U.S. patent application number 17/029815 was filed with the patent office on 2021-09-02 for method for identification of sensitivity of a patient to telomerase inhibition therapy.
The applicant listed for this patent is Geron Corporation. Invention is credited to Fabio Benedetti, Laurence Elias, Calvin B. Harley, Mark J. Ratain, Jennifer Smith.
Application Number | 20210269861 17/029815 |
Document ID | / |
Family ID | 1000005583400 |
Filed Date | 2021-09-02 |
United States Patent
Application |
20210269861 |
Kind Code |
A1 |
Harley; Calvin B. ; et
al. |
September 2, 2021 |
Method for Identification of Sensitivity of a Patient to Telomerase
Inhibition Therapy
Abstract
The invention provides methods for determining the
susceptibility of cancer patients to developing adverse reactions
if treated with a telomerase inhibitor drug by measurement of
telomere length in appropriate cells of the patient prior to
initiation of the telomerase inhibitor treatment.
Inventors: |
Harley; Calvin B.; (Murphys,
CA) ; Elias; Laurence; (Berkeley, CA) ; Smith;
Jennifer; (Menlo Park, CA) ; Ratain; Mark J.;
(Chicago, IL) ; Benedetti; Fabio; (San Francisco,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Geron Corporation |
Foster City |
CA |
US |
|
|
Family ID: |
1000005583400 |
Appl. No.: |
17/029815 |
Filed: |
September 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16818417 |
Mar 13, 2020 |
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17029815 |
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16227778 |
Dec 20, 2018 |
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16818417 |
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15445428 |
Feb 28, 2017 |
10196677 |
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16227778 |
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14505366 |
Oct 2, 2014 |
9617583 |
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15445428 |
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13124376 |
Jul 12, 2011 |
8877723 |
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PCT/US2009/060526 |
Oct 13, 2009 |
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14505366 |
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61106491 |
Oct 17, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 15/1137 20130101; C12Q 1/6883 20130101; G16B 30/00 20190201;
C12Q 2600/156 20130101; C12Q 1/6813 20130101; C12Q 2600/106
20130101 |
International
Class: |
C12Q 1/6813 20060101
C12Q001/6813; C12Q 1/6883 20060101 C12Q001/6883; G16B 30/00
20060101 G16B030/00; A61K 48/00 20060101 A61K048/00; C12N 15/113
20060101 C12N015/113 |
Claims
1. A method of monitoring a patient for an adverse event related to
telomerase inhibition therapy wherein the method comprises testing
a biological sample from the patient for the length or length
distribution of telomeres.
2.-33. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/124,376, filed Apr. 14, 2011, which is a 371 of PCT Application
Serial No. PCT/US09/60526, filed Oct. 13, 2009, which claims
priority to U.S. Ser. No. 61/106,491, filed Oct. 17, 2008, which
applications are hereby incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of
identification of patients who are sensitive to telomerase
inhibiting agents. In particular the invention provides methods for
detecting patients who should limit or modify the use of telomerase
inhibitors because they are in a risk category for developing
limiting sensitivity such as thrombocytopenia.
BACKGROUND
[0003] Telomeres are genetic elements located at the ends of all
eukaryotic chromosomes which preserve genome stability and cell
viability by preventing aberrant recombination and degradation of
DNA (McClintock, 1941, Genetics vol 26, (2) pp 234-282; Muller,
(1938) The collecting net, vol 13, (8) pp 181-198). In humans, the
telomeric sequence is composed of 10-20 kilobases of TTAGGG repeats
(Harley et al., 1990) Nature vol. 345 pp 458-460; Blackburn, (1991)
Nature vol. 350 pp 569-573; de Lange et al., (1990) Mol. Cell Biol.
Vol 10, (2) pp 518-527). There is increasing evidence that gradual
loss of telomeric repeat sequences (TTAGGG) may be a timing
("clock") mechanism limiting the number of cellular divisions in
normal human cells (Allsopp et al., (1992) Proc. Natl. Acad. Sci.
USA, vol. 89, pp. 10114-10118; Harley et al., (1990) Nature, vol.
345, pp. 458-460; Hastie et al., (1990) Nature, vol. 346, pp.
866-868; Vaziri et al., (1993) Amer. J. Hum. Genet., vol. 52, pp.
661-667). In contrast, immortal cells are capable of maintaining a
stable telomere length by upregulating or reactivating telomerase,
a ribonucleoprotein enzyme that is able to add telomeric repeats to
the ends of chromosomes (Greider and Blackburn, (1985) Cell, vol.
43, pp. 405-413; Greider and Blackburn, (1989) Nature, vol. 337,
pp. 331-337; Morin, (1989) Cell, vol. 59, pp. 521-529).
[0004] Telomerase is an enzyme which adds nucleotides to the
telomeres at the ends of chromosomes, helping to prevent telomeric
shortening to critical lengths. Structurally telomerase is a unique
macromolecular complex which incorporates a strand of RNA in its
active site. This RNA includes telomeric complementary sequence
(3'-AUCCCAAUC-5'), which functions both to anchor telomerase to the
telomere and as a template for adding repeats to the chromosome
end. Telomerase is active in essentially all cancers, but is
generally present at very low or non-detectable levels in normal
adult tissue. Thus, the average telomere length of normal cells
varies among individuals and declines with age (see FIG. 7).
Telomere shortening in normal tissues may also be accelerated by
oxidative, physiologic or immunologic stress and exposure to toxic
agents.
[0005] Cancer cells generally undergo repeated rounds of cell
division and have telomeres that are stable, but shorter than those
in normal cells. Telomerase activation is necessary for most cancer
cells to replicate indefinitely and thereby enables tumor growth
and metastasis. (Kim et al., Science vol. 266 pp 2011-2015; Greider
C W, Blackburn E H. Sci Am February:92-97, 1996; Shay J W and
Wright W E. "Senescence and immortalization: role of telomeres and
telomerase" Carcinogenesis 26:867-74, 2005). Therefore inhibition
of telomerase is considered a promising treatment strategy for a
broad variety of solid tumor types and hematological malignancies
(Harley C B, Nature Rev. Cancer, vol. 8 pp 167-179, 2008).
[0006] GRN163L is a thio-phosphoramidate oligonucleotide with a 5'
palmitoyl "tail". It inhibits the activity of intracellular
telomerase by binding to the template region of the RNA component
of the telomerase holoezyme. (Shea-Herbert et al Oncogene
24:5262-8, 2005) GRN163L has demonstrated telomerase inhibition and
cancer cell growth inhibition effects both in vitro and in vivo
(Dikmen Z G, et al. Cancer Res. 65:7866-73, 2005; Djojosobruto M W
et al. Hepatol 42:1-11, 2005; Hochreiter A E, et al. Clin Cancer
Res 12:3184-92 2006) GRN163L, is currently in clinical trials in
solid tumor and hematological cancers.
[0007] In any cancer treatment, chemotherapy-induced toxicity can
result in reductions in relative dose intensity of the
chemotherapy. Treatment-induced toxicities can include anemia,
neutropenia, leucopenia and thrombocytopenia. Thrombocytopenia is a
chemotherapy-induced toxicity that typically occurs in the first
round of chemotherapy treatment and may become more severe during
repeated rounds of treatment. Drugs that result in toxicities may
have limited applications because of reduced dose intensity (RDI),
dose delays and relative dose reductions. Such dose reductions,
reduced dose intensity or dose delays used as a means of reducing
toxicity may undermine disease control and overall survival,
particularly in patients with potentially curable malignancies. It
is generally recommended that in order to gain maximum benefit-risk
ratio from chemotherapy, the dose prescribed should be
individualized according to the goal of therapy and response.
[0008] Treatment of thrombocytopenia is determined by the etiology
and disease severity. The main concept in treating thrombocytopenia
is to eliminate the underlying problem, whether that means
discontinuing suspected drugs that cause thrombocytopenia, or
treating contributing immunologic or inflammatory factors. Patients
with severe thrombocytopenia may be managed with transfusions of
donor platelets for a period of time. In addition, Oprelvekin
(NEUMEGA.TM., Wyeth) is approved for the prevention of severe
thrombocytopenia following myelosuppressive chemotherapy in adult
patients with nonmyeloid malignancies. Another drug, Romiplostin
(NPLATE.TM., Amgen Inc.) has been approved for the treatment of
chronic idiopathic thrombocytopenic purpura (ITP).
[0009] In this context, a highly predictive test for patients who
are sensitive to developing telomerase inhibition therapy-induced
toxicity would provide significant reduction in the total burden of
toxicity associated with telomerase inhibition therapy, and allow
for the safer use of telomerase inhibition therapy without
inappropriate denial of access to its use.
[0010] The present invention seeks to present a method for
determining the susceptibility of cancer patients to developing
treatment limiting toxicities, such as thrombocytopenia, from
telomerase inhibition therapy.
SUMMARY OF THE INVENTION
[0011] The invention provides methods of determining the
susceptibility of cancer patients to develop toxicities if treated
with a telomerase inhibitor drug. The invention requires the
measurement of telomere lengths in appropriate cells of the patient
prior to initiation of the telomerase inhibitor treatment and the
correlation of the telomere length measurement with susceptibility
to thrombocytopenia. In one embodiment, an algorithm is provided to
assist with the correlation.
[0012] The invention provides a method of monitoring a patient for
an adverse event related to telomerase inhibition therapy wherein
the method comprises testing a biological sample from the patient
for the length or length distribution of telomeres. The method may
further comprise the step of identifying the likelihood that a
mammalian subject will exhibit an adverse reaction to treatment
with a telomerase inhibition therapy.
