U.S. patent application number 09/096335 was filed with the patent office on 2001-09-13 for methods of diagnosing renal salt wasting syndrome and alzheimer's disease and methods of treating the same.
Invention is credited to MAESAKA, JOHN K..
Application Number | 20010021508 09/096335 |
Document ID | / |
Family ID | 22256880 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021508 |
Kind Code |
A1 |
MAESAKA, JOHN K. |
September 13, 2001 |
METHODS OF DIAGNOSING RENAL SALT WASTING SYNDROME AND ALZHEIMER'S
DISEASE AND METHODS OF TREATING THE SAME
Abstract
A method is described to diagnose (1) renal salt wasting
syndrome and (2) Alzheimer's disease among dementia patients by
measuring a patient's level of prostaglandin D.sub.2 synthase.
Methods are also described to (1) treat renal salt wasting
syndrome, (2) inhibit the rate of apoptosis or (3) prevent the
onset of, or slow the rate of, progression of Alzheimer's disease.
These methods involve inhibiting the rate of
-.DELTA..sup.12prostaglandin J.sub.2 synthesis or by inhibiting the
activity of -.DELTA..sup.12prostaglandin J.sub.2.
Inventors: |
MAESAKA, JOHN K.; (NEW YORK,
NY) |
Correspondence
Address: |
OSTROLENK FABER GERB & SOFFEN
1180 AVENUE OF THE AMERICAS
NEW YORK
NY
100368403
|
Family ID: |
22256880 |
Appl. No.: |
09/096335 |
Filed: |
June 11, 1998 |
Current U.S.
Class: |
435/7.1 ;
435/810; 514/17.8; 514/18.9 |
Current CPC
Class: |
G01N 2800/2821 20130101;
G01N 33/68 20130101; G01N 33/6896 20130101; G01N 33/92 20130101;
Y10S 436/811 20130101 |
Class at
Publication: |
435/7.1 ; 514/2;
435/810 |
International
Class: |
A01N 037/18; A61K
038/00; G01N 033/53 |
Claims
What is claimed is:
1. A method of diagnosing or assessing the likelihood that a
patient is afflicted with renal salt wasting syndrome, said method
comprising measuring the level of prostaglandin D.sub.2 synthase in
a sample from said patient.
2. The method of claim 1, wherein the sample is plasma.
3. The method of claim 1, wherein the sample is urine.
4. The method of claim 1, wherein prostaglandin D.sub.2 synthase is
measured by contacting the samples with antibodies to prostaglandin
D.sub.2 synthase, and determining levels of immunocomplexes between
said antibodies and said prostaglandin D.sub.2 synthase.
5. The method of claim 1, wherein prostaglandin D.sub.2 synthase is
measured by immunoprecipitation or western blotting.
6. A method of diagnosing or assessing the likelihood that a
patient is afflicted with Alzheimer's disease, said method
comprising measuring the level of prostaglandin D.sub.2 synthase in
a sample from said patient.
7. The method of claim 6, wherein the sample is plasma.
8. The method of claim 6, wherein the sample is urine.
9. The method of claim 6, wherein prostaglandin D.sub.2 synthase is
measured by contacting said sample with antibodies to prostaglandin
D.sub.2 synthase, and determining levels of immunocomplexes between
said antibodies and said prostaglandin D.sub.2 synthase.
10. The method of claim 6, wherein prostaglandin D.sub.2 synthase
is measured by western blotting or immunoprecipitation.
11. A method of treating or reducing the risk of acquiring renal
salt wasting syndrome in a patient in need of such treatment or
reduction, said method comprising reducing
-.DELTA..sup.12prostaglandin J.sub.2 levels or activity thereof in
said patient.
12. The method of claim 11, wherein -.DELTA..sup.12prostaglandin
J.sub.2 biosynthesis is reduced.
13. The method of claim 12, wherein -.DELTA..sup.12prostaglandin
J.sub.2 biosynthesis is reduced by administering to said patient a
therapeutically effective amount of at least one agent selected
from the group consisting of cyclo-oxygenase inhibitor,
cyclo-oxygenase antibody, prostaglandin D.sub.2 synthase inhibitor,
prostaglandin D.sub.2 synthase antibody.
14. The method of claim 13, wherein said cyclo-oxygenase inhibitor
is indomethacin.
15. The method of claim 11 which comprises administering to said
patient a therapeutically effective amount of an inhibitor of
prostaglandin J.sub.2 activity.
16. A method of inhibiting the rate of apoptosis in a patient with
elevated prostaglandin D.sub.2 synthase in the plasma or urine,
said method comprising reducing -.DELTA..sup.12prostaglandin
J.sub.2 levels or activity in said patient.
17. The method of claim 16, wherein -.DELTA..sup.12prostaglandin
J.sub.2 biosynthesis is reduced.
18. The method of claim 17, wherein -.DELTA..sup.12prostaglandin
J.sub.2 biosynthesis is reduced by administering to said patient a
therapeutically effective amount of at least one agent selected
from the group consisting of cyclo-oxygenase inhibitor,
cyclo-oxygenase antibody, prostaglandin D.sub.2 synthase inhibitor,
prostaglandin D.sub.2 synthase antibody.
19. The method of claim 18, wherein said cyclo-oxygenase inhibitor
is indomethacin.
20. The method of claim 16 comprising administering to the patient
a therapeutically effective amount of an inhibitor of
-.DELTA..sup.12prostaglandin J.sub.2 activity.
21. A method of treating or reducing the risk of onset of
Alzheimer's disease in a patient in need of such treatment or
reduction, said method comprising reducing
-.DELTA..sup.12prostaglandin J.sub.2 levels or activity in said
patient, other than by administering a cyclo-oxygenase
inhibitor.
22. The method of claim 21, wherein biosynthesis of
-.DELTA..sup.12prostaglandin J.sub.2 is reduced.
23. The method of claim 22, wherein -.DELTA..sup.12prostaglandin
J.sub.2 biosynthesis is reduced by administering to said patient a
therapeutically effective amount of at least one agent selected
from the group consisting of cyclo-oxygenase antibody,
prostaglandin D.sub.2 synthase inhibitor and prostaglandin D.sub.2
synthase antibody.
24. The method of claim 21 which comprises administering to the
patient a therapeutically effective amount of an inhibitor of
-.DELTA..sup.12prostaglandin J.sub.2 activity.
25. A diagnostic kit for detecting the presence of prostaglandin
D.sub.2 synthase in a sample, said kit comprising antibodies to
said prostaglandin D.sub.2 synthase, and means for measuring
prostaglandin D.sub.2 synthase:anti-prostaglandin D.sub.2 synthase
immunocomplexes.
26. The kit of claim 25, wherein said measuring means comprises an
enzyme labelled anti-immunoglobulin and a chromogenic substrate for
said enzyme label.
27. The kit of claim 25, wherein said antibodies are immobilized on
a substrate.
28. The kit of claim 25, wherein said kit is for use in detection
of Alzheimer's disease, and further includes a comparison standard
for correlating said measurement to likelihood a patient from whom
said sample was taken is afflicted with Alzheimer's disease.
29. The kit of claim 25, wherein said kit is for detection of renal
salt wasting syndrome, and further includes a comparison standard
for correlating said measurement to likelihood a patient from whom
said sample was taken is afflicted with renal salt wasting
syndrome.
Description
[0001] Throughout this application, various references are referred
to within parentheses. Disclosure of the publications in their
entirety are hereby incorporated by reference into this application
to more fully describe the state of the art to which this invention
pertains.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of (1) diagnosing
or assessing the likelihood that a patient is afflicted with renal
salt wasting syndrome and (2) diagnosing or assessing the
likelihood that a patient is afflicted with or will develop
Alzheimer's disease. The present invention also relates to methods
of (1) treating, preventing the onset or slowing the rate of
progression of Alzheimer's disease, (2) treating or preventing
onset of renal salt wasting syndrome, and (3) inhibiting
apoptosis.
DESCRIPTION OF THE RELATED ART
[0003] A new medical syndrome, the renal salt wasting syndrome has
been described in patients suffering from pneumonia, cancers of the
lung, and brain diseases such as primary or secondary tumors, brain
hemorrhage, AIDS, and Alzheimer's disease (J. K. Maesaka et al.
Life Sci. 52:1875, 1993, J. K. Maesaka et al., J. Am. Ger. Soc.
41:501, 1993). Patients suffering from renal salt wasting syndrome
have low serum sodium (hyponatremia) and low serum uric acid levels
(hypouricemia). These patients share low serum uric acid
concentrations and a renal tubular transport defect for uric acid
which results in an increase in the fractional excretion of uric
acid. Renal salt wasting syndrome mimics the syndrome of
inappropriate secretion of antidiuretic hormone (SIADH) in many
clinical parameters except that renal salt wasting syndrome has
diminished total body water and sodium. Total body fluids are
increased in SIADH and decreased in the renal salt wasting
syndrome. Because it is extremely difficult to assess accurately
the fluid status of patients that do not suffer from edema, renal
salt wasting syndrome patients are frequently misdiagnosed as
having SIADH.
[0004] The importance of making a differentiation between renal
salt wasting syndrome and SIADH is the difference in treatment
modalities. SIADH is usually treated with water restriction whereas
the renal salt wasting syndrome patients require variable amounts
of fluid and salt supplementation depending on the extent of their
salt and water deficits. Moreover, large volumes of salt and fluid,
particularly water, actually exacerbate the hyponatremia in
patients with SIADH which can lead to coma and convulsions. On the
other hand, fluid restrictions, a common treatment for SIADH, could
worsen the clinical condition of the patient with renal salt
wasting syndrome because it exacerbates their underlying depletion
of body fluids.
