent apoptosis at dosages of 10 or 30 uMol/L
but not at 2 uMol/L. Given that vinpocetine failed to augment cAMP
accumulation and that we were unable to detect type 1 PDE activity
in CLL cells, we suspect that this drug may induce apoptosis by a
mechanism unrelated to cAMP. Consistent with this hypothesis, the
kinetics of CLL apoptosis were different when vinpocetine was
utilized with peak apoptosis by 24 rather than 48 hours (data not
shown). Nonetheless, we cannot rule out either a temporally
restricted or a topologically compartmentalized cAMP-mediated
apoptotic effect from this drug.
[0080] Given that CLL is a clinically heterogeneous disease, we
tested a total of 14 CLL patients of varying clinical stage,
treatment history and known resistance to chemotherapeutic agents
for the sensitivity of their cells to phosphodiesterase
inhibitor-mediated apoptosis. Patients were assessed for the
apoptotic sensitivity of their leukemic cells to rolipram,
forskolin or both drugs. In samples from ten "rolipram-sensitive
patients", treatment with 10 uMol/L rolipram induced apoptosis in
more than a third of the leukemic cells, with overall apoptosis
ranging from 44 to 80% (see Table 1 for tabulated results). Among
the seven rolipram-sensitive patients whose cells were treated with
both conditions, 40 uMol/L forskolin as a single agent induced less
apoptosis than rolipram alone, suggesting that blockade of cAMP
catabolism induced a more potent apoptotic signal than further
augmentation of adenylate cyclase activity (p<0.08, Wilcoxon
signed-ranks test for matched pairs). Among four relatively
rolipram-resistant patient samples (Pt#'s 11-14: Table 1), the
absolute increase in apoptotic cells was less than 33%, with
overall apoptosis ranging from 14 to 40%. Nonetheless, addition of
forskolin to rolipram augmented apoptosis (68% and 69%) in two of
the three patient samples examined within this group.
1TABLE 1 Drug Pt. Rai Res. Basal Roli Fsk R/Fs dbcAMP 1. III -- 47
80 ND ND ND 2. IV Ch, Cy 18 .+-. 2 79 46 89 ND 3. IV Ch, CH 38 77
.+-. 1 59 .+-. 0 79 .+-. 1 71 .+-. 3 4. I -- 31 .+-. 2 76 .+-. 2 ND
ND ND 5. IV -- 18 57 .+-. 2 ND ND ND 6. IV Ch 17 .+-. 4 54 .+-. 1
30 .+-. 4 60 .+-. 2 39 .+-. 4 7. II Ch 13 .+-. 2 47 .+-. 2 32 .+-.
0 58 .+-. 0 32 .+-. 4 8. 0 -- 5 46 22 55* ND 9. IV Ch, Cy, 4 .+-. 0
44 .+-. 3 17 .+-. 4 77 .+-. 2 46 .+-. 1 Fl 10. I -- 11 44 .+-. 0 26
.+-. 4 56 .+-. 0 ND 11. 0 -- 24 .+-. 4 40 .+-. 0 40 .+-. 3 69 .+-.
0 39 .+-. 2 12. IV Ch, Fl, 0 .+-. 4 38 .+-. 1 43 .+-. 1 68 .+-. 0
ND 2C 13. 0 -- 2 .+-. 0 34 .+-. 0 ND ND 23 .+-. 6 14.** I -- 13
.+-. 0 14 .+-. 0 19 .+-. 3 37 .+-. 3 22 .+-. 0 Apoptosis of CLL
patients' leukemic cells following treatment with 10 uMol/L
rolipram, 40 uMol/L forskolin or 100 uMol/L dbcAMP. Cultures were
performed for three days and the percentage of apoptotic cells
measured by Hoechst 3342 flow cytometry. Drugs: Ch = chlorambucil,
CH = CHOP, Cy = cyclophosphamide, Fl = fludarabine, 2C =
2-chlorodeoxyadenosine. SEMs are shown for samples done in
triplicate; the remainder of the values are the mean of duplicate
cultures. *Rolipram was at 1 uMol/L. **This patient had a
significant population of cells with prolymphocyte morphology.