[0013] The invention includes a method for identifying the
likelihood that a mammalian subject will exhibit an adverse
reaction to telomerase inhibition therapy comprising,
(a) determining the average or median telomere length in a
biological sample comprising cells obtained from the mammalian
subject prior to or at the time of treatment with a telomerase
inhibition therapy and multiplying the average or median telomere
length by a coefficient to arrive at a telomere length component;
(b) multiplying the intended treatment dosage by a coefficient to
arrive at a dosage component; (c) calculating the sum of the
telomere component, the dosage component and a constant; and (d)
determining the expected likelihood of an adverse reaction in the
mammalian subject from treatment with the telomerase inhibition
therapy.
[0014] In one aspect of the method, the mammalian subject is a
human.
[0015] In one aspect of the method, the adverse reaction is
selected from thrombocytopenia, anemia, leucopenia, or
neutropenia.
[0016] The method wherein the adverse reaction is thrombocytopenia
and the sum of the telomere component, the dosage component and the
constant determines the percentage decrease of the mammalian
subject's platelet number from the subject's baseline platelet
number prior to treatment. The method wherein the adverse reaction
is any grade of thrombocytopenia. The method wherein the adverse
reaction is grades 3 or 4 thrombocytopenia.
[0017] In an aspect of the method, the biological sample is blood
cells obtained from the mammalian subject. In one aspect the blood
cells are white blood cells. The method wherein the white blood
cells are selected from granulocytes or lymphocytes. In one aspect
the blood cells are granulocytes. The granulocytes are selected
from neutrophils, basophils or eosinophils. In another aspect the
blood cells are lymphocytes. In another aspect the blood cells are
monocytes or macrophages.
[0018] In an aspect of the method, the mammalian subject is being
treated with the telomerase inhibitor to treat cancer. In an aspect
of the method, the telomerase inhibitor is an oligonucleotide. In
an aspect of the method, the telomerase inhibitor is GRN163L.
[0019] In an aspect of the method, the cancer is selected from the
group consisting of breast cancer, colon cancer, lung cancer,
prostate cancer, testicular cancer, gastric cancer,
gastrointestinal cancer, pharynx cancer, rectal cancer, pancreatic
cancer, cervical cancer, ovarian cancer, liver cancer, bladder
cancer, cancer of the urinary tract, thyroid cancer, renal cancer,
skin cancer, brain cancer, leukemia, myeloma and lymphoma.
[0020] In an aspect of the method, the telomere length component is
a positive component when calculating the percentage change.
[0021] In an aspect of the method, the dosage component is a
negative component when calculating the percentage change.
[0022] In an aspect of the method, the method further comprises the
step of assigning to the subject the likelihood of having an
adverse reaction to telomerase inhibitor treatment.
[0023] In an aspect of the method, the shorter baseline telomere
lengths are associated with an increased risk of an adverse
reaction.
[0024] In an aspect of the method, the increased dosage is
associated with an increased risk of an adverse reaction.
[0025] In an aspect of the method, the baseline telomere length is
determined by FISH analysis, Southern blot analysis, PCR analysis
or STELA analysis.
[0026] In an aspect of the method, the baseline telomere length is
determined by FISH analysis.
[0027] According to another aspect of the present invention there
is provided a method of determining the likelihood that a mammalian
subject will experience thrombocytopenia related to telomerase
inhibition therapy wherein the method comprises,
(a) determining the average or median telomere length in a
biological sample comprising cells obtained from the mammalian
subject prior to or at the time of treatment with a telomerase
inhibition therapy and multiplying the average or median telomere
length by a coefficient to arrive at a telomere length component;
(b) multiplying the intended treatment dosage by a coefficient to
arrive at a dosage component; (c) calculating the sum of the
telomere component and the dosage component and the log of the
subject's baseline platelet number to determine the predicted
platelet nadir during the first weeks of treatment; and (d)
determining the expected likelihood of thrombocytopenia in the
mammalian subject from treatment with the telomerase inhibition
therapy.
[0028] According to another aspect of the present invention there
is provided a method to identify a patient potentially requiring a
telomerase inhibitor dose level below the maximum recommended dose
level, in which the method comprises
(a) determining the average or median telomere length in a
biological sample comprising cells obtained from the mammalian
subject prior to or at the time of treatment with a telomerase
inhibition therapy and multiplying the average or median telomere
length by a coefficient to arrive at a telomere length component;
(b) multiplying the intended treatment dosage by a coefficient to
arrive at a dosage component; (c) calculating the sum of the
telomere component and the dosage component and the log of the
subject's baseline platelet number to determine the predicted
platelet nadir during the first weeks of treatment; (d) determining
the expected likelihood of thrombocytopenia in the mammalian
subject from treatment with the telomerase inhibition therapy; and
(e) administering a reduced dose of the telomerase inhibitor or a
reduced dosage regimen of the telomerase inhibitor.
[0029] According to another aspect of the present invention there
is provided a method to identify a mammalian subject requiring an
ameliorating pharmaceutical administered in conjunction with a
telomerase inhibitor in which the method comprises
(a) determining the average or median telomere length in a
biological sample comprising cells obtained from the mammalian
subject prior to or at the time of treatment with a telomerase
inhibition therapy and multiplying the average or median telomere
length by a coefficient to arrive at a telomere length component;
(b) multiplying the intended treatment dosage by a coefficient to
arrive at a dosage component; and (c) calculating the sum of the
telomere component and the dosage component and the log of the
subject's baseline platelet number to determine the predicted
platelet nadir during the first weeks of treatment; and (d)
determining the expected likelihood of thrombocytopenia in the
mammalian subject from treatment with the telomerase inhibition
therapy (e) administering an appropriate dosage of an ameliorating
pharmaceutical in conjunction with the telomerase inhibitor.
[0030] According to another aspect of the invention there is
provided a method for identifying a mammalian subject on telomerase
inhibition therapy that requires adverse event monitoring
comprising testing a non-cancerous biological sample from the
mammalian subject for telomere length prior to telomerase
inhibition therapy.
[0031] Preferably the method further comprises monitoring the
mammalian subject for an adverse reaction relating to treatment
with the telomerase inhibitor.
[0032] According to another aspect of the invention there is
provided a computer-accessible medium comprising a database that
includes a plurality of records, wherein each record associates (a)
information that identifies a mammalian subject, with (b)
information that indicates whether the subject has shortened
telomeres and wherein each record further associates (a) with (c)
information that identifies the presence or absence of an adverse
event in the subject resulting from administration of a telomerase
inhibitor to the subject.
[0033] According to another aspect of the invention there is
provided a method for administration of GRN163L which comprises
administration of about 1.6 mg/kg to about 20 mg/kg of GRN163L on
day 1 and on approximately day 8 of a 21 day cycle.
[0034] According to another aspect of the invention there is
provided a method for administration of GRN163L which comprises
administration of about 1.6 mg/kg to about 20 mg/kg of GRN163L on
day 1 and on approximately day 15 of a 28 day cycle.
[0035] According to another aspect of the invention there is
provided a method for administration of GRN163L which comprises
administration of about 1.6 mg/kg to about 20 mg/kg of GRN163L two
times in the first week in a 14 day cycle.
[0036] Other aspects and advantages of the invention will become
more fully apparent when the following detailed description of the
invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a graph showing platelet levels over time for
individual patients in cohorts 1-3 of the study. Broken horizontal
lines show the ranges for different levels of thrombocytopenia.
Circles at time points indicate that the patients received a dose
of GRN163L and platelets were collected.
[0038] FIG. 2 is a graph showing platelet levels over time for
individual patients in cohort 4 of the study. Broken horizontal
lines show the ranges for different levels of thrombocytopenia.
Circles at time points indicate that the patients received a dose
of GRN163L and platelets were collected.
[0039] FIG. 3 is a graph showing platelet levels over time for
individual patients in cohort 5 of the study. Broken horizontal
lines show the ranges for different levels of thrombocytopenia.
Circles at time points indicate that the patients received a dose
of GRN163L and platelets were collected.
[0040] FIG. 4 is a graph showing platelet levels over time for
individual patients in cohort 6 of the study. Broken horizontal
lines show the ranges for different levels of thrombocytopenia.
Circles at time points indicate that the patients received a dose
of GRN163L and platelets were collected.
[0041] FIG. 5 is a graph showing the change in platelet levels at 5
week nadir versus the baseline granulocyte telomere length in the
study patients. Circles indicate patients dosed with 0.4, 0.8 and
1.5 mg/kg. Triangles indicate patients dosed with 4.8 mg/kg.
[0042] FIG. 6 is a graph showing the change in baseline granulocyte
telomere length at 5 week nadir versus the number of cytotoxic
regimens experienced by the patients prior to enrollment in this
study.
[0043] FIG. 7 is a graph showing the change in telomere length as a
function of age.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described, including the addition of
other risk factor components which may be relevant to different
patient populations or combinations of telomerase inhibition
therapy with other treatments. It is to be understood that the
invention includes all such variations and modifications. The
invention also includes all of the steps, features, compositions
and compounds referred to or indicated in the specification,
individually or collectively, and any and all combinations or any
two or more of the steps or features.
[0045] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purpose of exemplification only. Functionally equivalent products,
compositions and methods are clearly within the scope of the
invention as described herein.