[0005] Volume depletion and persistence of the hypouricemia and
increased fractional excretion (FE) of urate by the kidneys after
correction of the hyponatremia distinguish renal salt wasting
syndrome from the SIADH. Since assessment of extracellular volume
(ECV) which is necessary to determine volume depletion has been
shown to be inaccurate in non-edematous and non-ascitic cases (H.
M. Chung et al., Am. J. Med. 83:905, 1987), it was postulated that
it might be possible to differentiate renal salt wasting syndrome
from inappropriate secretion of antidiuretic hormone by
scrutinizing urate metabolism and response of the patient to saline
infusion. However, the necessary salt balance studies are believed
to be less practical than the simple determination described
herein.
[0006] The plausibility of a salt wasting syndrome in patients with
neurosurgical or possibly active brain diseases lies in the
demonstration of natriuretic-apoptotic factor(s) circulating in the
plasma of patients with neurosurgical and Alzheimer's diseases by
Maesaka et al. (Life Sci. 52:1875, 1993; J. Am. Ger. Soc. 41:501,
1993). There was a fourfold or greater increase in apoptosis in
cultured LLC-PK1 cells that have been exposed to Alzheimer plasma
as compared to normal and multi-infarct dementia (MID) plasma (J.
K. Maesaka et al., J. Am. Soc. Nephrol. 6:740, 1995 (abst.)).
However, the identity of this factor is not known and the testing
of its presence based on an increase in apoptosis in tissue
cultured cells is impractical.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide
effective methods and kits for diagnosing Alzheimer's disease and
renal salt wasting syndrome.
[0008] It is another object of the invention to provide a method of
treating, reducing the risk of onset of, or slowing the rate of
progression of, Alzheimer's disease.
[0009] It is yet another object of the invention to provide a
method to treat or reduce onset of renal salt wasting syndrome.
[0010] It is yet another object of the invention to provide a
method to inhibit the rate of apoptosis.
[0011] It is yet another object of the invention to provide a
clinical kit for the quantification of prostaglandin D.sub.2
synthase levels, preferably for aiding diagnosis of Alzheimer's
disease and/or renal salt wasting syndrome.
[0012] In one embodiment, the invention provides a method of
diagnosing or assessing the likelihood that a patient is afflicted
with renal salt wasting syndrome, said method comprising measuring
the level of prostaglandin D.sub.2 synthase in a sample from said
patient.
[0013] In another embodiment, the invention provides a method of
diagnosing or assessing the likelihood that a patient is afflicted
with Alzheimer's disease, said method comprising measuring the
level of prostaglandin D.sub.2 synthase in a sample from said
patient.
[0014] In yet another embodiment, the invention provides a method
of treating or reducing the risk of acquiring renal salt wasting
syndrome in a patient in need of such treatment or reduction, said
method comprising reducing -.DELTA..sup.12prostaglandin J.sub.2
levels or activity thereof in said patient.
[0015] In yet another embodiment, the invention provides a method
of inhibiting the rate of apoptosis in a patient with elevated
prostaglandin D.sub.2 synthase in the plasma or urine, said method
comprising reducing -.DELTA..sup.12prostaglandin J.sub.2 levels or
activity in said patient.
[0016] In yet another embodiment, the invention provides the method
of treating or reducing the risk of onset of Alzheimer's disease in
a patient in need of such treatment or reduction, said method
comprising reducing -.DELTA..sup.12prostaglandin J.sub.2 levels, or
activity thereof, in said patient, other than by administering a
cyclo-oxygenase inhibitor.
[0017] In yet another embodiment, the invention provides a
diagnostic kit for detecting the presence of prostaglandin D.sub.2
synthase in a sample, said kit comprising antibodies to said
prostaglandin D.sub.2 synthase, and means for measuring
prostaglandin D.sub.2 synthase:anti-prostaglandin D.sub.2 synthase
immunocomplexes.
[0018] The term "inhibitor" as used herein means any agent which
reduces the normal physiological effect of an already-formed agent,
e.g. by action on the agent itself or by antagonistic effect on a
receptor for that agent. EXCEPTION: As used herein, the term
"cyclo-oxygenase inhibitor" does not include cyclo-oxygenase
antibodies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a transmission electron micrograph of LLC-PK1
cells that have been exposed to AD plasma for 2 hours prior to
fixation. Note condensed and black nuclei (black arrows).
Arrowheads represent small residual nuclear bodies engulfed by a
neighboring cell. White arrows show a section fold. Magnification:
A=2200.times., B=4500.times.. This shows that LLC-PK1 cells undergo
apoptosis after exposure to AD plasma.
[0020] FIGS. 2A and 2B illustrate a TUNEL assay with ApoDetek Kit
(Enzo). FIG. 2A is a light micrograph of a TUNEL assay performed on
LLC-PK1 cells exposed to control plasma for 2 hours showing normal
pale staining nuclei. FIG. 2B is a light micrograph of a TUNEL
assay performed on LLC-PK1 cells exposed to plasma of patients with
Alzheimer's disease for 2 hours showing condensed, dark nuclei
(large arrowheads) and the normal oval pale nuclei (small
arrowheads). Magnification=300.times.. This shows that LLC-PK1
cells undergo apoptosis after exposure to AD plasma.
[0021] FIGS. 3A and 3B show a dose response (A) and Time course (B)
of apoptosis in LLC-PK1 cells exposed to plasma of patients with
Alzheimer's disease as measured by TUNEL assay. This shows that AD
plasma contains a component that causes LLC-PK1 cells to undergo
apoptosis.
[0022] FIG. 4 shows internucleosomal DNA cleavage in LLC-PK1 cells
that had been exposed to plasma of patients with Alzheimer's
disease (AD) for intervals 2, 3, 4, and 5 days. Maximum DNA
fragmentation was 4 days after exposure to AD plasma and showed the
characteristic 180 bp spacing. Control (NC) plasma did not exhibit
a ladder even after incubation for 5 days. The DNA laddering
indicates that apoptosis occurs in LLC-PK1 cells exposed to AD
plasma.
[0023] FIG. 5 is an elution profile of pooled plasma from patients
with Alzheimer's disease (4.5 mL) chromatography on (1.times.8 cm)
Affi-Gel Blue Gel Agarose column (20 mM phosphate Buffer, pH 7.1)
(Flow Rate=1 mL/min.) (15 mL Load and Wash, 25 mL 0.5 M NaCl
fraction, 10 mL 2M NaCl fraction). Line represents active
fraction.
[0024] FIGS. 6A and 6B show the results of iso-electric focusing.
FIG. 6A depicts fractionation of 0.5 M NaCl eluate from Affi-Blue
run on Rotofor (BioRad) using pH 3-10 gradient. FIG. 6B depicts
fractionation of the active pool from FIG. 6A (pI 4.4-5.7) on
Rotofor, using pH 4-6 gradient. Arrow points to fraction 2, line
represents the active fraction.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In accordance with the invention, prostaglandin D.sub.2
synthase is a marker for Alzheimer's disease and is also a marker
for renal salt wasting syndrome. Levels of Prostaglandin D.sub.2
synthase are elevated in the blood and urine of patients suffering
from Alzheimer's disease as compared to normal, non-demented age
and gender-matched controls and comparably demented patients with
multi-infarct dementia. The levels of prostaglandin D.sub.2
synthase are also elevated in the blood and urine of patients
suffering from renal salt wasting syndrome and not in patients with
the syndrome of inappropriate secretion of antidiuretic hormone
(SIADH), a common cause of hyponatremia.
[0026] The bioassay of the present invention to determine the
presence of prostaglandin D.sub.2 synthase provides a simple means
of differentiating renal wasting syndrome from SIADH. The bioassay
of the present invention also provides simple means of
differentiating Alzheimer's disease from multi-infarct dementia.
These clinical differentiations are often difficult to make. The
importance of making a differentiation between both renal salt
wasting syndrome and SIADH is the difference in treatment
modalities.
[0027] Clinical differentiation between Alzheimer's disease and
other dementia type of diseases such as multi-infarct dementia is
also very important particularly at the earliest stages of the
disease when diagnosis is very difficult. Early diagnosis of
Alzheimer's disease may be particularly helpful because it might
lead to early treatment before more damage is done to the
brain.
Diagnosis of Alzheimer's Disease and Renal Salt Wasting Syndrome by
Detecting Prostaglandin D.sub.2 Synthase
[0028] A sample is normally taken from a subject suspected of
having renal salt wasting syndrome or Alzheimer's disease. This
sample is then tested to measure the level of prostaglandin D.sub.2
synthase. An elevated level of prostaglandin D.sub.2 synthase over
control samples (e.g. one or two standard deviations above normal,
and especially levels more than twice the normal level) is an
indication of renal salt wasting syndrome or of Alzheimer's
disease. The method of the invention and detection kits in
accordance with the invention, preferably include comparison
standards derived from previously tested control samples. The
method of the invention may be practiced by comparing measured
levels of prostaglandin D.sub.2 synthase (in a test sample) to the
comparison standards. Likelihood that the patient suffers from
Alzheimer's disease or renal salt wasting syndrome derived from
correlation of measured levels to the comparison standards. The
comparison standards may be any well known in the art, e.g. color
change, phosphorescence, enzymatic activity or any other parameter
common in the art. Some examples are set forth below in the section
entitled "Methods of Detection of Prostaglandin D.sub.2 Synthase".