EXAMPLE 5
[0081] This example describes the effect of PDE4 inhibition on
normal B and T cells. Given that cAMP has been reported to be
cytocidal to specific normal lymphocyte subsets, it was determined
whether rolipram also induced apoptosis in normal circulating human
B and T cell populations. Specifically, one million WMC were
cultured with the indicated concentration of rolipram with or
without the addition of 40 uMol/L forskolin for 72 hours in the
presence of 2 units/mL IL-2. Apoptosis was determined by Hoechst
33342 flow cytometry. It was found that IL-2 cultured WMC (>90%
CD3+T cells by flow cytometry) were resistant to even high doses of
rolipram and forskolin (FIG. 8, upper panel). In contrast, magnetic
bead purified CD19+B cells were sensitive to rolipram, although the
increment in apoptosis observed was superimposed on a high basal
apoptosis rate that has previously been reported in cultured human
B cells. Given that crosslinking of cell surface immunoglobulin on
resting B cells has been reported to reduce basal and
forskolin-induced apoptosis in culture,-we also stimulated
CD19+cells with a polyclonal Fab'2 anti-IgM/IgG reagent 30 minutes
prior to the addition of the phosphodiesterase inhibitor. Prior
stimulation through surface Ig markedly reduced both basal
apoptosis and the sensitivity of the B cells to rolipram (FIG. 8,
middle panel). In contrast, anti-Ig stimulation of CLL cells
derived from two patients failed to protect these cells from
rolipram-induced apoptosis, a result that is consistent with
reported defects in CLL cells in either surface m heavy chain
expression or mutations in the B29 (CD79b) B cell receptor
accessory protein (FIG. 8, bottom panel).
[0082] The alteration in rolipram sensitivity in anti-Ig stimulated
B cells was not due to a change in the ability of this drug to
augment cAMP levels at two hours in these cells, as rolipram raised
cAMP levels equivalently in unstimulated or stimulated CD19+B cells
(FIG. 9). In order to determine whether the rolipram-sensitive cell
populations had a more prolonged elevation of cAMP than the
insensitive cell populations following drug treatment, we measured
cAMP levels 6 or 24 hours after addition of rolipram, times at
which levels of apoptosis were still low even in sensitive
populations. For each of the four cell populations, two or six
hours after drug treatment, cAMP levels were higher for
rolipram/forskolin treated cells than for forskolin only treated
cells (test: one tailed significance level<0.05) (FIG. 9). By 24
hours, there was no longer significant rolipram-induced
augmentation of forskolin-stimulated cAMP accumulation in any of
the four cell populations (FIG. 9). Thus, the degree of cAW
augmentation by rolipram did not predict the sensitivity of cell
populations to induction of apoptosis by this drug at any time
point tested.
EXAMPLE 6
[0083] In this example, the sensitivity of four populations to the
cell permeable cAMP analog, dibutyryl cAMP was examined.
Specifically, 1 million CLL cells, resting or anti-Ig activated
CD19+cells, or IL-2 supplemented WMC were cultured for 72 hours
with 10 uMol/L rolipram, 40 uMol/L forskolin or the dbcAMP
concentration indicated in FIG. 10 (in uMol/L) prior to measurement
of apoptosis by Hoechst 33342 flow cytometry. The SEMs of
triplicate cultures are indicated. As shown in FIG. 10, a strong
correlation was found between rolipram and dbcAMP induced
apoptosis. For CLL cells and resting B cells, the percentage of
apoptotic cells increased significantly relative to control cells
after treatment with rolipram and forskolin or concentrations of
dbcAMP greater than or equal to 30 mMol/L (t test: one tailed
significance level<0.05). For anti-Ig stimulated B cells or IL-2
cultured WMC, comparable treatments did not significantly increase
the percentage of apoptotic cells. Consistent with these results,
in the seven CLL patients studied thus far, there has also been a
correlation between sensitivity to rolipram and sensitivity to
dbcAMP (see Table 1). These data indicate that type 4 PDE is the
relevant target for rolipram in its induction of apoptosis in CLL
cells.
EXAMPLE 7
[0084] It is not intended that the present invention be limited to
only one particular inhibitor. The present invention contemplates
the treatment of patients with chronic lymphocytic leukemia (CLL)
with a variety of inhibitors that specifically inhibit Type 4
cyclic adenosine monophosphate phosphodiesterase. For example, FIG.