[0046] All references cited, including patents or patent
applications are hereby incorporated by reference. No admission is
made that any of the references constitute prior art.
A. Definitions
[0047] The terms below have the following meanings unless indicated
otherwise.
[0048] A `mammalian subject", "subject" or "patient" refers to a
mammal. For the purposes of this invention, mammals include humans;
agriculturally important mammals, such as cattle, horses, sheep;
and/or veterinary mammals, such as cats, rabbits, rodents and dogs.
A "patient" means a subject who is receiving medical or veterinary
treatment.
[0049] A "dose" means quantity to be administered at one time, such
as a specified amount of medication. For GRN163L, the adult
starting dose is from about 0.8 mg/kg to about 50 mg/kg; from about
1.6 mg/kg to about 20 mg/kg. The adult dose for GRN163L is from
about 1.6 mg/kg; or about 3.2 mg/kg; or about 4.8 mg/kg; or about
6.2 mg/kg; or about 7.2 mg/kg; or about 9 mg/kg; or about 12 mg/kg;
to about 20 mg/kg. The dose may be administered twice weekly, once
weekly or at other rates of administration. Higher doses may be
required to produce the desired remission in some patients. The
doses may be administered by a 2-24 hour infusion, more preferably
by a 2-4 hour infusion.
[0050] The term "baseline telomere length" or "average or median
baseline telomere length" means the average or median length of the
patient's telomeres in the appropriate cells prior to or at the
same time that the patient receives the first treatment of the
telomerase inhibitor.
[0051] The term "platelet nadir during the first weeks of
treatment" means the number of platelets present in the patient or
subjects blood at the lowest point in the first weeks after
treatment. The first weeks of treatment means in weeks 1-12 of
treatment, preferably 1-8 of treatment, preferable 1-6 of
treatment, preferably 1-4 of treatment.
[0052] "Adverse event" or "adverse reaction" means the development
of an undesirable medical condition or the deterioration of a
pre-existing medical condition following or during exposure to a
pharmaceutical product. An adverse reaction can be selected from
thrombocytopenia, anemia, leucopenia, or neutropenia. Where the
adverse reaction is thrombocytopenia and the sum of the telomere
component and the dosage component and a constant determines the
percentage decrease of the mammalian subject's platelet count from
the subjects platelet count prior to treatment. Where the adverse
reaction is thrombocytopenia, the adverse reaction may be any grade
of thrombocytopenia. The adverse reaction may be grades 3 or 4
thrombocytopenia.
[0053] Thrombocytopenia has been classified into different grades
depending on the number of platelets in the mammalian subject's
blood.
TABLE-US-00001 Grade of Thrombocytopenia Number of
platelets/microliter Grade 1 75-150,000 Grade 2 50-75,000 Grade 3
25-50,000 Grade 4 <25,000
[0054] The term "neutropenia means the presence of abnormally small
numbers of neutrophils in the blood.
[0055] The term "leucopenia" means an abnormally low number of
white blood cells in the blood.
[0056] The term "anemia" means a deficiency in the oxygen-carrying
component of the blood, measured in unit volume concentrations of
hemoglobin, red blood cell volume or red blood cell number.
[0057] The term "number of baseline platelets" means the number of
platelets per microliter of the mammalian subject's blood prior to
treatment with the telomerase inhibitor.
[0058] "Benefit risk ratio" means the relation between the risks
and benefits of a given treatment or procedure. An acceptable risk
relates to the potential for suffering disease or injury that will
be tolerated by an individual in exchange for the benefits of using
a substance or process that will cause such disease or injury.
Acceptability of risk depends on scientific data, social and
economic factors, and on the perceived benefits arising from a
chemical or drug that creates the risk(s) in question.
[0059] A "biological sample" is a blood sample or tissue sample
from the mammalian subject. In one aspect the biological sample is
blood containing white blood cells. In one aspect the white blood
cells are granulocytes. Granulocytes are one or more of
neutrophils, basophils or eosinophils. In another aspect the white
blood cells are one or more of lymphocytes, monocytes or
macrophages. Preferably the cells arc non-cancerous or normal
cells.
[0060] A "telomerase inhibitor" is a compound that directly or
indirectly inhibits or blocks the expression or activity of
telomerase. A telomerase inhibitor is said to inhibit or block
telomerase if the activity of the telomerase in the presence of the
compound is less than that observed in the absence of the compound.
Preferably the telomerase is human telomerase. Preferably the
telomerase inhibitor is an active site inhibitor. More preferably,
the telomerase inhibitor is an hTR template antagonist.
[0061] A "polynucleotide" or "oligonucleotide" refers to a ribose
and/or deoxyribose nucleoside subunit polymer or oligomer having
from about 2 to about 200 contiguous subunits, from about 5 to
about 20 contiguous subunits, from about 10 to about 15 subunits.
The nucleoside subunits can be joined by a variety of intersubunit
linkages, including, but not limited to, phosphodiester,
phosphotriester, methylphosphonate, P3'.fwdarw.N5' phosphoramidate,
N3'.fwdarw.P5' phosphoramidate, N3.fwdarw.P5' thiophosphoramidate,
and phosphorothioate linkages. The term also includes such polymers
or oligomers having modifications, known to one skilled in the art,
to the sugar (e.g., 2' substitutions), the base (see the definition
of "nucleoside" below), and the 3' and 5' termini. In embodiments
where the oligonucleotide moiety includes a plurality of
intersubunit linkages, each linkage may be formed using the same
chemistry, or a mixture of linkage chemistries may be used. When an
oligonucleotide is represented by a sequence of letters, such as
"ATGUCCTG," it will be understood that the nucleotides are in
5'.fwdarw.3' order from left to right. Representation of the base
sequence of the oligonucleotide in this manner does not imply the
use of any particular type of internucleoside subunit or
modifications in the base component or elsewhere in the
oligonucleotide.
[0062] The term "nucleoside" includes the natural nucleosides,
including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in
Komberg and Baker, DNA Replication, 2nd Ed. (Freeman, San
Francisco, 1992), and analogs. "Analogs", in reference to
nucleosides, includes synthetic nucleosides having modified
nucleobase moieties (see definition of "nucleobase" below) and/or
modified sugar moieties, e.g., described generally by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980). Such analogs
include synthetic nucleosides designed to enhance binding
properties, e.g., stability, specificity, or the like, such as
disclosed by Uhlmann and Peyman (Chemical Reviews 90:543-584,
1990). An oligonucleotide containing such nucleosides, and which
typically contains synthetic nuclease-resistant internucleoside
linkages, may itself be referred to as an "analog".
[0063] A "nucleobase" includes (i) native DNA and RNA nucleobases
(uracil, thymine, adenine, guanine, and cytosine), (ii) modified
nucleobases or nucleobase analogs (e.g., 5-methylcytosine,
5-bromouracil, or inosine) and (iii) nucleobase analogs. A
nucleobase analog is a compound whose molecular structure mimics
that of a typical DNA or RNA base.
[0064] An oligonucleotide having "nuclease-resistant linkages"
refers to one whose backbone has subunit linkages that are
substantially resistant to nuclease cleavage, in non-hybridized or
hybridized form, by common extracellular and intracellular
nucleases in the body; that is, the oligonucleotide shows little or
no nuclease cleavage under normal nuclease conditions in the body
to which the oligonucleotide is exposed. The N3'.fwdarw.P5'
phosphoramidate (NP) or N3'.fwdarw.P5' thiophosphoramidate (NPS)
linkages described below are nuclease resistant.
[0065] The term "lipid" is used broadly herein to encompass
substances that are soluble in organic solvents, but sparingly
soluble, if at all, in water. The term lipid includes, but is not
limited to, hydrocarbons, oils, fats (such as fatty acids and
glycerides), sterols, steroids and derivative forms of these
compounds. Preferred lipids are fatty acids and their derivatives,
hydrocarbons and their derivatives, and sterols, such as
cholesterol.
[0066] Fatty acids usually contain even numbers of carbon atoms in
a straight chain (commonly 12-24 carbons) and may be saturated or
unsaturated, and can contain, or be modified to contain, a variety
of substituent groups. For simplicity, the term "fatty acid" also
encompasses fatty acid derivatives, such as fatty or esters.
[0067] The term "hydrocarbon" encompasses compounds that consist
only of hydrogen and carbon, joined by covalent bonds. The term
encompasses open chain (aliphatic) hydrocarbons, including straight
chain and branched hydrocarbons, and saturated as well as mono-and
poly-unsaturated hydrocarbons. The term also encompasses
hydrocarbons containing one or more aromatic rings.
[0068] As used herein, the term "lipid" also includes amphipathic
compounds containing both lipid and hydrophilic moieties.
[0069] The term "tumor" as used herein, refers to all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0070] An "ameliorating pharmaceutical" is a pharmaceutical which
can lessen or remove the risk of developing the adverse reaction.
For example, Oprelvekin (NEUMEGA.TM., Wyeth) is approved for the
prevention of severe thrombocytopenia following myelosuppressive
chemotherapy in adult patients with nonmyeloid malignancies.
Another drug, Romiplostin (NPLATE.TM., Amgen Inc.) has been
approved for the treatment of chronic idiopathic thrombocytopenic
purpura (ITP).