Naturally, the comparison standards should reflect control levels
measured by the same measurement technique as will be utilized for
measuring prostaglandin D.sub.2 synthase in the patient sample. It
is preferred that the comparison standard show any age and
gender-based variations.
[0029] Preferably, the samples to be tested are body fluids such as
blood, plasma, urine, tears, saliva and the like. Both medical and
veterinary applications are contemplated. In addition to human
samples, samples may be taken from other mammals such as non-human
primates, horses, swine, etc. In some instances it may be possible
or even desirable to dilute the sample prior to testing. Plasma,
when used as the sample, may be diluted, for example, with one or
more fluids selected from the group consisting of
phosphate-buffered saline, pH 7.0-7.4 (hereinafter "PBS"),
PBS-containing TWEEN 20 (hereinafter "PBS T"), PBS T with
thimerosal (hereinafter "PBS TT"), PBS TT (gelatin) (hereinafter
"PBS TTG").
[0030] Preferred diluents and dilution ratios may vary in a known
manner according to the sample being tested. In some instances, it
can be desirable to concentrate a sample that is initially too
dilute. Prior to testing a sample whose pH is outside of the
preferred pH for antibody function (e.g. urine), the pH of the
sample is preferably adjusted to between about 7.0 and 7.4, the
preferred pH for antibody function.
Prostaglandin D.sub.2 Antibody Preparation
[0031] (i) Polyclonal Antibodies
[0032] Polyclonal antibodies to prostaglandin D.sub.2 or
prostaglandin D.sub.2 fragments can generally be raised in animals
by multiple subcutaneous (sc), intradermal (id), or intraperitoneal
(ip) injections of natural or recombinant prostaglandin D.sub.2
synthase or prostaglandin D.sub.2 synthase fragment or synthetic
peptide and an adjuvant. It may be useful to conjugate
prostaglandin D.sub.2 synthase or a fragment containing the target
amino acid sequence to a protein that is immunogenic in the species
to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, or SOCl2.
[0033] Animals can be immunized against the prostaglandin D.sub.2
synthase protein or a fragment thereof, immunogenic conjugates, or
derivatives by combining 1 mg or 1 .mu.g of the peptide or
conjugate (for rabbits or mice, respectively) with 3 volumes of
Freund's complete adjuvant or other adjuvant and injecting the
solution intradermally at multiple sites. Four to five weeks later
the animals are boosted with 1/5 to {fraction (1/10)} the original
amount of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites or intradermal injection
at multiple sites of an equivalent amount of natural or recombinant
prostaglandin D.sub.2 synthase. Seven to 14 days later the animals
are bled and the serum is assayed for prostaglandin D.sub.2
synthase or prostaglandin D.sub.2 synthase fragment antibody titer.
Animals are boosted until the titer plateaus. Preferably, the
animal is boosted with purified natural or recombinant
prostaglandin D.sub.2 synthase, the conjugate of the same
prostaglandin D.sub.2 synthase or prostaglandin D.sub.2 synthase
fragment, but conjugated to a different protein and/or through a
different cross-linking reagent. Conjugates also can be made in
recombinant cell culture as protein fusions Also, aggregating
agents such as alum may be used to enhance the immune response.
[0034] (ii) Monoclonal Antibodies
[0035] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. For
example, the prostaglandin D.sub.2 synthase monoclonal antibodies
of the invention may be made using the hybridoma method (Nature,
256: 495 (1975), or may be made by known recombinant DNA
methods.
[0036] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the prostaglandin D2
synthase or prostaglandin D.sub.2 synthase fragment used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103
[Academic Press, 1986]).
[0037] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0038] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. U.S.A., and SP-2 cells available from the
American Type Culture Collection, Rockville, Md. U.S.A.
[0039] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
prostaglandin D.sub.2 synthase. Preferably, the binding specificity
of monoclonal antibodies produced by hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0040] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson and
Pollard, Anal. Biochem., 107: 220 (1980).
[0041] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. Suitable culture media for this purpose include,
for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma
cells may be grown in vivo as ascites tumors in an animal.
[0042] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxyapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0043] DNA encoding the monoclonal antibodies of the invention is
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). Once isolated, the DNA may be placed into expression
vectors. Host cells are then transformed or transfected with said
vectors. Suitable host cells include but are not limited to E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. Review articles on recombinant expression in bacteria
of DNA encoding an antibody include Skerra et al., Curr. Opinion in
Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130:
151-188 (1992).
[0044] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (Morrison, et
al., Proc. Nat. Acad. Sci., 81: 6851 [1984]), or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. In that
manner, "chimeric" or "hybrid" antibodies are prepared that have
the binding specificity of an anti-prostaglandin D.sub.2 synthase
monoclonal antibody herein.
[0045] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody of the
invention, or they are substituted for the variable domains of one
antigen-combining site of an antibody of the invention to create a
chimeric bivalent antibody comprising one antigen-combining site
having specificity for a prostaglandin D.sub.2 synthase and another
antigen-combining site having specificity for a different
antigen.
[0046] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may be
constructed using a disulfide-exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
[0047] (iii) Human Antibodies
[0048] Human monoclonal antibodies can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described,
for example, by Kozbor, J. Immunol. 133, 3001 (1984); Brodeur, et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et
al., J. Immunol., 147: 86-95 (1991).
[0049] It is now possible to produce transgenic animals (e.g. mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J[H]) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array in such germ-line mutant mice will result
in the production of human antibodies upon antigen challenge. See,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551
(1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann
et al., Year in Immuno., 7: 33 (1993).
[0050] Alternatively, phage display technology (McCafferty et al.,
Nature, 348: 552-553 [1990]) can be used to produce human
antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
According to this technique, antibody V domain genes are cloned
in-frame into either a major or minor coat protein gene of a
filamentous bacteriophage, such as M13 or fd, and displayed as
functional antibody fragments on the surface of the phage particle.
Because the filamentous particle contains a single-stranded DNA
copy of the phage genome, selections based on the functional
properties of the antibody also result in selection of the gene
encoding the antibody exhibiting those properties. Thus, the phage
mimics some of the properties of the B-cell. Phage display can be
performed in a variety of formats; for their review see, e.g.,
Johnson, Kevin S. and Chiswell, David J., Current Opinion in
Structural Biology, 3: 564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature,
352: 624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol., 222: 581-597 (1991), or Griffith et al., EMBO
J., 12: 725-734 (1993).
[0051] In a natural immune response, antibody genes accumulate
mutations at a high rate (somatic hypermutation). Some of the
changes introduced will confer higher affinity, and B cells
displaying high-affinity surface immunoglobulin are preferentially
replicated and differentiated during subsequent antigen challenge.
This natural process can be mimicked by employing the technique
known as "chain shuffling" (Marks et al., Bio/Technol., 10: 779-783
[1992]). In this method, the affinity of "primary" human antibodies
obtained by phage display can be improved by sequentially replacing
the heavy and light chain V region genes with repertoires of
naturally occurring variants (repertoires) of V domain genes
obtained from unimmunized donors. This technique allows the
production of antibodies and antibody fragments with affinities in
the nM range. A strategy for making very large phage antibody
repertoires has been described by Waterhouse et al., Nucl. Acids
Res., 21: 2265-2266 (1993).
[0052] Gene shuffling can also be used to derive human antibodies
from rodent antibodies, where the human antibody has similar
affinities and specificities to the starting rodent antibody.
According to this method, which is also referred to as "epitope
imprinting", the heavy or light chain V domain gene of rodent
antibodies obtained by phage display technique is replaced with a
repertoire of human V domain genes, creating rodent-human chimeras.
Selection on antigen results in isolation of human variable capable
of restoring a functional antigen-binding site, i.e. the epitope
governs (imprints) the choice of partner. When the process is
repeated in order to replace the remaining rodent V domain, a human
antibody is obtained (see PCT WO 93/06213, published Apr. 1, 1993).
Unlike traditional humanization of rodent antibodies by CDR
grafting, this technique provides completely human antibodies,
which have no framework or CDR residues of rodent origin.
Methods of Detection of Prostaglandin D.sub.2 Synthase
Detection with Antibodies
[0053] For diagnostic applications (i.e. detection of prostaglandin
D.sub.2 synthase), antibodies against prostaglandin D.sub.2
synthase typically will be labeled with a detectable moiety. The
detectable moiety can be any one which is capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, 35S or .sup.125I; a fluorescent or
chemiluminescent compound (Melegos et al., Clin. Chem. 42:12
(1996)), such as fluorescein isothiocyanate, rhodamine, or
luciferin; radioactive isotopic labels, such as, e.g., .sup.125I,
.sup.32P, .sup.14C, or .sup.3H; or an enzyme, such as alkaline
phosphatase, beta-galactosidase, or horseradish peroxidase.
[0054] Any method known in the art for separately conjugating the
antibody to the detectable moiety may be employed, including those
methods described by Hunter et al., Nature, 144: 945 (1962); David
et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol.
Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30:
407 (1982).
