U.S. patent application number 10/136807 was filed with the patent office on 2003-01-09 for treatment of diseases involving cyst formation.
Invention is credited to Joly, Alison, Schreiner, George F., Stanton, Lawrence W., White, R. Tyler.
Application Number | 20030008864 10/136807 |
Document ID | / |
Family ID | 26810615 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008864 |
Kind Code |
A1 |
Schreiner, George F. ; et
al. |
January 9, 2003 |
Treatment of diseases involving cyst formation
Abstract
The invention concerns the use of ligands of peripheral-type
benzodiazepine receptors (PTBR) in the diagnosis and treatment of
diseases involving cyst formation and in particular polycystic
kidney disease. The invention further concerns the treatment of
hypertension accompanying polycystic kidney disease, and
pharmaceutical compositions and articles of manufacture for the
treatment or diagnosis of the target disease or condition.
Inventors: |
Schreiner, George F.; (Los
Altos Hills, CA) ; Joly, Alison; (San Mateo, CA)
; Stanton, Lawrence W.; (Redwood City, CA) ;
White, R. Tyler; (Fremont, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
26810615 |
Appl. No.: |
10/136807 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10136807 |
Apr 30, 2002 |
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09461910 |
Dec 15, 1999 |
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6380183 |
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60113008 |
Dec 18, 1998 |
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60136208 |
May 26, 1999 |
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Current U.S.
Class: |
514/221 ;
514/449 |
Current CPC
Class: |
G01N 33/948 20130101;
A61K 31/5513 20130101; A61K 31/5513 20130101; A61K 45/06 20130101;
A61P 13/12 20180101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 38/212 20130101; A61K 38/212 20130101 |
Class at
Publication: |
514/221 ;
514/449 |
International
Class: |
A61K 031/5513; A61K
031/337 |
Claims
What is claimed is:
1. A pharmaceutical composition for the treatment of a disease
involving cyst formation, comprising an effective amount of a
ligand of a peripheral-type benzodiazepine receptor (PTBR) in
admixture with pharmaceutically acceptable carrier.
2. The composition of claim 1 wherein said disease is a polycystic
kidney disease (PKD).
3. The composition of claim 2 wherein said composition additionally
comprises taxol or a taxol derivative.
4. An article of manufacture comprising a container, an effective
amount of a ligand of a peripheral-type benzodiazepine receptor
within said container, and a label or package insert with
instructions for administering said ligand for the treatment of a
disease involving cyst formation.
5. The article of manufacture of claim 4 wherein said disease is a
polycystic kidney disease.
Description
FIELD OF THE INVENTION
[0001] The present invention concerns the treatment of diseases
involving cyst formation, such as polycystic kidney disease. The
present invention also concerns various endogenous and exogenous
ligands of peripheral-type benzodiazepine receptors, and in
particular, their use in the prevention or treatment of cyst
formation.
BACKGROUND OF THE INVENTION
[0002] A. Diseases Involving Cyst Formation
[0003] There are several human diseases that result in the
formation of cysts containing either semi-solid or fluid material.
Benign cysts can occur, for example, in the ovary, spleen, lungs,
kidney and liver, where they are often hereditary. Cysts can be
acquired, as in diverticulosis of the intestines, or acquired as a
secondary cause of an inherited disease, as in cystic fibrosis, or
can be directly inherited, as in polycystic disease of the kidney,
which can also affect the liver and brain.
[0004] Renal cysts arise in the renal parenchyma, and begin as
dilations or outpouchings from existing nephrons or collecting
ducts or from the developmental counterparts of these structures.
Renal cysts contain a fluid that presumably derives from their
parent nephron and/or is a local secretion. They may be hereditary,
developmental, or acquired, and may occur in the cortex, medulla or
both, and may or may not be associated with other renal or systemic
abnornalities. For further details see, for example, Brenner &
Rector, The Kidney, Fourth Edition, 1991, Vol. I, pp.
1657-1659.
[0005] Polycystic kidney disease (PKD) is a subset of renal cystic
disorders in which cysts are distributed throughout the cortex and
medulla of both kidneys. PKD is usually the hallmark of a unique
autosomal dominant (autosomal dominant polycystic kidney disease,
ADPKD) or autosomal recessive (autosomal recessive polycystic
kidney disease, ARPKD) disorder but may also be found in
association with a variety of clinical conditions or acquired at
some point of life by a patient with an underlying, noncystic renal
disease. PKD is the most prevalent hereditary renal disorder,
accounting for over 5 percent of patients on chronic
hemodialysis.