[0071] A "cancer" may be a malignant tumor. At least 80% of all
cancers are carcinomas, and include but are not limited to breast
cancer, both ductal and lobular carcinomas of the breast; colon
cancer, lung cancer, prostate cancer, testicular cancer, gastric
cancer, gastrointestinal cancer, pharynx cancer, rectal cancer,
pancreatic cancer, cervical cancer, ovarian cancer; liver cancer
(including hepatocellular carcinoma), bladder cancer, cancer of the
urinary tract, thyroid cancer, renal cancer, skin cancer (including
basal-cell carcinoma, the most common non-melanoma skin cancer and
squamous cell carcinoma, a common form of skin cancer), and brain
cancer. The cancer cells making up a carcinoma are referred to as
"carcinoma cells." Also included in the term "cancer" are cancers
of the blood cells such as leukemias, lymphomas and myelomas and
cancers of other types of tissue such as sarcomas, mesothelioma,
gliomas, melanoma, neuroblastoma, etc . . . .
[0072] A "prognosis" is used herein to refer to the prediction of
the likelihood of an adverse reaction to treatment with a
telomerase inhibitor. The term "prediction" is used herein to refer
to the likelihood that a mammalian subject or patient will respond
either favorably or unfavorably to a drug or a set of drugs, and
also the extent of those responses.
[0073] In term "adjuvant therapy" is generally used to refer to
treatment that is given in addition to a primary (initial)
treatment. In cancer treatment, the term "adjuvant therapy" is used
to refer to chemotherapy, hormonal therapy and/or radiation therapy
following surgical removal of the tumor, with the primary goal of
reducing the risk of cancer recurrence.
B. Detailed Description
[0074] The practice of the present invention will employ, unless
otherwise indicated convention techniques of molecular biology,
microbiology, cell biology and biochemistry which are within the
skill of the art. Such techniques are explained fully in the
literature, such as "Molecular Cloning: A Laboratory Manual,
2.sup.nd edition (Sambrook et al., 1989); Oligonucleotide
Synthesis: A practical Approach (M. J. Gait ed, 1984); "Current
Protocols in Molecular Biology" (F. M. Ausubel et al., eds. 1987)
and "PCR: The Polymerase Chain reaction" (Mullis et al., eds.
1994).
[0075] The present invention provides an algorithm for determining
the likelihood of an adverse reaction to treatment with a
telomerase inhibitor. The method is based on the identification of
(1) the average or median telomere length in a cells from a patient
and the dosage of telomerase inhibitor received can serve to
determine the likelihood of the patient suffering an adverse
reaction to therapy with the telomerase inhibitor, (2) certain
weights assigned to the average or median telomere length and the
dosage reflect their value in predicting the response to therapy
and used in a formula; and (3) determination of threshold values
used to divide patients into groups with varying degrees of risk to
developing an adverse reaction, such as low, medium and high risk
groups or groups in which the likelihood of an adverse reaction to
telomerase inhibitors is low, medium or high. The algorithm yields
a numerical score which can be used to make treatment decisions
concerning the therapy of cancer patients.
1. Techniques for Determination of Telomere Length.
[0076] Several methods are available for measuring the length of
telomere repeats in cells. Generally the cells whose telomeres are
to be measured are isolated from the biological sample of the
patient. The DNA is isolated from the cells by methods known in the
art, such as for example, proteinase K, RNAse A and
phenol/chloroform protocols (Sambrook et al. Molecular Cloning: a
Laboratory Manual 2nd ed. (1989) or use of commercially available
DNA purification kits.
A. Southern Blot Analysis
[0077] One method for the analysis of telomere lengths is to
measure the length of the terminal restriction fragment (TRF) by
Southern Blot analysis. In this method cellular DNA is digested
with the restriction enzymes such as Hinf1 and RsaI and run in
agarose gels for transfer to Nytran filters. The filters are
hybridized with a telomere specific probe such as (TTAGGG).sub.3.
Autoradiographs are generated without an intensifying screen using
the linear response range of the film and scanned with a
densitometer. Output is digitized. Mean telomere length is defined
as .SIGMA.(OD.sub.i)/.SIGMA.(OD.sub.i/L.sub.i) where OD.sub.i is
the densitometer output (arbitrary units) and Li is the length of
the DNA at position i. Sums are calculated over the range of 3-17
KB. This calculation assumes that DNA transfers at equal efficiency
from all points in the gel and that the number of target sequences
(telomere repeats) per DNA fragment is proportional to DNA length.
The signal from the gels may be normalized to the signal from other
Southern blots using a control probe in order to estimate the total
amount of telomeric DNA as well as its length. (Harley et al.,
Nature 345:458-460 (1990), Englehardt et la., Leukemia 12:13-24
(1998)). This method also gives the size distribution of telomere
lengths in the cell population from which the DNA was isolated.
Since short telomeres are particularly susceptible to telomere
dysfunction, modifications to the current invention could include
calculations based on the mean or median telomere length of the
short telomeres (e.g. for the shortest quartile of telomeres).
B. Polymerase Chain Reaction
[0078] Polymerase Chain Reaction (PCR) methods have been developed
to measure average and chromosome specific telomere lengths. The
first method provides a measure of telomeric DNA relative to
genomic DNA (typically a single-copy gene) as a single ration value
of a sample of genomic DNA (Cawthon R M, Nuc. Acids Res. Vol 30, pp
e47).
[0079] In the Single Telomere Length Analysis (STELA) the telomere
lengths from individual chromosomes are determined (Baird et al.,
Nature Genetics 33 203-207 (2003)). In this process, the DNA is
digested with a restriction enzyme such as EcoRI and quantitated by
Hoechst fluorometry. A linker or "telorette" comprising several
bases complementary to the TTAGGG single-stranded region of the
chromosome, preceded at the 5' end by a 20 nucleotides of unique
sequence DNA. This telorette is annealed to the TTAGGG overhang at
the end of the telomere and ligated to the 5'end of the
complementary C-rich strand of the chromosome. This effectively
tags the end of the telomere with a telorette tail that has a
unique sequence capable of binding one of the PCR primers.
Polymerase chain reaction (PCR) is then performed using a primer
(`teltail") that is complementary to the telorette tail, together
with a primer that is corresponds to the chromosome region adjacent
to the telomere. The primer corresponding to the chromosome region
adjacent to the telomere can also be made chromosome specific by
exploiting chromosomal polymorphisms. After the PCR. The DNA
fragments are resolved with agarose gel electrophoresis and
detected y Southern blot hybridization with a random primed
telomere adjacent probe The size of the hybridized fragments can be
determine from size standards on the gel and used to calculate the
length of individual telomeres. This method also gives the size
distribution of the telomeres from the specific chromosome targeted
in the cell population from which the DNA was isolated. Since PCR
biases amplification of short DNA fragments, STELA is particularly
useful for analysis of the shortest telomeres in a cell. This has
application to the current invention as described above.
C. Flow Cytometry and FISH Analysis
[0080] The average or median length of telomere repeats in calls
can also be determined using fluorescent in situ hybridization
(FISH) with labeled peptide nucleic acid (PNA) probes specific for
telomere repeats in combination with fluorescence measurements by
flow cytometry (flow FISH). (See Baerlocher et al., Nature
Protocols vol. 1 2365-2376 (2006) incorporated by reference in its
entirety). The advantage of Flow FISH is in providing
multi-parameter information on the length of telomere repeats in
thousands of individual cells. Automated multicolor flow FISH is
one of the fastest and most sensitive methods available to measure
the average or median telomere length in granulocytes, naive T
cells, memory T cells, B cells and natural killer (NK) cells in
human blood. (Baerlocher and Lansdorp, Methods in Cell boil. 75,
719-750 (2004).
[0081] In flow FISH whole blood is centrifuged, red cells lysed and
the red cell lysate separated from the cell pellet consisting of
granulocytes, monocytes, lymphocytes, platelets and any remaining
red cells. The white blood cell pellet is resuspended in a
hybridization buffer and counted. The nucleated human blood cells
are mixed with bovine thymocytes, included as an internal control
as these cells are easily obtained and because the telomere length
in bovine thymocytes is about 2-3 times longer than is typically
measured in human cells. Accordingly these control cells are easily
distinguished from the human test cells and provide a reference
point for telomere fluorescence measurements. The mixture of human
cells and bovine thymocytes is hybridized with the Cy5 or
flourescein labeled PNA (peptide nucleic acid) probe which is
complementary to the telomere repeat sequence. A second mixture of
the cells is not hybridized with the probe. This latter is required
to measure the level of autofluorescence in the cells of interest
and to enable telomere length to be calculated from specific PNA
hybridization. The fluorescein or Cy5 PNA probe is commercially
available. After hybridization, the cells are pelleted and the cell
pellet washed. The cells may be counterstained with non-saturating
concentrations of a DNA dye and various antibodies. The cell
samples are run on a flow cytometer.
[0082] The first step in the subsequent analysis is to identify
cells using forward light and side scatter in a bivariate dot plot.
Three cell populations can be observed. The bovine thymocytes can
be distinguished from the human lymphocytes which in turn can be
distinguished from the granulocytes. By combining the fluorescence
in the contour plots fluorescence histograms of the different cell
populations can be obtained, which are used for subsequence
calculations of telomere length. Antibodies specific for CD45RA and
CD20 cells can be used to perform telomere length analysis of
specific populations within the lymphocyte population. The average
telomere length can be determined by subtracting the fluorescence
of the unstained white blood cell populations from the level of
fluorescence of the PNA stained cells. This method collects an
average telomere signal from each individual cell, thus the
telomere size distribution of the overall population can be
obtained, and one could analyze the subset of cells with short
telomeres. This has application to the current invention as
described above.