[0055] The antibodies used for diagnostic purposes in the present
invention may be employed in any known assay method, such as
competitive binding assays, direct and indirect sandwich assays,
and immunoprecipitation assays. Zola, Monoclonal Antibodies: A
Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
[0056] Competitive binding assays rely on the ability of a labeled
standard (which may be prostaglandin D.sub.2 synthase or an
immunologically reactive portion thereof) to compete with the test
sample for binding with a limited amount of antibody. The amount of
prostaglandin D.sub.2 synthase in the test sample is inversely
proportional to the amount of standard that becomes bound to the
antibodies. To facilitate determining the amount of standard that
becomes bound, the antibodies generally are insolubilized before or
after the competition, so that the standard and prostaglandin
D.sub.2 synthase from the tested sample that are bound to the
antibodies may conveniently be separated from the unbound
material.
[0057] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein (prostaglandin D.sub.2 synthase) to be detected. In
a sandwich assay, the test sample protein (prostaglandin D.sub.2
synthase) is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
protein, thus forming an insoluble three-part complex. David and
Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay (Enzyme Linked
immunoabsorbent assay), in which case the detectable moiety is an
enzyme (e.g., horseradish peroxidase).
[0058] Prostaglandin D.sub.2 synthase antibodies are useful in
diagnostic assays for prostaglandin D.sub.2 synthase, e.g., its
production in specific cells or tissues, or its presence in urine
or serum. The antibodies are labeled and/or are immobilized on an
insoluble matrix. In one embodiment, an antibody that binds to
prostaglandin D.sub.2 synthase is immobilized on an insoluble
matrix, the test sample is contacted with the immobilized antibody
composition to adsorb all prostaglandin D.sub.2 synthase, and then
the immobilized prostaglandin D.sub.2 synthase molecules are
contacted with antibodies that recognize different antigenic sites
on prostaglandin D.sub.2 synthase, these antibodies being
identifiable by a unique label such as discrete fluorophores or the
like. By determining the presence and/or amount of the unique
label, the amount of prostaglandin D.sub.2 synthase can be
determined.
[0059] Competitive assays rely on the ability of a tracer (i.e.
labelled) analogue to compete with the test sample prostaglandin
D.sub.2 synthase for a limited number of binding sites on a common
binding partner. The binding partner generally is insolubilized
before or after the competition and then the tracer and
prostaglandin D.sub.2 synthase bound to the binding partner are
separated from the unbound tracer and prostaglandin D.sub.2
synthase. This separation is accomplished by decanting (where the
binding partner was preinsolubilized) or by centrifuging (where the
binding partner was precipitated after the competitive reaction).
The amount of test sample prostaglandin D.sub.2 synthase is
inversely proportional to the amount of bound tracer as measured by
the amount of marker substance. Dose-response curves with known
amounts of prostaglandin D.sub.2 synthase are prepared and compared
with the test results to quantitatively determine the amount of
prostaglandin D.sub.2 synthase present in the test sample. These
assays are called ELISA systems when enzymes are used as the
detectable markers.
[0060] Another species of competitive assay, called a "homogeneous"
assay, does not require a phase separation. Here, a conjugate of an
enzyme with prostaglandin D.sub.2 synthase is prepared and used
such that when anti-prostaglandin D.sub.2 synthase binds to the
prostaglandin D.sub.2 synthase, the presence of the
anti-prostaglandin D.sub.2 synthase modifies the enzyme activity.
In this case, prostaglandin D.sub.2 synthase or its immunologically
active fragments are conjugated with a bifunctional organic bridge
to an enzyme such as peroxidase. Conjugates are selected for use
with anti-prostaglandin D.sub.2 synthase so that binding of the
anti-prostaglandin D.sub.2 synthase inhibits or potentiates the
enzyme activity of the label. This method per se is widely
practiced under the name of EMIT.
[0061] Sandwich assays particularly are useful for the
determination of prostaglandin D.sub.2 synthase. In sequential
sandwich assays an immobilized binding partner is used to adsorb
test sample prostaglandin D.sub.2 synthase, the test sample is
removed as by washing, the bound prostaglandin D.sub.2 synthase is
used to adsorb labeled binding partner, and bound material is then
separated from residual tracer. The amount of bound tracer is
directly proportional to test sample prostaglandin D.sub.2
synthase. In "simultaneous" sandwich assays the test sample is not
separated before adding the labeled binding partner. A sequential
sandwich assay using an anti-prostaglandin D.sub.2 synthase
monoclonal antibody as one antibody and a polyclonal
anti-prostaglandin D.sub.2 synthase antibody as the other is useful
in testing samples for prostaglandin D.sub.2 synthase presence.
Detection with Assay for Apoptosis
[0062] Applicant demonstrated the presence of a factor (isolated
and identified as prostaglandin D.sub.2 synthase) in the plasma of
patients with Alzheimer's disease that increases apoptosis in
cultured LLC-PK1 cells when compared to plasma from control
subjects (C) and subjects suffering from multi-infarct dementia
(MID). To verify this result, applicant also demonstrated (infra)
that addition of -.DELTA..sup.12prostaglandin J.sub.2 to cultured
LLC-PK1 cells increases the rate of apoptosis.
[0063] The determination of apoptosis can be done in a variety of
ways such as a TUNEL assay, demonstration of a nucleosomal ladder
by agarose gel electrophoresis, and electron micrographic analysis
showing typical morphology of apoptosis.
[0064] The conclusion that apoptosis results from the presence of a
factor (i.e. prostaglandin D.sub.2 synthase) in the plasma of
patients suffering from Alzheimer's disease is based on observed
DNA degradation in nuclei of affected cells. The degree of
apoptosis was dose and time dependent, continually increasing up to
at least 8 h with renewed sampling of Alzheimer's plasma every 2 h.
The apoptotic ladder seen by agarose gel electrophoresis results
from the double-stranded endonucleolytic cleavage of DNA which
occurs at the linker regions of nucleosomes to produce fragments of
multiples of about 180 bp. This fragmentation of DNA appears
coincident with condensation of nuclear chromatin prior to cell
death and is considered a characteristic biochemical feature of
apoptosis (Y. Gavrieli et al., J. Cell. Biol. 119:493-501, 1992).
Demonstration of this repeat pattern was, therefore, used as an
indicator of apoptosis. The 3'OH ends of this cleaved DNA can also
serve as substrate for deoxynucleotidyl terminal transferase TdT,
which led to the development of TdT-mediated dUTP-biotin nick end
labeling (TUNEL) (Y. Gavrieli et al., J. Cell. Biol. 119:493-501,
1992). This technique results in the labeling of nuclei in-situ,
prior to the appearance of the ladder by gel electrophoresis. TUNEL
staining of DNA fragments occurs not only in histologically-defined
apoptotic cells but also in intact cells during the early stages of
apoptosis (Y. Gavrieli et al., J. Cell. Biol. 119:493-501, 1992).
Electron microscopy of the nuclei of LLC-PK1 cells shows
chromosomal fragmentation (nuclear condensation) upon exposure to
prostaglandin D.sub.2 synthase.
[0065] Prostaglandin D.sub.2 synthase may also be detected in an
enzymatic assay according to the method described by Urade et al.
(J. Bio. Chem. 270: 1422-1428; 1995).
[0066] The foregoing are merely exemplary diagnostic assays for
detection of prostaglandin D.sub.2 synthase in accordance with the
invention. Because it is the level of prostaglandin D.sub.2
synthase that is relevant, any other technique that effectively
measures prostaglandin D.sub.2 synthase is also included within the
scope hereof.
Treatment of Alzheimer's Disease and Renal Salt Wasting
Syndrome
[0067] Prostaglandin D.sub.2 synthase plays a role in the synthesis
pathway of .DELTA..sup.12prostaglandin J.sub.2. Arachidonic acid is
initially converted by cyclo-oxygenase to prostaglandin H.sub.2.
Prostaglandin D.sub.2 synthase, is an enzyme that converts
prostaglandin H.sub.2 to prostaglandin D.sub.2. Prostaglandin
D.sub.2 then spontaneously converts to .DELTA..sup.12prostaglandin
J.sub.2, presumably the biologically active metabolite of this
pathway.
[0068] Without intending to be bound by theory, it is believed that
the presence of prostaglandin D.sub.2 synthase in the urine and
blood of patients suffering from Alzheimer's disease or renal salt
wasting syndrome is an indication of an excess of this enzyme at
least in some regions of the patient's body which evidently results
in excess production of -.DELTA..sup.2prostaglandin J.sub.2. Along
this line, more prostaglandin H.sub.2 gets converted into
prostaglandin D.sub.2 which is spontaneously (i.e. immediately and
without the need of an enzyme) converted to prostaglandin J.sub.2
and then to -.DELTA..sup.12prostagland- in J.sub.2.
[0069] Applicant found that prostaglandin D.sub.2 synthase
increased apoptosis of human kidney proximal tubule cells in
culture. However, -.DELTA..sup.12prostaglandin J.sub.2 was the only
prostaglandin in the above pathway that induces apoptosis.
-.DELTA..sup.12Prostaglandin J.sub.2 increased apoptosis to the
same degree as prostaglandin D.sub.2 synthase. The addition of
prostaglandin D.sub.2 synthase and indomethacin, which inhibits
cyclo-oxygenase and reduces the prostaglandin synthesis downstream
did not increase apoptosis above baseline. The inhibition of
prostaglandin D.sub.2 synthase by N-Ethyl Maleimide inhibited
apoptosis. In addition, combination of indomethacin, prostaglandin
D.sub.2 synthase and -.DELTA..sup.12prostadlandin J.sub.2 increased
apoptosis. Furthermore, addition of -.DELTA..sup.12prostadlandi- n
J.sub.2 to indomethacin increased apoptosis. All these results
indicate that prostaglandin D.sub.2 synthase causes apoptosis by
helping to produce more of -.DELTA..sup.12prostaglandin J.sub.2.