11 graphically shows an increase in the percent apoptotic cells
with increasing doses of the inhibitor XX5. One million purified
CLL cells were cultured for three days in media (control), 40 uM
forskolin (F) and/or the indicated concentrations of the PDE4
inhibitor XX5. Cells were then harvested and analyzed for apoptosis
using the Hoechst 33342 FACS assay. SEM of triplicate cultures is
shown.
EXAMPLE 8
[0085] In this example, it is demonstrated that the specific
inhibitors of the present invention augment apoptosis induced by
commonly used drugs (e.g., doxorubicin, chlorambucil and
fludarabine). FIG. 12 graphically shows that rolipram augments
fludarabine-induced apoptosis in CLL cells. One million CLL cells
were incubated for three days in the presence of media (control),
theophylline (50 ug/nmL), forskolin (40 uMol/L), rolipram (10
uMol/L), fludarabine (0.3-3.0 uMol/L) or combinations of these
drugs. The percentage of apoptotic cells was determined by Hoechst
33342 FACS analysis. SEM of triplicate cultures is shown. The right
and left graphs I1 represent data derived from the cells of two
different CLL patients.
[0086] FIG. 13 graphically shows that rolipram augments
chlorambucil-induced apoptosis in CLL cells. CLL cells were
incubated for three days with media (control), chlorambucil
(indicated concentration in uMol/L), rolipram (10 uMol/L) or a
combination of chloramnbucil and rolipram. The percentage of
apoptotic cells was then 20 determined by Hoechst 33342 FACS
analysis. SEM of triplicate samples are shown.
[0087] FIG. 14 graphically shows that rolipram augments
doxorubicin-induced apoptosis in CLL cells. One million CLL cells
were cultured for three days with theophylline (50 ug/mL), rolipram
(10 uMol/L), forskolin (F: 40 uMol/L), doxorubicin (0.03 or 0.1
uMol/L) or combinations of these drugs as indicated. The 25
percentage of apoptotic cells was determined by Hoechst 33342 FACS
analysis. SEM of triplicate cultures is shown. Consequently, the
present invention specifically contemplates the use of the
inhibitors in combination with other drugs, including but not
limited to cytotoxic drugs.
[0088] From the above, it should be clear that, inhibition of type
4 cAMP phosphodiesterase activity is a novel means by which to
trigger apoptosis in chronic lymphocytic leukemia cells in vitro.
Treatment of CLL cells with the PDE4 family-specific inhibitor
rolipram raised cAMP levels and induced apoptosis in a dose and
time-dependent manner, an effect which correlated with apoptosis
induced by dbcAMP. Treatment is possible even where patients
demonstrated clinical resistance to chlorambucil, cyclophosphamide
and/or fludarabine (see Table 1).
[0089] If PDEs are to be a useful pharmacologic target in the
therapy of CLL, drug dosages which trigger apoptosis in leukemic
cells in vivo must have clinically tolerable effects on other
tissues. Despite their widespread tissue distribution, type 4
inhibitors have been used effectively as anti-inflammatory drugs in
animal models of asthma, inhibiting pulmonary eosinophil
accumulation after allergen challenge of sensitized animals.
Rolipram has been studied extensively as an antidepressant in
humans and is well tolerated, although higher dosages are
emetogenic.
[0090] An effort has been made here to determine whether rolipram's
ability to induce apoptosis in the leukemic cells of some CLL
patients is unique to this malignant population or shared by normal
circulating lymphocytes. We observed that IL 2 cultured WMC and
sIg-triggered B cells were largely insensitive to both rolipram and
dbcAMP-induced apoptosis, while treatment of non-stimulated B cells
with rolipram or dbcAMP induced a moderate increase in apoptosis,
albeit superimposed on considerable basal apoptosis.
[0091] BCL-2 and related proteins are likely to regulate
sensitivity to apoptosis in CLL and are also potential targets for
cAMP-mediated signal-transduction. Although less than 10% of CLL
patients have chromosomal translocations involving BCL-2,
hypomethylation and high level BCL-2 transcription is common.
* * * * *