2. Algorithm to Predict the Platelet Levels, or Changes after
Telomerase Inhibition Therapy and to Generate Likelihood of Adverse
Reaction
[0083] An aspect of the present invention is to use the measured
average length of the telomeres in the cells from the patient to
provide information regarding the likelihood of an adverse reaction
to a telomerase inhibitor prior to administration of a telomerase
inhibitor.
[0084] In the next step the measured average telomere length is
multiplied by a coefficient reflecting its relative contribution to
the risk of the adverse reaction to treatment with a telomerase
inhibitor to determine the telomere length component.
[0085] The next step is to take the intended dosage of the
telomerase inhibitor and multiply the dosage by a coefficient
reflecting its relative contribution to the risk of the adverse
reaction to treatment with a telomerase inhibitor to determine the
dosage component.
[0086] The telomere length component and the dosage component are
added with an intercept factor to determine the likelihood of the
adverse reaction.
[0087] For example, the equation to describe the predicted number
of platelets at platelet nadir in a patient after 4 complete weeks
of treatment is as follows:
Predicted # of platelets=baseline platelets number-(baseline
platelet number x% change in platelets/100).
% change in platelet #=(-73.8)-6.6.times.inhibitor dose
(mg/kg)+11.2.times.average telomere length (kbp)
[0088] The equation to describe the predicted number of platelets
at platelet nadir in a patient during first 4 weeks of treatment is
as follows:
Predicted # of platelets=e.sup.[(-0.38)-0.13.times.inhibitor dose
(mg/kg)+0.25.times.average telomere length (kbp)+0.80.times.log of
baseline platelet number]
[0089] Prediction intervals may be calculated for example, to
predict likely percent change in platelet levels or the platelet
nadir for patients or subjects with a particular set of baseline
and treatment values, for example, telomere length, baseline
platelets and dose level. The regression equation provides the
expected value for a future individual with specified covariates
(J. Neter et al. Applied linear statistical models: regression,
analysis of variance, and experimental designs, 3.sup.rd edition
pp. 81-83 (1990)). However, due to sampling distribution error as
well as interindividual variability, a patient may have platelet
levels that fall above or below that predicted value. A series of
prediction intervals may be created with decreasing coverage. For
example, a 99% prediction interval with upper and lower bounds
P.sub.U99 and P.sub.L99 may be created and would, on average,
contain 99% of the patients' observed platelet levels. A 90%
prediction interval with upper and lower bounds P.sub.U99
(<P.sub.U99) and P.sub.L90 (>P.sub.L99) may be created and
would, on average, contain 90% of future observed platelet levels.
This allows one to determine the likelihood that the patient would
develop a grade 3 or 4 thrombocytopenia.
[0090] The likelihood of risk of an adverse reaction, as determined
by the algorithm of the present invention, provides valuable tools
for the practicing physician to make critical treatment decisions.
Thus if the risk of a particular patient is low, the physician
might decide that following surgical removal of the cancer the
patient can be treated aggressively with high doses and high
frequency of administration of the telomerase inhibitor. If, on the
other hand the level of risk is determined to be high, this
information can be used to decide the level of dose of the
telomerase inhibitor to administer, the regimen of dosing to use,
including the use of weeks with no dose administered, so called
"resting" weeks. The physician may decide to monitor the patient
more closely for adverse reactions, such as thrombocytopenia. The
physician may decide to administer an ameliorating pharmaceutical
concurrently with the telomerase inhibitor. If the risk of the
patient for an adverse reaction is high, other treatment modalities
may be used to combat cancer in that particular patient. Other
treatment modalities for a particular cancer include, for example,
other chemotherapies such as anthracycline and/or taxane based
treatments, HER inhibitors, EGFR inhibitors and/or other treatment
options, such as radiation therapy alone, before or after
chemotherapy.
3. Telomerase Inhibitors and Treatment of Cancer with a Telomerase
Inhibitor
[0091] Telomerase is a ribonucleoprotein that catalyzes the
addition of telomeric repeat sequences (having the sequence
5'-TTAGGG-3' in humans) to chromosome ends. A variety of cancer
cells have been shown to be telomerase-positive, including cells
from cancer of the skin, connective tissue, adipose, breast, lung,
stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon,
prostate, central nervous system (CNS), retina and hematologic
tumors (such as myeloma, leukemia and lymphoma). Targeting of
telomerase can be effective in providing treatments that
discriminate between malignant and normal cells to a high degree,
avoiding many of the deleterious side effects that can accompany
chemotherapeutic regimens which target dividing cells
indiscriminately.
[0092] Inhibitors of telomerase identified to date include
oligonucleotides, preferably oligonucleotides having nuclease
resistant linkages, as well as small molecule compounds.
A. Small Molecule Compounds
[0093] Small molecule inhibitors of telomerase include, for
example, BRACO19
((9-(4-(N,N-dimethylamino)phenylamino)-3,6-bis(3-pyrrolodino
propionamido)acridine (see Mol. Pharmacol. 61(5):1154-62, 2002);
DODC (diethyloxadicarbocyanine), and telomestatin. These compounds
may act as G-quad stabilizers, which promote the formation of an
inactive G-quad configuration in the RNA component of telomerase.
Other small molecule inhibitors of telomerase include BIBR1532
(2-[(E)-3-naphthen-2-yl but-2-enoylamino]benzoic acid) (see Ward
& Autexier, Mol. Pharmacol. 68:779-786, 2005; also J. Biol.
Chem. 277(18):15566-72, 2002); AZT and other nucleoside analogs,
such as ddG and ara-G (see, for example, U.S. Pat. Nos. 5,695,932
and 6,368,789), and certain thiopyridine, benzo[b]thiophene, and
pyrido[b]thiophene derivatives, described by Gaeta et al. in U.S.
Pat. Nos. 5,767,278, 5,770,613, 5,863,936, 5,656,638 and 5,760,062.
One example is
3-chlorobenzo[b]thiophene-2-carboxy-2'-[(2,5-dichlorophenyl
amino)thia]hydrazine, described in U.S. Pat. No. 5,760,062.
B. Oligonucleotide Based Telomerase Inhibitors: Sequence and
Composition
[0094] The genes encoding both the protein and RNA components of
human telomerase have been cloned and sequenced (see U.S. Pat. Nos.
6,261,836 and 5,583,016, respectively, both of which are
incorporated herein by reference). Oligonucleotides can be targeted
against the mRNA encoding the telomerase protein component (the
human form of which is known as human telomerase reverse
transcriptase, or hTERT) or the RNA component of the telomerase
holoenzyme (the human form of which is known as human telomerase
RNA, or hTR). U.S. Pat. Nos. 5,583,016; 5,776,679; 5,837,857 which
are incorporated herein by reference.
[0095] The template sequence of the RNA component of telomerase is
located within the region defined by nucleotides 46-56
(5'-CUAACCCUAAC-3')(SEQ ID NO:1), which is complementary to a
telomeric sequence composed of about one-and-two-thirds telomeric
repeat units. The template region functions to specify the sequence
of the telomeric repeats that telomerase adds to the chromosome
ends and is essential to the activity of the telomerase enzyme (see
e.g. Chen et al., Cell 100: 503-514, 2000; Kim et al., Proc. Natl.
Acad. Sci. USA 98 (14):7982-7987, 2001). The design of antisense,
ribozyme or small interfering RNA (siRNA) agents to inhibit or
cause the destruction of mRNAs is well known (see, for example,
Lebedeva, I, et al. Annual Review of Pharmacology and Toxicology,
Vol. 41: 403-419, April 2001; Macejak, D, et al., Journal of
Virology, Vol. 73 (9): 7745-7751, September 1999, and Zeng, Y. et
al., PNAS Vol. 100 (17) p. 9779-9784, Aug. 19, 2003) and such
agents may be designed to target the hTERT mRNA and thereby inhibit
production of hTERT protein in a target cell, such as a cancer cell
(see, for example, U.S. Pat. Nos. 6,444,650 and 6,331,399).
[0096] Oligonucleotides targeting hTR (that is, the RNA component
of the enzyme) act as inhibitors of telomerase enzyme activity by
blocking or otherwise interfering with the interaction of hTR with
the hTERT protein, which interaction is necessary for telomerase
function. See, for example, Villeponteau et al., U.S. Pat. No.
6,548,298.
[0097] A preferred target region of hTR is the template region,
spanning nucleotides 30-67 of the RNA component of human
telomerase. Oligonucleotides targeting this region are referred to
herein as "hTR template inhibitors" (see e.g. Herbert et al.,
Oncogene 21(4):638-42 (2002).) Preferably, such an oligonucleotide
includes a sequence which is complementary or near-complementary to
some portion of the 11-nucleotide region having sequence
5'-CUAACCCUAAC-3' (SEQ ID NO:1), spanning nucleotides 46-56 of the
RNA component of human telomerase (hTR).
[0098] Another preferred target region is the region spanning
nucleotides 137-179 of human telomerase (hTR) (see Pruzan et al.,
Nucl. Acids Research, 30:559-568, 2002). Within this region, the
sequence spanning 141-153 is a preferred target. PCT publication WO
98/28442 describes the use of oligonucleotides of at least 7
nucleotides in length to inhibit telomerase, where the
oligonucleotides are designed to be complementary to accessible
portions of the hTR sequence outside of the template region,
including nucleotides 137-196, 290-319, and 350-380 of hTR.