Thus, the present invention seeks to reduce
-.DELTA..sup.12prostaglandin J.sub.2 levels.
[0070] Furthermore, it is believed that Alzheimer's disease is the
result of neuronal brain cells undergoing apoptotic cell death. It
is also believed that renal salt wasting syndrome might be the
result of apoptotic cell death by kidney tubule cells. Therefore,
inhibiting the rate of apoptosis which, at least in part, is caused
by elevated -.DELTA..sup.12prostaglandin J.sub.2 levels is expected
to be an effective treatment for both Alzheimer's disease and renal
salt wasting syndrome.
[0071] Accordingly, the present invention provides methods of (1)
treating or reducing risk of onset of renal salt wasting syndrome,
(2) inhibiting the rate of apoptosis, and (3) reducing the risk of
onset, or treating (e.g. by slowing the rate of progression of)
Alzheimer's disease. The methods inhibit the effect of, or reduce
the levels of -.DELTA..sup.12prostaglandin J.sub.2 levels.
Reduction of -.DELTA..sup.12prostaglandin J.sub.2 Activity
[0072] The reduction of -.DELTA..sup.12prostaglandin J.sub.2 levels
can be accomplished in a wide variety of ways, for example those
set forth below.
[0073] 1) Inhibiting the rate of synthesis of
-.DELTA..sup.12prostaglandin J.sub.2. This can be accomplished by
administering at least one agent selected from the group consisting
of cyclo-oxygenase inhibitor, cyclo-oxygenase antibody,
prostaglandin D.sub.2 synthase inhibitor, prostaglandin D.sub.2
synthase antibody. The cyclo-oxygenase inhibitor can be for example
indomethacin and prostaglandin D.sub.2 synthase inhibitor can be
N-ethyl maleimide.
[0074] 2) Increasing the rate of degradation or elimination of
-.DELTA..sup.12prostaglandin J.sub.2. This can be accomplished for
example by adding an agent that increases the rate of catabolism or
the rate of turnover of -.DELTA..sup.12prostaglandin J.sub.2.
[0075] 3) Administering to the subject an inhibitor of
-.DELTA..sup.12prostaglandin J.sub.2 (e.g. a receptor
antagonist).
Pharmaceutical Administration
[0076] In accordance with one aspect of the invention, once
Alzheimer's disease or renal salt wasting syndrome is diagnosed, at
least one agent selected from the group of cyclo-oxygenase
inhibitor, cyclo-oxygenase antibody, prostaglandin D.sub.2 synthase
inhibitor, prostaglandin D.sub.2 synthase antibody, and
.DELTA..sup.12prostaglandin J.sub.2 inhibitor, is(are) administered
at a dosage sufficient to reach the affected location (for example,
the brain or kidney) and reduce the rate of apoptosis. Non-limiting
examples of methods of administration and dosages which apply to
both treatment and prevention are detailed below. Dosages will be
the same when the invention is used prophylactically, preferably
for patients at higher risk than the general population of
acquiring the disease in question. Risk factors are known in the
art. As used herein, a "patient" may be a human or other mammalian
patient. Veterinary use of the inventions herein are
appropriate.
[0077] As used in the invention, any of the above-identified agents
may be administered with or without additional carrier or diluent
by the oral, systemic, percutaneous, transmucosal, or other typical
route. In a pharmaceutical composition for oral administration, an
agent as described above is preferably present in a concentration
between 5 and 99% by weight relative to total weight of the
composition, more preferably between 50 and 99 percent, especially
between 80 and 99 percent.
[0078] When prepared for percutaneous administration, an agent is
preferably present in a concentration between 2 and 20% by weight
relative to the total weight of the composition, more preferably
between 5 and 15%, especially between 5 and 10%.
Oral Administration
[0079] When administered by the oral route, the agent described
hereinabove may be formulated with conventional pharmaceutical
excipients, e.g. spray dried lactose and magnesium stearate, into
tablets or capsules for oral administration at concentrations
providing easy dosage in a range from 1 ng to 10 g, preferably,
from 1-10 mg per day per kg of body weight.
[0080] The active substance can be worked into tablets or dragee
cores by being mixed with solid, pulverulent carrier substances,
such as sodium citrate, calcium carbonate or dicalcium phosphate,
and binders such as polyvinyl pyrrolidone, gelatin or cellulose
derivatives, possibly by adding also lubricants such as magnesium
stearate, sodium lauryl sulfate, "Carbowax" or polyethylene glycol.
Of course, taste-improving substances can be added in the case of
oral administration forms. The active substance can be also
administered in solid dispersion state in appropriate carriers.
Such carriers may, for example, be chosen from the group consisting
of polyethylene glycols of molecular weight varying from 1,000 to
20,000 daltons and polyvinylpyrrolidone (e.g., Povidone from
American Chemicals Ltd., Montreal, Canada).
[0081] As further forms, one can use plug capsules, e.g. of hard
gelatin, as well as closed soft-gelatin capsules comprising a
softener or plasticizer, e.g. glycerine. The plug capsules contain
the active substance preferably in the form of granulate, e.g. in
mixture with fillers, such as lactose, saccharose, mannitol,
starches such as potato starch or amylopectin, cellulose
derivatives or highly dispersed silicic acids. In soft-gelatin
capsules, the active substance is preferably dissolved or suspended
in suitable liquids, such as vegetable oils or liquid polyethylene
glycols.
Topical Administration
[0082] For the treatment of conditions associated with apoptosis of
the skin, the preferred mode of administration is topical. Any
pharmaceutically acceptable base typically used in the art for
preparing formulations in the form of topical gels, ointments,
lotions, or the like may be used as the base. The agent described
above is preferably provided at a concentration of 0.001-10%, more
preferably 0.1-1% by weight of the total formulation. One to two
applications per day to the affected area are recommended.
Transdermal Delivery
[0083] When the composition of the present invention is formulated
as an ointment, lotion, gel, cream or the like, for transdermal
administration, the active compound is admixed with a suitable
carrier which is compatible with human skin or mucosa and which
enhances transdermal or transmucosal penetration of the compound
through the skin or mucosa. Suitable carriers are known in the art
and include but are not limited to Klucel HF and Glaxal base which
is available from Glaxal Canada Limited. Other suitable vehicles
can be found in Koller and Buri, S.T.P. Pharma 3(2), 115-124, 1987.
The carrier is preferably one in which the active ingredient(s)
is(are) soluble at ambient temperature at the concentration of
active ingredient that is used. The carrier should have sufficient
viscosity to maintain the precursor on a localized area of skin or
mucosa to which the composition has been applied, without running
or evaporating for a time period sufficient to permit substantial
penetration of the precursor through the localized area of skin.
The carrier is typically a mixture of several components, e.g.
pharmaceutically acceptable solvents and a thickening agent. A
mixture of organic and inorganic solvents can aid hydrophilic and
lipophilic solubility, e.g. water and an alcohol such as ethanol.
Desirably, the carrier is one which, if applied twice daily in an
amount providing 1 ng to 10 g, preferably 1 mg to 1 g, and more
preferably 100 mg 1 g of agent to the afflicted area, will provide
blood serum levels sufficient to reduce apoptosis in the effected
tissues.
[0084] The carrier may include various additives commonly used in
ointments, lotions, gels, and creams and well known in the cosmetic
and medical arts. For example, fragrances, antioxidants, perfumes,
gelling agents, thickening agents such as carboxymethylcellulose,
surfactants, stabilizers, emollients, coloring agents and other
similar agents may be present.
[0085] The lotion, ointment, gel or cream should be thoroughly
rubbed into the skin so that no excess is plainly visible, and the
skin would not be washed in that region until most of the
transdermal penetration has occurred, preferably, at least 15
minutes and, more preferably, at least 30 minutes after
application.
[0086] A transdermal patch may be used to deliver the composition
of the present invention in accordance with known techniques. It is
typically applied for a long period, e.g. 0.5 to 4 days, but
typically contacts active ingredients to a smaller surface area,
allowing a slow and constant delivery of active ingredient.
[0087] A number of transdermal drug delivery systems that have been
developed, and are in use, are suitable for delivering the active
ingredient of the present invention. The rate of release is
typically controlled by a matrix diffusion, or by passage of the
active ingredient through a controlling membrane.
[0088] Mechanical aspects of transdermal devices are well known in
the art, and are explained, for example, in U.S. Pat. Nos.
4,162,037, 5,154,922, 5,135,480, 4,666,441, 4,624,665, 3,742,951,
3,797,444, 4,568,343, 4,064,654, 5,071,644, 5,071,657, the
disclosures of which are incorporated herein by reference.
Additional background is provided by European Patent 0279982 and
British Patent Application 2185187.
[0089] The device may be any of the general types known in the art
including adhesive matrix and reservoir-type transdermal delivery
devices. The device may include drug-containing matrixes
incorporating fibers which absorb the active ingredient and/or
carrier. In a reservoir-type device, the reservoir may be defined
by a polymer membrane impermeable to the carrier and to the active
ingredient.