[0099] The region of the therapeutic oligonucleotide that is
targeted to the hTR sequence is preferably exactly complementary to
the corresponding hTR sequence. While mismatches may be tolerated
in certain instances, they are expected to decrease the specificity
and activity of the resultant oligonucleotide conjugate. In
particular embodiments, the base sequence of the oligonucleotide is
thus selected to include a sequence of at least 5 nucleotides
exactly complementary to the hTR target, and enhanced telomerase
inhibition may be obtained if increasing lengths of complementary
sequence are employed, such as at least 8, at least 10, at least
12, at least 13 or at least 15 nucleotides exactly complementary to
the hTR target. In other embodiments, the sequence of the
oligonucleotide includes a sequence of from at least 5 to 20, from
at least 8 to 20, from at least 10 to 20 or from at least 10 to 15
nucleotides exactly complementary to the hTR target sequence.
[0100] Optimal telomerase inhibitory activity may be obtained when
the full length of the oligonucleotide is selected to be
complementary to the hTR target sequence. However, it is not
necessary that the full length of the oligonucleotide is exactly
complementary to the target sequence, and the oligonucleotide
sequence may include regions that are not complementary to the
target sequence. Such regions may be added, for example, to confer
other properties on the compound, such as sequences that facilitate
purification. Alternatively, an oligonucleotide may include
multiple repeats of a sequence complementary to an hTR target
sequence.
[0101] The method includes administering to the subject an
oligonucleotide telomerase inhibitor of the type composed of an
oligonucleotide having nuclease-resistant intersubunit linkages and
an oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR. Preferably, the amount
of the telomerase inhibitor is effective to inhibit the
proliferation of cancer cells in the subject when the telomerase
inhibitor is administered alone.
[0102] The oligonucleotide may be 10-20 bases in length.
Preferably, the oligonucleotide is 13-20 bases in length and
includes the sequence (5'-TAGGGTTAGACAA-3') (SEQ ID NO:2). An
exemplary telomerase inhibitor is the compound identified as
GRN163L, or an analog thereof. This compound has (i) N3'.fwdarw.P5'
thiophosphoramidate internucleoside linkages; (ii) the sequence
5'-TAGGGTTAGACAA-3'(SEQ ID NO:2); and (iii) a palmitoyl (C16)
moiety linked to the 5' end of the oligonucleotide through a
glycerol or aminoglycerol linker.
[0103] The internucleoside linkages in the oligonucleotide may
include any of the available oligonucleotide chemistries, e.g.
phosphodiester, phosphotriester, methylphosphonate, P3'.fwdarw.N5'
phosphoramidate, N3'.fwdarw.P5' phosphoramidate, N3'.fwdarw.P5'
thiophosphoramidate, and phosphorothioate. Typically, but not
necessarily, all of the internucleoside linkages within the
oligonucleotide will be of the same type, although the
oligonucleotide component may be synthesized using a mixture of
different linkages.
[0104] In preferred embodiments, the oligonucleotide has at least
one N3'.fwdarw.P5' phosphoramidate (NP) or N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkage, which linkage may be represented
by the structure: 3'-(-NH--P(.dbd.O)(--XR)--O-)-5', wherein X is O
or S and R is selected from the group consisting of hydrogen,
alkyl, and aryl; and pharmaceutically acceptable salts thereof,
when XR is OH or SH. More preferably, the oligonucleotide includes
all NP or, most preferably, all NPS linkages.
[0105] A particularly preferred sequence for an hTR template
inhibitor oligonucleotide is the sequence complementary to
nucleotides 42-54 of the hTR. The oligonucleotide having this
sequence (TAGGGTTAGACAA) (SEQ ID NO:2) and N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkages is designated herein as GRN163.
See, for example, Asai et al., Cancer Research 63:3931-3939 (2003);
Gryaznov et al., Nucleosides Nucleotides Nucleic Acids
22(5-8):577-81 (2003).
[0106] These compounds may be prepared as described, for example,
in McCurdy et al., Tetrahedron Letters 38:207-210 (1997) or
Pongracz & Gryaznov, Tetrahedron Letters 49:7661-7664 (1999).
The starting 3'-amino nucleoside monomers may be prepared as
described in Nelson et al., J. Org. Chem. 62:7278-7287 (1997) or by
the methods described in Gryaznov et al., US Appn. Pubn. No.
2006/0009636.
[0107] A variety of synthetic approaches can be used to conjugate a
lipid moiety L to the oligonucleotide, depending on the nature of
the linkage selected; see, for example, Mishra et al., Biochim. et
Biophys. Acta 1264:229-237 (1995), Shea et al., Nucleic Acids Res.
18:3777-3783 (1995), or Rump et al., Bioconj. Chem. 9:341-349
(1995). Typically, conjugation is achieved through the use of
suitable functional groups at an oligonucleotide terminus. For
example, the 3'-amino group present at the 3'-terminus of the NP
and NPS oligonucleotides can be reacted with carboxylic acids, acid
chlorides, anhydrides and active esters, using suitable coupling
catalysts, to form an amide linkage. Thiol groups are also suitable
as functional groups (see Kupihar et al., Bioorg. Med. Chem.
9:1241-1247 (2001)). Various amino- and thiol-functionalized
modifiers of different chain lengths are commercially available for
oligonucleotide synthesis.
[0108] Specific approaches for attaching lipid groups to a terminus
of an NP or NPS oligonucleotide include those described in US Appn.
Pubn. No. 2005/0113325, which is incorporated herein by reference.
In addition to the amide linkages noted above, for example, lipids
may also be attached to the oligonucleotide chain using a
phosphoramidite derivative of the lipid, to produce a
phosphoramidate or thiophosphoramidate linkage connecting the lipid
and the oligonucleotide. The free 3'-amino group of the fully
protected support-bound oligonucleotide may also be reacted with a
suitable lipid aldehyde, followed by reduction with sodium
cyanoborohydride, which produces an amine linkage.
[0109] The oligonucleotide GRN163 administered alone has shown
inhibitory activity in vitro in cell culture, including epidermoid
carcinoma, breast epithelium, renal carcinoma, renal
adenocarcinoma, pancreatic, brain, colon, prostate, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells.
[0110] The oligonucleotide GRN163 has also been tested and shown to
be therapeutically effective in a variety of animal tumor models,
including ovarian and lung, both small cell and non-small cell.
C. Lipid-Oligonucleotide Conjugates
[0111] Preferably, the oligonucleotide-based enzyme inhibitor
includes at least one covalently linked lipid group (see US
Publication. No. 2005/0113325, which is incorporated herein by
reference). This modification provides superior cellular uptake
properties, such that an equivalent biological effect may be
obtained using smaller amounts of the conjugated oligonucleotide
compared to the unmodified form. When applied to the human
therapeutic setting, this may translate to reduced toxicity risks,
and cost savings.
[0112] The lipid group L is typically an aliphatic hydrocarbon or
fatty acid, including derivatives of hydrocarbons and fatty acids,
with examples being saturated straight chain compounds having 14-20
carbons, such as myristic (tetradecanoic) acid, palmitic
(hexadecanoic) acid, and stearic (octadeacanoic) acid, and their
corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane
and octadecane. Examples of other suitable lipid groups that may be
employed are sterols, such as cholesterol, and substituted fatty
acids and hydrocarbons, particularly polyfluorinated forms of these
groups. The scope of the lipid group L includes derivatives such as
amine, amide, ester and carbamate derivatives. The type of
derivative is often determined by the mode of linkage to the
oligonucleotide, as exemplified below.
[0113] In one exemplary structure, the lipid moiety is palmitoyl
amide (derived from palmitic acid), conjugated through an
aminoglycerol linker to the 5' thiophosphate group of an NPS-linked
oligonucleotide. The NPS oligonucleotide having the sequence shown
for GRN163 and conjugated in this manner (as shown below) is
designated GRN163L herein. In a second exemplary structure, the
lipid, as a palmitoyl amide, is conjugated through the terminal 3'
amino group of an NPS oligonucleotide.
##STR00001##
[0114] For attachment of a lipid to the 5' terminus, as also
described in US Appn. Pubn. No. 2005/0113325, the oligonucleotide
can be synthesized using a modified, lipid-containing solid
support. Reaction of 3-amino-1,2-propanediol with a fatty acyl
chloride (RC(O)CI), followed by dimethoxytritylation of the primary
alcohol and succinylation of the secondary alcohol, provides an
intermediate which is then coupled, via the free succinyl carboxyl
group, to the solid support. An example of a modified support is
shown below, where S-- represents a long chain alkyl amine CPG
support, and R represents a lipid.
##STR00002##
[0115] This procedure is followed by synthesis of the
oligonucleotide in the 5' to 3' direction, as described, for
example, in Pongracz & Gryaznov (1999), starting with
de-protection and phosphitylation of the -ODMT group. This is
effective to produce, for example, the following structure, after
cleavage from the solid support:
##STR00003##
[0116] The structure above, when --R is --(CH.sub.2).sub.14CH.sub.3
(palmitoyl), is designated herein as GRN163L.
IV. Administration
[0117] The cancer should also be one that is responsive to
cancer-cell inhibition by telomerase inhibition. As noted above,
oligonucleotide telomerase inhibitors, as exemplified by GRN163 and
GRN163L, have shown inhibitory activity in vitro against human
kidney, lung, pancreatic, brain, colon, prostate, breast, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells, and in vivo, via local and systemic delivery, against human
brain, prostate, lymphoma, myeloma, cervical, lung, and liver
cancer cells. Other preferred targets include small cell lung,
esophageal, head and neck, and stomach cancers.