[0090] In a transdermal device, the device itself maintains active
ingredient in contact with the desired localized skin surface. In
such a device, the viscosity of the carrier for active ingredient
is of less concern than with a cream or gel. A solvent system for a
transdermal device may include, for example, oleic acid, linear
alcohol lactate and dipropylene glycol, or other solvent systems
known in the art. The active ingredient may be dissolved or
suspended in the carrier.
[0091] For attachment to the skin, a transdermal patch may be
mounted on a surgical adhesive tape having a hole punched in the
middle. The adhesive is preferably covered by a release liner to
protect it prior to use. Typical material suitable for release
includes polyethylene and polyethylene-coated paper, and preferably
silicone-coated for ease of removal. For applying the device, the
release liner is simply peeled away and the adhesive attached to he
patient's skin. In U.S. Pat. No. 4,135,480, the disclosure of which
is incorporated by reference, Bannon et al. described an
alternative device having a non-adhesive means for securing the
device to the skin.
Intravenous Injection
[0092] Sterile solutions can also be administered intravenously.
The active ingredient may be prepared at a final dose of 1 ng to 10
g, preferably 1 mg to 1 g per Kg of body weight as a sterile solid
composition which may be dissolved or suspended at the time of
administration using sterile water, saline, or other appropriate
sterile injectable medium. Carriers are intended to include
necessary and inert binders, suspending agents, lubricants,
flavorants, sweeteners, preservatives, dyes, and coatings.
Preferred Uses of the Invention
[0093] The invention is applicable to both diagnostic, prevention
and treatment purposes. A non-exclusive list of diagnostic uses is
set forth in column 1 of Table 1 below. Columns 2-4 set forth, for
each use, preferences regarding the manner in which certain
diagnostic tests may be varied for best results.
1TABLE 1 Preferred Diagnostic test/Detection of Preferred Preferred
Method of Prostaglandin D.sub.2 sythase Population Sample Detection
Renal Salt Wasting General, Urine or ELISA Syndrome especially
Plasma symptomatic patients Alzheimer's Disease Patients with Urine
or ELISA dementia Plasma
[0094] A non-exclusive list of treatment uses is set forth in
column 1 of Table 2 below. Columns 2-4 set forth, for each use,
preferences regarding the preferred pharmaceutical agent(s) to be
used, the dosage and the manner of admistration.
2TABLE 2 Pharmaceutical Treatment Agent Dosage Administration Renal
Salt Wasting 1) Cyclo-oxygenase 50 mg Oral, three times Syndrome
inhibitors such as daily indomethacin 2) Prostaglandin D.sub.2 240
mg Intravenous, once synthase daily monoclonal antibodies
Alzheimer's Disease 1) Postaglandin D.sub.2 240 mg Intravenous,
once synthase daily monoclonal antibodies.
Experimental Details
[0095] Patient Selection. Patients were randomly recruited at the
Division of Geriatric psychiatry, UMDNJ-Robert Wood Johnson Medical
School based on their willingness to participate in the study. All
subjects were examined by a board certified geriatric
neuropsychiatrist who established the diagnosis of dementia. The
bioassay was performed at Winthrop University Hospital. The
protocol for these studies was approved by the respective
institutional review boards of both institutions. Consent from
demented patients was obtained from their legal guardian on all
cases. Seventeen subjects with Alzheimer's disease met NINCDS-ADRDA
criteria for probable Alzheimer's disease (G. McKhann et al.,
Neurol. 34:939-944, 1984) and 11 multi-infarct dementia (MID)
subjects met DSM-IIIR criteria for the diagnosis of MID and had
Hachinski Ichemia Scale scores greater than 7 (American Psychiatric
Association. Diagnostic and Statistical manual of mental disorder.
4th edition (1994). Am. Psychiatric Assoc. Washington, D.C.
Dementia Work Group: Gary J. Tucker, Chairperson; V. C. Hachinski
et al., Arch. Neurol. 32:632-637, 1975). Nine subjects of the same
age and gender distribution served as normal controls (C). In
addition to the routine testing, all patients received either a CT
scan or magnetic resonance imaging of brain and Mini-Mental State
Examination (MMSE) score (M. F. Folstein et al., J. Psychiatric
Res. 12:189-198, 1975). Heparinized whole blood from all subjects
was centrifuged at 1500 g for 10 minutes at 4.degree. C. within 30
min. after collection; the plasma was then transferred to a new
plastic tube and stored at -70.degree. C. all samples were stored
at -70.degree. C. until time of bioassay, except during overnight
shipping on dry ice.
[0096] Cell Culture and Assay Protocol. LLC-PK1, a pig kidney
epithelial cell line was plated at a density of 10.sup.3 cells per
well into eight-well Permanox plastic chamber slides (NUNC,
Naperville, Ill.). The cells were cultured at 37.degree. C. in 5%
CO.sub.2 in humidified incubators and grown for 3 days to 70-80%
confluency in DMEM-F12 that was supplemented with 10% fetal calf
serum, 7.5% sodium bicarbonate, 15 mM HEPES, 200 mM L-Glutamate,
100 u penicillin and 0.1 ug/ml streptomycin (Life Technologies,
Gaithersburg, Md.). The culture fluid was then removed and cells
were exposed to plasma from control individuals, Alzheimer's
disease patients or multi-infarct dementia patients diluted 1:5 in
fresh DMEM-F12 media, supplemented as above, for 2 h at 37.degree.
C. The cells were then rinsed in PBS, and fixed in 4% formaldehyde
in PBS for 10 min., permeabilized with 0.5% Triton X-100 (Sigma
Chemical CO., St. Louis, Mo.) for 5 min. and washed in 4 changes of
distilled water. A positive control was obtained by exposing cells
to 0.6 mM H.sub.2O.sub.2 diluted in DMEM-F12 for 2 h.
[0097] Apoptosis Assay (TUNEL): Nuclear DNA fragmentation
consistent with apoptosis was determined by the method of
TdT-mediated dUTP-biotin nick-end labeling (TUNEL) (Y. Gavrieli et
al., J. Cell. Biol. 119:493-501, 1992). The ApopDetek cell death
assay kit (Enzo, Farmingdale, N.Y.) was used utilizing terminal
deoxynucleotide transferase to incorporate Bio-16-dUTP onto the
3'-OH termini in the DNA of apoptotic cells, subsequent binding
with streptavidin-horseradish peroxidase, and visualization after
conversion of the substrate and chromagen (hydrogen peroxide and
aminoethylcarbazole) into a localized brick red precipitate. A blue
counter stain was also used. Slides were then observed for
morphologically irregular and condensed nuclei which contain dark
red precipitate to indicate TUNEL-positive cells using a Nikon
(Nikon, Inc., Melville, N.Y.) Optiphot microscope. Five to six
random field totaling approximately 1,000 to 1,500 cells were
counted per slide. Apoptotic index (AI), defined as the percent of
cells undergoing apoptosis, is calculated by dividing the number of
positive nuclei by the total number of nuclei counted multiplied by
100.
[0098] Dose and Time-Response Studies. The TUNEL assay was
performed in LLC-PK1 cells that were exposed to different dilutions
of plasma of patients with Alzheimer's disease and control plasma
at different intervals of time. Alzheimer's disease and control
plasma were diluted with DMEM-F12 at 1:100, 1:20, 1:10; 1:5, 1:3
and 1:2 and added to 70-80% confluent LLC-PK1 cells for 2 h;
conversely, Alzheimer's disease and control plasma were diluted 1:5
with DMEM-F12 and exposed to LLC-PK1 cells for 60, 90, 120 and 180
min. The selection of 2 h exposure in the dilution studies and 1:5
dilution of plasma in the time response studies were based on
maximum apoptotic index noted with the respective studies.
[0099] Electron Microscopy. LLC-PK1 cells were plated at 10.sup.3
cells per 35 mm plastic petri dish, exposed to Alzheimer's disease
or control plasma at 1:5 dilution in DMEM-F12 for 2 h and fixed
with 2.5% glutaraldehyde in 0.1M sodium cacodylate, pH 7.2, for 1 h
at 4.degree. C. The cells were then postfixed in 1% buffered osmium
tetroxide, dehydrated in a graded series of ethanol, and embedded
in LX112 (Ladd Research Industries, Burlington, Vt.). En fac and
cross-sectional thin sections were stained with uranyl acetate and
lead citrate and examined on a Zeiss EM10 transmission electron
microscope.
[0100] DNA Ladder Assay. DNA ladder was observed using a
modification of the procedure described by Eastman (Eastman, A.
"Assays for Features Associated with Apoptosis" in Meth. Cell Biol.
46:41-55 edited by L. M. Schwartz and B. A. Osborne, Academic
Press). LLC-PK1 cells (10.sup.6) were seeded into T75 flasks
(Falcon) containing 10 ml of DMEM-F12--10% fetal calf serum
supplemented with 0.12% NaHCO.sub.3, 5 mM glutamine, 15 mM HEPES
and 1% pen/strep. Cells were allowed to attach overnigh at
37.degree. C. in 5% humidified CO.sub.2. Five ml of medium were
withdrawn and 0.5 ml of test plasma added. Cells in the medium and
adherent cells (0.05% trypsin in 0.53 mM EDTA, GIBCO, 3 min.,
37.degree. C.) were harvestedon days 2, 3, 4, and 5 by
centrifugation at 142.times.g for 3 min. at room temperature. The
cellpellet was warmed to 50.degree. C. for 2-3 min. and resuspended
in 2% Sea Plaque agarose (FMC, Rockland, Me.) in 0.125 M EDTA pH
7.4, and dispensed into a precooled (4.degree. C.) mold. The
agarose plugs were incubated at 50.degree. C. for 2 h in 0.5 M EDTA
pH 8.0, 1% sarcosine (Sigma), and 1 mg/ml of proteinase K
(Boehringer Mannheim). Plugs were then incubated at 37.degree. C.
for 30 min. in 10.times. volume of 10 mM TrisHCl pH 7.5, 50 mM
EDTA. The buffer was exchanged with TE (10 mM TrisHCl pH 7.5, 1 mM
EDTA), RNase A, previously boiled for 15 min (Sambrook, J., E. F.
Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual",
Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press,
1989), was added to a final concentration of 250 .mu.g/ml, and the
plugs incubated an additional 50 min. at 37.degree. C. DNA in the
plugs was subjected to electrophoresis through a 2% SeaKem (FMC)
agarose gel in TAE buffer (Sambrook, J., E. F. Fritsch, and T.
Maniatis, "Molecular Cloning: A Laboratory Manual", Cold Spring
Harbor, N.Y., Cold Spring Harbor Laboratory Press, 1989) at 2V/cm
for 14 h and visualized as described (Eastman, A. "Assays for
Features Associated with Apoptosis" in Meth. Cell Bio. 46:41-55
edited by L. M. Schwartz and B. A. Osborne, Academic Press).
[0101] Partial Protein Purification and Heating of Plasma. Pooled
plasma from Alzheimer's disease and control subjects were dialyzed
in a 10 kDa m.w. cut-off membrane in 20 mM phosphate buffer, pH
7.1, and centrifuged at 13,000 g for 15 min. The clear supernatant
was loaded into 10 ml of an Affi-Gel Blue Gel affinity column
(Bio-Rad Laboratories, Hercules, Calif.). The column was then
washed with loading buffer until protein levels were not
detectable, followed by the sequential elution with 0.5 M and 2 M
NaCl in buffer. Protein concentration was monitored by UV
absorbance at 280 nm. The three protein fractions (load and wash,
0.5 M NaCl and 2 M NaCl) were dialyzed in a 10 kDa m.w. cut-off
membrane, concentrated over a bed of PEG 8000 in a 1 kDa m.w.
cut-off membrane and dialyzed in a 10 kDa m.w. cut-off membrane
against 10 mM phosphate buffer, pH 7.1. Cultured LLC-PK1 cells were
then exposed to 30-100 ug of the two pooled fractions for 2 h at
37.degree. C. and a TUNEL assay performed.
[0102] In separate experiments, Alzheimer's disease and control
plasma were heat-treated at 56.degree. C. for 30 min. In some
experiments, plasma was boiled at 100.degree. C. for 5 min. and the
denatured protein aggregates removed by sedimentation at 1,000 g
for 1 min. prior to testing by TUNEL assay as noted above. In
separate experiments, Alzheimer's disease plasma was alternately
frozen at -70.degree. C. and thawed to room temperature at least 3
times and a TUNEL assay performed in LLC-PK1 cells after a 2 h
exposure to a 1:5 dilution of the plasma with DMEM-F12 at
37.degree. C.
[0103] Isoelectric Focusing. The active fraction from the Affi-Gel
Blue-Gel run (0.5 M NaCl) was further fractionated by isoelectric
focusing (IEF) using Fotofor (Bio-Rad). This active fraction was
run at a pH gradient of 3-10, using Bio-Lyte ampholyte 3/10, at a
constant power of 15 W at 4.degree. C. for 4 h. Fractions were
pooled according to their protein profile and assayed for apoptotic
activity. Fractions with highest apoptotic index were pooled,
dialyzed and refractionated by IEF using a narrow pH gradient of
4-6, at the same settings, utilizing Bio-Lyte ampholyte 4/6 and
3/10, 80:20%, respectively (Bio-Rad Laboratories, Hercules,
Calif.).
[0104] Effect of Protein Synthesis on Apoptotic Activity. LLC-PK1
cells were exposed to Alzheimer's disease and control plasma in the
absence and presence of cycloheximide (0.2-200 uM) (Sigma, St.
Louis, Mo.). Apoptotic index was measured in these cells by TUNEL
assay.
[0105] Effect of Calcium Depletion on Factor Activity. The TUNEL
assay was performed in the usual manner except for substituting
DMEM-F12 with calcium-free DMEM, supplemented with dialyzed 10%
fetal calf serum (Life Technologies, Grand Island, N.Y.) and 0.6 mM
EGTA to chelate calcium. Dialyzed Alzheimer's disease and control
plasma were then added to the calcium-free media for 2 h at
37.degree. C. and a TUNEL assay performed.
[0106] Contribution of Known Apoptotic Inducers. To test the
possibility that the apoptotic factor in Alzheimer's disease plasma
was .beta. amyloid, TNF-.alpha. or myeloma light chain, the TUNEL
assay was repeated as above after a 2 h incubation at 37.degree. C.
with 0.10-50 mM .beta. amyloid (Peninsula Laboratories, Inc.,
Belmont, Calif.), 5 pM-3 nM TNF-.alpha. (Quantikine, Minneapolis,
Minn.) and 3-60 ug .lambda. myeloma light chain, kindly supplied by
Dr. Vecihi Batuman, Tulane University School of Medicine, New
Orleans, La. To eliminate the possibility that a protease in
Alzheimer's disease plasma is responsible for the apoptotic
activity, the effect of a broad spectrum protease inhibitor
cocktail (Boehringer Mannheim) was studied on apoptotic index using
the TUNEL assay. LCC-PK1 cells were incubated with Alzheimer's
disease plasma with and without the inhibitor cocktail and assayed
as detailed above. This cocktail inhibits a large spectrum of
serine, cysteine, and metalloproteases as well as calpains. It
consists of aprotinin, leupeptin, EDTA, and pefabloc.
[0107] Statistical Analysis. All TUNEL assays were performed in
triplicate and the data expressed as the mean.+-.SEM. An unpaired
Student's T test was used to compare one set of experiments from
the other and a P<0.05 was deemed significant. A multivariate
analysis was used to determine whether any medications taken by the
patients might affect the results or if there was a correlation
between apoptotic index and MMSE score.
EXAMPLE 1
A Factor in the Plasma of Patients with Alzheimer's Disease Causes
Apoptosis; Partial Purification and Characterization of the
Factor
[0108] Electron microscopy of LLC-PK1 cells after incubation with
Alzheimer's disease plasma (see Experimental Details) illustrates
the distinct pattern of apoptotic cells. Apoptotic cells have
condensed, black nuclei and some cells are noted to be shrunken and
engulfed by neighboring cells, FIG. 1. Light microscopic view of
these cells that had been labeled in situ by TUNEL method are
depicted in FIG. 2.
[0109] Table 3 summarizes the results of the exposure of LLC-PK1
cells to plasma from control individuals, patients with Alzheimer's
disease and multiple infarct dementia. There was a nearly fourfold
increase in apoptotic index in LLC-PK1 cells that were exposed to
Alzheimer's disease plasma (25.6.+-.8.8%) as compared to control
plasma (6.0.+-.2.4%), P<0.001, and multiple infarct dementia
plasma (6.5.+-.2.3%), p<0.001. There was no significant
difference in apoptotic index between control plasma and multiple
infarct dementia plasma, p>0.05. As noted in FIG. 3B, apoptotic
index increased progressively as the time of incubation with
Alzheimer's disease plasma increased, peaking at 2 h with an
apoptotic index of 16.1.+-.0.3%. Diluting Alzheimer's disease
plasma in a range of 1:2 to 1:100 revealed a maximum apoptotic
index of 12.4.+-.0.2% at 1:5 dilution of plasma with medium (FIG.
3A). There was no correlation between apoptotic index and the
medications the patients had been taking at the time of study or
the MMSE scores in Alzheimer's disease.
3TABLE 3 Apoptotic index in LLC-PK1 cells exposed to control,
Multi-Infarct dementia, and Alzheimer's disease patient's plasma AI
(%) Alzheimer's Plasma 25.6 .+-. 8.8 MID Plasma 6.5 .+-. 2.3
Control Plasma 6.0 .+-. 2.4
[0110] FIG. 4 shows internucleosomal DNA cleavage in LLC-PK1 cells
that had been exposed to plasma from patients with Alzheimer's
disease (AD) for intervals of 2, 3, 4, and 5 days. Maximum DNA
fragmentation was 4 days after exposure to AD plasma and showed the
characteristic 180 bp spacing. Control (NC) plasma did not exhibit
a ladder even after incubation for 5 days.
[0111] As noted in Table 4, elimination of calcium from the
incubating medium, fetal bovine serum and plasma or incubation with
200 uM cycloheximide resulted in inhibition of apoptosis by
Alzheimer's disease plasma, suggesting that apoptosis in the system
is dependent on the level of extracellular calcium and protein
synthesis. There was no inhibition of apoptosis at the lower
concentrations of cycloheximide. In a separate group of
experiments, heating Alzheimer's disease and control plasma at
56.degree. C. for 30 min., which deactivated complement, did not
alter the apoptotic activity, table 4. However, boiling the
Alzheimer's disease plasma at 100.degree. C. for 5 min. resulted in
AI that was not different from control plasma. Moreover, freezing
and thawing the plasma from -70.degree. C. to room temperature at
least three times decreased apoptotic activity, table 4. These data
suggest that the apoptotic factor in Alzheimer's disease plasma is
a protein.