[0118] The dose administered and the dosing schedule will follow,
for example, known or recommended doses for the inhibitor employed,
as indicated, for example, in the drug product insert or published
clinical or animal-model data. For GRN163L, the adult starting dose
is from about 0.8 mg/kg to about 50 mg/kg; from about 1.6 mg/kg to
about 20 mg/kg. The adult dose for GRN163L is from about 1.6 mg/kg;
or about 3.2 mg/kg; or about 4.8 mg/kg; or about 6.2 mg/kg; or
about 7.2 mg/kg; or about 9 mg/kg; or about 12 mg/kg; to about 20
mg/kg The dose may be administered twice weekly, once weekly or at
other rates of administration. Higher doses may be required to
produce the desired remission in some patients.
[0119] GRN163L may be administered to a patient at a dose of at
least about 4.8 mg/kg of GRN163L on day 1 and on approximately day
8 of a 21 day cycle. Alternatively it may be administered at a dose
of at least about 4.8 mg/kg of GRN163L on day 1 and on
approximately day 15 of a 28 day cycle. Alternatively, GRN163L may
be administered to a patient at a dose of at least about 1.6 mg/kg
of GRN163L two times in the first week of a 14 day cycle.
[0120] The therapeutic protocol for administering the telomerase
inhibitor in the therapy will depend on various factors including,
but not limited to, the type of cancer, the age and general health
of the patient, the aggressiveness of disease progression, the
telomere length and telomerase activity of the diseased cells to be
treated, and the ability of the patient to tolerate the agents that
comprise the combination, which may depend upon telomerase activity
and telomere length in various normal cells, particularly normal
cells in highly proliferative tissues, particularly, but not
limited to, the bone marrow.
[0121] In general, treatment of all cancer and hematological
malignancy types is contemplated. In selected embodiments, the
target disease comprises a solid tumor; in other embodiments, the
target disease comprises a hematological malignancy. An exemplary
course of treatment involves multiple doses. Sequence of
combination treatments will be determined by clinical compliance
criteria and/or preclinical or clinical data supporting dose
optimization strategies to augment efficacy or reduce toxicity of
the combination treatment. The time between dosages may be for a
period from about 1-6 hours, to about 6-12 hours, to about 12-24
hours, to about 1-2 days, to about 1-2 wk or longer following the
initiation of treatment. During a course of treatment, the need to
complete the planned dosings may be re-evaluated.
[0122] The compounds may be administered by direct injection of a
tumor or its vasculature. Alternatively, the tumor may be infused
or perfused with the therapeutic compounds using any suitable
delivery vehicle. The compounds may be administered locally to an
affected organ. Systemic administration may also be performed.
Continuous administration may be applied where appropriate; for
example, where a tumor is excised and the tumor bed is treated to
eliminate residual disease. Delivery via syringe or catheterization
is preferred. Such continuous perfusion may take place for a period
from about 1-6 hours, to about 6-12 hours, to about 12-24 hours, to
about 1-2 days, to about 1-2 weeks or longer following the
initiation of treatment. Generally, the dose of the therapeutic
composition via continuous perfusion will be equivalent to that
given by a single or multiple injections, adjusted over a period of
time during which the perfusion occurs.
[0123] The therapeutic agents are administered to a subject, such
as a human patient, in a formulation and in an amount effective to
achieve a clinically desirable result. For the treatment of cancer,
desirable results include reduction in tumor mass (as determined by
palpation or imaging; e.g., by radiography, radionucleotide scan,
CAT scan, or MRI), reduction in the rate of tumor growth, reduction
in the rate of metastasis formation (as determined e.g., by
histochemical analysis of biopsy specimens), reduction in
biochemical markers (including general markers such as ESR, and
tumor specific markers such as serum PSA), and improvement in
quality of life (as determined by clinical assessment, e.g.,
Karnofsky score), increased time to progression, disease-free
survival and overall survival.
[0124] The amount of each agent per dose and the number of doses
required to achieve such effects will vary depending on many
factors including the disease indication, characteristics of the
patient being treated and the mode of administration. Typically,
the formulation and route of administration will provide a local
concentration at the disease site of between 1 nM and 100 .mu.M of
each agent. The physician will be able to vary the amount of the
compounds, the carrier, the dosing frequency, and the like, taking
into consideration such factors as the particular neoplastic
disease state and its severity; the overall condition of the
patient; the patient's age, sex, and weight; the mode of
administration; the suitability of concurrently administering
systemic anti-toxicity agents; monitoring of the patient's vital
organ functions; and other factors typically monitored during
cancer chemotherapy. In general, the compounds are administered at
a concentration that affords effective results without causing
excessive harmful or deleterious side effects.
[0125] Modes of administration and formulation may be dependent on
the drug and its approved mode of administration. When the
telomerase inhibitor is GRN163L, formulation in 0.9% sodium
chloride (normal saline) and administration by i.v. is a preferred
route, preferably via infusion over 1 to 24 hours, more preferably
over 2 to 8 hours, e.g. a 6 hr infusion. While the lipid-conjugated
oligonucleotides described herein, such as GRN163L, have superior
characteristics for cellular and tissue penetration, these and
other compounds may be formulated to provide further benefit in
this area. Other useful adjuvants include substrates for
transendothelial migration, such as glucose uptake systems for
facilitated egress from the vascular space to the tumor
microenvironment.
[0126] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
Example 1: Study of Various Parameters in Patients Prescribed
Telomerase Inhibitors
[0127] A study was designed and conducted to determine the
likelihood of development of thrombocytopenia in a population of
solid tumor cancer patients being treated with the telomerase
inhibitor GRN163L. GRN163L is a 13-mer oligonucleotide inhibitor of
telomerase activity. The study utilized archived blood cells as a
source of cellular telomere length before the study and matched
archived patient records.
Study Design
[0128] The patents accepted were adults with refractory, advanced
solid tumors and treated with GRN163L in a phase T clinical trail.
The GRN163L was given by continuous weekly i.v. dosing. In addition
interim data are presented on a cohort 6 treated with an
alternative dosing regimen of designed to reduce the potential for
thrombocytopenia. This was a multicenter Phase I clinical trial
with sequential cohorts of dose escalation. Patients were enrolled
in successive cohorts at 0.4 to 4.8 mg/kg. Cohorts 1-5 received 2
hr intravenous infusions of GRN163L weekly. Cohort 6 received an
intermittent dosing schedule of weekly iv infusions of GRN163L (4.8
mg/kg) X2, followed by a 13 day rest. Completion of 1 cycle (4
weekly infusions) was required for Dose Limiting Toxicity (DLT)
evaluation
[0129] Patients were excluded from the study if they had a primary
malignancy or active metastasis in the Central Nervous System;
hematologic malignancies; hemoglobin<9.0 g/dL; ANC<1,500/mm3;
platelet count<100,000/mm3; or a serum chemistry abnormality
(bilirubin, AST, ALT, albumin, creatinine).
[0130] The patient population included 28 patients. Patients had
received up to 9 prior therapies for this tumor; more than half
received 4 or more. See Table 1
TABLE-US-00002 TABLE 1 Demographics at Baseline # of Patients 28
Primary Tumor Site Primary Tumor Site Male 20 Lung Other Female 8
Lung 3 Bone 1 Age; Median 63 Pleura 2 Breast 1 (years) Range 31 76
Gastro Oropharnyx 1 Karnofsky Status Esophagus 1 Parathyroid 1
70-80 6 Stomach 1 Prostate 1 90-100 12 Pancreas 4 Skin 1 Stage 3 1
Liver 2 Testicular 1 Stage 4 26 Colon 5 Unknown 1 Rectal 3
[0131] 28 patients in Cohorts 1-5 received at least 1 infusion of
GRN163L. A total of 177 doses were administered. See Table 2. All
28 patients discontinued the study; reasons for discontinuation
included progressive disease (22/28; 78%), death (3/28; 11%) and
thrombocytopenia (3/28; 11%).
TABLE-US-00003 TABLE 2 Dosing Cohorts 1 2 3 4 5 Total Dose (mg/kg)
0.4 0.8 1.6 3.2 4.8 -- # of Patients 2 2 2 8 14 28 Median # of
Cycles/patient 3.0 3.0 1.5 1.5 2.0 2.0 Median # of Doses/patient
11.5 12.0 5.5 5.5 5.5 7.0 Percent of Doses Received 100% 100% 100%
81% 84% 88%
[0132] Blood samples were taken from the patients at different
times during the study. FIGS. 1-4 show the platelet levels in the
patients over time in the various cohorts.
Materials and Methods:
[0133] Blood samples were collected from each patient prior to
starting treatment. Median telomere length was determined by Repeat
Diagnostics, Vancouver Canada using the Flow-FISH method, with
gating on granulocyte and lymphocyte populations by the method
described in Baerlocher et al., "Flow cytometry and FISH to measure
the average length of telomeres (flow FISH) Nature Protocols Vol.
1, No. 5: 2365-2376 (2006) which is incorporated by reference
herein in its entirety
[0134] Briefly, whole blood from the 28 patients were. The
supernatant was aspirated without disturbing the white blood cell
or red blood cell pellet. The cells were mixed with cold NH.sub.4Cl
to lyse the red blood cells. The cells were centrifuged. The
supernatant was aspirated and the red cell lysate removed. The
resulting white blood cells were suspended in Hybridization Buffer
(5% dextrose/10 mM Hepes/0/1% BSA). The cells were counted and
diluted to approximately 5.times.10.sup.6 cells/200 .mu.l of
Hybridization Buffer.