4TABLE 4 Characteristics of Apoptotic Factor Alzheimer's Plasma
Control Plasma AI (%) AI (%) No treatment 25.6 .+-. 8.8 6.0 .+-.
2.4 56.degree. C., 30 min. 19.5 .+-. 2.7 8.3 .+-. 2.5 100.degree.
C., 5 min. 4.7 .+-. 2.2 8.7 .+-. 1.2 Cycloheximide (200 uM) 7.7
.+-. 1.6 6.3 .+-. 1.5 (0.2-20 uM) 28.7 .+-. 4.0 5.0 .+-. 0.0
Ca.sup.++ free medium 6.3 .+-. 1.6 4.0 .+-. 0.7 Freeze and thaw 6.0
.+-. 2.4 5.3 .+-. 1.5
[0112] To exclude the possibility that the factor is
.beta.-amyloid, cells were incubated with 0.1, 10 and 50 uM of
.beta.-amyloid dissolved in media. No detectable apoptotic activity
was observed even at 50 uM, AI=6.1.+-.3.1%. To eliminate the
possibility that TNF-.alpha. might be the apoptotic factor, control
and Alzheimer's disease plasma were quantified for the presence of
TNF-.alpha. by ELISA (Quantikine). The levels of TNF-.alpha. in
control and Alzheimer's disease plasma were less than the lowest
level of detection by the ELISA kit of 0.3 pM. We achieved a
standard curve with the ELISA with TNF-.alpha. standards and
blocked the reaction, utilizing TNF-.alpha. antibody. We also
tested the effect of TNF-.alpha. on LLC-PK1 cells at 5, 50, 500,
and 3000 pM for 2 h and found that doses as high as 50 pM yielded
background levels of apoptosis, AI=6%. On a Western blot both the
control and Alzheimer's disease plasma resulted in no signal, while
a positive control of 100 ng TNF-.alpha. yielded a positive signal.
A similar situation occurred with interleukin-1.beta.. Both control
and Alzheimer's disease plasma had undetectable levels of
interleukin 1.beta. by ELISA.
[0113] FIG. 5 depicts the protein profile from each individual step
of purification on a Affi-Gel-Blue-Gel column. The highest AI of
21% was found in the 0.5 M NaCl eluate. No activity was found in
the load and wash fraction and only a modest activity was noted in
the 2M NaCl eluate, AI of 6% vs. 9%, respectively. The 2M NaCl
eluate was mainly composed of albumin. The active fraction (0.5 M
NaCl eluate) was dialyzed overnight in a 10 kDa m.w. cut-off
membrane at 4.degree. C. against 10 mM phosphate buffer, pH 7.1 to
remove salt. Isoelectric focusing was performed on this protein
fraction at a pH gradient of 3-10, FIG. 6A. Fractions within
clearly defined protein peaks were pooled and dialyzed to remove
ampholyte, followed by a TUNEL assay to monitor for apoptotic
activity. Dialysis with a 10 kDa m.w. cut-off membrane demonstrated
retention of apoptotic activity in the dialysis bag, suggesting
that the size of the protein exceeded 10 kDa. The highest AI of
29.4% was noted in fraction 2 of the pooled samples and isoelectric
focusing repeated only on this active fraction at a narrow range pH
gradient of 4-6, FIG. 6B. The active fraction with an AI of 22% was
noted in fraction 2 of this additional purification step. The pI
range of both fractions was 4.7-5.5.
EXAMPLE 2
Isolation and Identification of Prostaglandin D.sub.2 Synthase
[0114] Eleven liters of urine were collected from a patient
suffering from renal salt wasting syndrome. The protein was
precipitated from the urine with 80% ammonium sulfate and
centrifuged to get a pellet. A portion of the pellet was dissolved
in 25 mM Tris.HCl at pH 7.5 and then dialyzed overnight in the same
buffer in a 10 kDa cutoff membrane. The dialyzed proteins were then
loaded onto a High-Trap Q Sepharose column. The proteins were
eluted off this column with 0.5M NaCl and 1.0 M NaCl in several
fractions. These fractions were then dialyzed in a phosphate buffer
at pH 7.1. Subsequently the fractions were assayed for their
ability to induce apoptosis (see Experimental Details) and the
activity was found in the 0.5M NaCl. Following this, isoelectric
focusing (see Experimental Details) was performed from pH 3 to 10
and fractions were collected. The active fraction (pH 4.8-5.5) was
further purified by HPLC-C.sub.18 column. The active fraction from
this column was found in a single peak. The active fraction was
placed on SDS PAGE gel and proteins with molecular weight of 29,
32, 33, and 42 Kd were eluted from the gel and assayed for
activity. The activity was found to be associated with the 32 Kd
band.
[0115] Following the above procedure, the 32 Kd band was sequenced
and found to contain 2 proteins, one of which is .alpha..sub.1
microglobulin. Since .alpha..sub.1 microglobulin was found to have
no apoptotic activity, it was absorbed on a protein A column to
which the .alpha..sub.1 microglobulin-specific antibody was
attached. The result was a pure 23-29 kD.sub.2 band protein as seen
on an SDS PAGE gel. The single 23-29 kD.sub.2 protein was
transferred from SDS-PAGE gel to a protein sequencing membrane and
sequenced. With two separate analyses based on the first 20 N
terminal amino acids, the apoptotic factor was positively
identified as prostaglandin D.sub.2 synthase which sequence was
described by Nagata et al. (Proc. Natl. Acad. Sci. USA,
88:4020-4024; 1991) .
EXAMPLE 3
Modulation of the Synthesis of -.DELTA..sup.12Prostaglandin
J.sub.2
[0116] Prostaglandin D.sub.2 synthase is an enzyme involved in the
-.DELTA..sup.12Prostaglandin J.sub.2 synthesis pathway. The
following is an illustration of this pathway. 1
[0117] Prostaglandin D.sub.2 Synthase increased apoptosis of human
kidney proximal tubule cells in culture about four times above
control (see Experimental Details). To find out which prostaglandin
is responsible for inducing apoptosis, the different prostaglandins
were tested for their ability to induce apoptosis in kidney
proximal tubule cells. -.DELTA..sup.12Prostaglandin J.sub.2 was
found to be the only prostaglandin listed above that induces
apoptosis. It induced apoptosis to the same degree as Prostaglandin
D.sub.2 Synthase. Also, Prostaglandins E, H and D did not increase
apoptosis.
[0118] Adding Indomethacin, which blocks cyclo-oxygenase reduces
the prostaglandins downstream and inhibited apoptosis to baseline.
Furthermore, the simultaneous addition of Indomethacin and
Prostaglandin D.sub.2 Synthase did not increase apoptosis above
baseline. In addition, the deactivation of Prostaglandin D.sub.2
Synthase by N-Ethyl Maleimide inhibited apoptosis. The combination
of Indomethacin, Prostaglandin D.sub.2 Synthase and
-.DELTA..sup.12Prostaglandin J.sub.2 increased apoptosis and so did
the addition of .DELTA..sup.12Prostaglandin J.sub.2 to Indomethacin
increased apoptosis.
[0119] In combination, these results indicate that prostaglandin
D.sub.2 synthase increases apoptosis by increasing the production
of prostaglandin D.sub.2 which necessarily results in the
production of -.DELTA..sup.12Prostaglandin J.sub.2. It is therefore
the activity of -.DELTA..sup.12prostaglandin J.sub.2 that clinical
techniques should seek to reduce. Indirect methods such as reducing
activity of prostaglandin D.sub.2 synthase or reducing any
"upsteam" synthesis of -.sup.12prostaglandin J.sub.2, or a
precursor thereto, is quite useful. Increased catabolism of
-.DELTA..sup.12prostaglandin J.sub.2, its upstream precursors, or
enzymes involved in its synthesis is also expected to be effective.
Naturally, direct inhibition of -.DELTA..sup.12prostaglandin
J.sub.2 activity (e.g. using a -.DELTA..sup.12prostaglandin
inhibitor, e.g. a receptor antagonist) may also provide therapeutic
effect.
EXAMPLE 4
Production of Antibodies to Prostaglandin D.sub.2 Synthase
[0120] EST's homologous to mRNA for the glutathione independent
PGD2S were obtained from ATCC and assembled into a full-length cDNA
and a premature stop codon mutation corrected. The full-length cDNA
was inserted into a bacterial expression vector, pMAL-C2, joining
the vector encoded carrier protein to the PGD2S coding sequence at
the signal peptidase cleavage site. Purified recombinant fusion
protein was purified by affinity chromatography, cleaved with
factor Xa, and the carrier protein separated from the recombinant
factor by ion exchange chromatography. One milligram of purified
recombinant factor was mixed with Titermax adjuvant and injected
intradermally in a New Zealand white rabbit. Five weeks later, the
rabbit was boosted with another milligram of recombinant factor in
adjuvant and serum collected 10 days later. Polyclonal antisera
from this rabbit was able to detect 1 nanogram of reduced
recombinant factor in a Western blot. This antisera also reacted in
a Western blot with PGD2S from a natural source and which had the
same MW as described in the literature.
[0121] Although the present invention has been described in
relation to particular embodiments thereof, many other variations
and modifications and other uses will become apparent to those
skilled in the art. The present invention therefore is not limited
by the specific disclosure herein, but only by the appended
claims.
* * * * *