Bovine thymocytes were isolated from fresh bovine thymus and fixed
in formaldehyde. The fixed bovine thymocytes were mixed with
Hybridization buffer (Tris, NaCl, BSA, dionized formamide). For the
unlabeled hybridization control, unlabeled hybridization mix stock
was added to the cells. For the Labeled Hybridization Mixture,
hybridization Peptide nucleic acid (PNA) probe (Cy5 or flourescein
labeled CCC TAA CCC TAA CCC TAA) (SEQ ID NO:3) was added to the
cells. The cells were hybridized with the PNA probe. Then the cells
were centrifuged and the cell pellet was washed to remove unbound
probe.
[0135] The flow cytometer was calibrated and the cells run on the
flow cytometer.
[0136] From the flow cytometry data analysis software program, the
flow FISH analysis template was used to calculate the fluorescence
of the various cell populations. The fluorescence was used to
calculate the average telomere length.
Results
[0137] Univariate and multivariate analyses were undertaken to
explore predictive factors for post-treatment decreases in platelet
levels and baseline telomere length. Factors included age, sex,
baseline platelet counts, time since cancer diagnosis, number of
prior cytotoxic or myelosuppressive chemotherapy regimens, prior
radiation therapy, and baseline granulocyte and lymphocyte telomere
length.
[0138] Infusions of GRN163L were generally well tolerated. Adverse
events (AE) that were considered to be related or possibly/probably
related to treatment were reported in 16/28 (57.1%) patients. See
Table 3. Most possibly/probably related adverse events were
reversible and Grade 1-2.
[0139] No dose limiting toxicities (DLT) were observed in Cohorts
1-3. In Cohort 4 (3.2 mg/kg), a patient who was tolerating therapy
well died of unknown cause after the 4th dose, and this was
considered a DLT. In Cohort 5 (4.8 mg/kg), two DLTs were
observed--both thrombocytopenia (one grade 4 and one grade 2
causing a >2 week delay in treatment).
TABLE-US-00004 TABLE 3 Reported adverse events (Possibly/Probably)
Related to Treatment in >1 patient.dagger. Dose (m/kg) 0.4-1.6
3.2 4.8 Total # of Patients 6 8 14 28 Reported at least 2 (33.3%) 8
(100%) 13 (92.9%) 23 (82.1%) 1 AE Activated partial thromboplastin
time prolonged Grade 1-2 0 6 (75%) 6 (42.9%) 12 (42.9%) Grade 3 0 0
6 (42.9%) 6 (21.4%) Thromboeytopenia.dagger-dbl. Grade 1-2 0 1
(12.5%) 3 (21.4%) 4 (14.3%) Grade 3-4 0 1 (12.5%) 2 (14.3%) 3
(10.7%) Anemia-Grade 2-3 0 1 (12.5%) 2 (14.3%) 3 (10.7%)
Leukopenia- 0 0 2 (14.3%) 2 (7.1%) Grade 1-3 Neutropenia- 0 0 2
(14.3%) 2 (7.1%) Grade 2-3 .dagger.Adverse events in 1 patient only
included photophobia, peripheral neuropathy, sedimentation rate
elevated, increased alkaline phosphotase, lymphopenia, tenderness
at neck above port site, burning on urination, candidiasis, chest
tightness, confusion, dehydration, elevated AST and death.
.dagger-dbl.Not all lab readings consistent with thrombocytopenia
were reported as adverse events.
[0140] Platelet levels over time for individual patients receiving
weekly infusions of GRN163L are shown by dose cohort in FIGS. 1-3.
Circled data points indicate that the patient received treatment at
that visit. Platelet levels over time for 5 of 6 patients on the
intermittent 4.8 mg/kg dosing schedule (Cohort 6) are shown in FIG.
4.
[0141] To better understand patient and treatment factors
potentially influencing platelet declines (or increases), a model
was developed to identify predictors of percent change in platelet
levels at 4 weeks.
[0142] The change in platelet levels after 4 weeks of treatment
with GRN163L was plotted relative to the baseline median telomere
length in each patient in FIG. 5.
[0143] The first model included 20/28 patients. Dose and baseline
granulocyte telomere length were significant predictors. No dose by
telomere length interaction was found.
TABLE-US-00005 TABLE 4 Multivariate Analysis Predictor Reression
Coefficient P Value Dose -7 0.034 Baseline Granulocyte 8 0.023
Telomere length
[0144] The model was extended to include 8 additional patients (all
28 patients in Cohorts 1-5). The relationships detected in the
earlier model remained significant. Baseline GRN163L dose level and
median baseline Granulocyte telomere length were both predictive of
the % decrease of platelet levels during the first 4 weeks of
treatment
TABLE-US-00006 TABLE 5 Multivariate Analysis Predictor Regression
Coefficient P Value Intercept 73.8 0.004 Dose -6.6 0.033 Baseline
Granulocyte 11.2 0.005 Telomere length Model: R-squared =
0.319853
[0145] FIG. 5 shows the percent change from baseline to the 4-week
nadir in platelet levels as a function of baseline granulocyte
telomere length. The lines indicate the percentage change as a
function of the dose level of GRN163L.
[0146] The equation to describe the predicted number of platelets
in a patient during the first 4 weeks of treatment is as
follows;
Predicted # of platelets=baseline platelets number-(baseline
platelet number x % change in platelets/100).
% change in platelet #=(-73.8)-6.6.times.inhibitor dose
(mg/kg)+11.2.times.average telomere length (kbp)
[0147] In the univariate analyses of predictors of percent change
in platelet levels, only granulocyte telomere length was
significant (p=0.024).
TABLE-US-00007 TABLE 6 Univariate Analysis Predictor Regression
coefficient p Value Intercept -84.54302 0.0018 Baseline granulocyte
9.17580 0.0241 telomere length
[0148] Another model was then developed to identify predictors of
log nadir platelet levels during the first 4 weeks. This model
resulted in a higher R.sup.2 value indicating a better fit to the
data.
TABLE-US-00008 TABLE 7 Multivariate Analysis Regression Predictor
coefficient (b) p Value Intercept -0.38 0.725 Dose (mg/kg) -0.13
0.017 Baseline Granulocyte Telomere 0.25 0.001 Length Log of
Baseline Platelets 0.80 <0.001 Model: R-squared = 0.645071
[0149] The equation to describe the predicted number of platelets
in a patient at nadir during the first 4 weeks of treatment is as
follows:
Predicted # of platelets=e.sup.[(-0.38)-0.13.times.inhibitor dose
(mg/kg)+0.25.times.average telomere length (kbp)+0.80.times.log of
baseline platelet number]
[0150] In the univariate analyses of predictors of log nadir
platelet levels, granulocyte telomere length, alone, was
significant (p=0.004).
TABLE-US-00009 TABLE 8 Univariate Analysis Regression Predictor
coefficient p Value | Intercept 3.42818 <.0001 Baseline
granulocyte telomere 0.27189 0.0040 length
[0151] Prediction intervals may be calculated, for example, to
predict likely percent change in platelet levels or the platelet
nadir for patients or subjects with a particular set of baseline
and treatment values, for example, telomere length, baseline
platelets and dose level. The regression equation provides the
expected value for a future individual with specified covariates
(J. Neter et al. Applied linear statistical models: regression,
analysis of variance, and experimental designs, 3.sup.rd edition
pp. 81-83 (1990)). However, due to sampling distribution error as
well as interindividual variability, a patient may have platelet
levels that fall above or below that predicted value. A series of
prediction intervals may be created with decreasing coverage. For
example, a 99% prediction interval with upper and lower bounds
P.sub.U99 and P.sub.L99 may be created and would, on average,
contain 99% of the patients' observed platelet levels. A 90%
prediction interval with upper and lower bounds P.sub.U90
(<P.sub.U99) and P.sub.L90 (>P.sub.L99) may be created and
would, on average, contain 90% of future observed platelet levels.
This allows one to determine the likelihood that the patient would
develop a grade 3 or 4 thrombocytopenia.
[0152] The average telomere length of normal cells differs among
individuals and declines with age as shown by the percentiles in
FIG. 7. Granulocyte telomere lengths in the 28 patients in this
study are generally shorter than normal, which is consistent with
the effects of physiologic stress and chemotherapy. Although prior
chemotherapy history as tabulated here was not predictive of
platelet decreases, it was highly correlated with baseline telomere
lengths in a univariate analysis. The telomere length measurement
reflects prior treatment effects along with other hereditary or
acquired influences with high precision. Age was also a significant
predictor of telomere length.
[0153] The results from this study demonstrate a close
correspondence between baseline telomere length values as
determined by the described test and the risk of the patient
developing dose limiting thrombocytopenia.
[0154] Although the invention has been described with respect to
particular embodiments and applications, those skilled in the art
will appreciate the range of applications and methods of the
invention disclosed herein and the invention is not limited to such
embodiments.
[0155] All references cited throughout the disclosure are hereby
expressly incorporated by reference herein in their entireties.
Sequence CWU 1
1
3111RNAArtificial SequenceSynthetic oligonucleotide 1cuaacccuaa c
11213DNAArtificial SequenceSynthetic oligonucleotide 2tagggttaga
caa 13318DNAArtificial SequenceSynthetic oligonucleotide
3ccctaaccct aaccctaa 18
* * * * *