[0006] ADPKD, the most common dominantly inherited kidney disease
usually appears in midlife, and is characterized morphologically be
massive cyst enlargement, moderate interstitial infiltration with
mononuclear cells, and extensive fibrosis. Characteristic symptoms
include proteinuria, abdominal pain and palpable kidneys, followed
by hematuria, hypertension, pyuria, uremia and calculi. In about
15% of patients, death is due to cerebral aneurysm. ADPKD is caused
by mutations in one of three genes: PKD1 on chromosome 16 accounts
for approximately 85% of cases whereas PKD2 on chromosome 4
accounts for approximately 15%. Mutations in the so far unmapped
PKD3 gene are rare. (Reeders et al, Nature 317:542-544 [1985];
Kimberling et al, Genomics 18:467-472 [1993]; Daoust et al,
Genomics 25:733-736 [1995]; Koptides et al., Hum. Mol. Genet.
8:509-513 [1999]).
[0007] ARPKD is a rare inherited disorder which usually becomes
clinically manifest in early childhood, although presentation of
ARPKD at later ages an survival into adulthood have also been
observed in many cases. ARPKD was first studied in C57BL/6J mice in
whom it arises spontaneously (Preminger et al., J. Urol.
127:556-560 [1982]). The cpk mutation characteristic of this
disease has been mapped to mouse chromosome 12 (Davisson et al.,
Genomics 9:778-781 [1991]). The gene responsible for ARPKD in
humans has been mapped to chromosome 6p. More recently, fine
mapping of the autosomal recessive polycystic kidney disease locus
(PKHD 1) has been reported (Mucher et al., Genomics 48:40-45
[1998]).
[0008] It has been reported that taxol and taxol derivatives
inhibit the progression of PKD and prolongs the survival of
polycystic cpk mice (Woo et al, Nature 368:750-753 [1994]; PCT
publication WO 94/08041; U.S. Pat. No. 5,882,881). Since taxol
specifically induces the expression of TNF-.alpha. in macrophages
and lymphocytes, it has also been suggested that TNF-.alpha. is
useful in the treatment of PKD (U.S. Pat. No. 5,750,495).
[0009] In APKD, the renal cysts remain small for 30-40 years. They
then start to expand, progressively replacing normally functioning
renal parenchyma. Factors involved in cyst expansion include loss
of epithelial differentiation, increased proliferation and
apoptosis, secretion of chloride and other ions into the cyst fluid
and the development of inflammation around the outer circumference
of the cyst wall (Grantham, J, Am J. Kid. Dis. 28:788-803
[1996]).
[0010] There is a need for the identification of endogenous and
exogenous factors that are suitable for the prevention and
treatment of diseases involving cyst formation and cyst expansion.
In view of the severity and frequency of occurrence of PKD, there
is a particular need for finding therapeutic agents useful in the
prevention and treatment of this disease.
[0011] B. Ligands of Peripheral-Type Benzodiazepine Receptors
(PTBR)
[0012] Ligands of PTBR's have been known for many years and
anti-anxiety CNS effects of PTBR
[0013] agonists (e.g. Valium) are widely known. With respect to
benzodiazepine receptors outside the CNS (PTBR) most of what is
known concerns the role of such receptors in mediating muscle
relaxation, particularly smooth muscle relaxation. Vagal tone has
been found to decrease following intravenous administration of
diazepam. (Adinoff et al., Psychiatry Research 41:89-97 [1992]).
There is evidence for control of cardiac vagal tone by
benzodiazepine receptors (DiMicco, Neuropharmacology 26:553-559
[1987]). PTBR ligands Ro5-4864 and PK1195, but not diazepam, have
been described to depress cardiac function in an isolated working
rat heart model (Edoute et al., Pharmacology 46:224-230 [1993]).
Ro5-4864 has also been reported to increase coronary flow in an
isolated perfused Langendorf rat heart without affecting heart rate
and left ventricular contractility. PK11195 did not antagonize this
vasodilatory effect (Grupp et al., Eur. J. Pharm. 143:143-147
[1987]). In an isolated rat heart preparation, diazepam induced a
transient negative inotropic effect followed by a positive
inotropic response. The positive inotropy was antagonized by
PK11195. (Leeuwin et al., Eur. J. Pharm. 299:149-152 [1996]).
Diazepam increased contractile force in Langendorf rat heart.
(Leeuwin et al., Arch. Int. Pharmacodyn. 326:5-12 [1993]). Ro5-4864
has been shown to have a small (20%) depressant effect on the
contraction amplitude (negative inotropic effect) of human atrial
strips that was no antagonized by PK11195 (Shany et al., Eur.J.
Pharm. 253:231-236 [1994]). In a guinea pig heart preparation
Ro5-4864 decreased the duration of intracellular action potential
and contractility. Diazepam was less effective and clonazepam
ineffective. The effects of Ro5-4864 were reversed by PK11195 but
not by a specific antagonist of the CNS BZR. (Mestre et al., Life
Sciences 35:953-962 [1984]). The presence of PTBR binding sites in
the hearts of dogs and humans was demonstrated in vivo by positron
emission tomography using [.sup.11C]-PK11195. (Charmonneau et al.,
Circulation 73:476-483 [1986]). It has also been reported that
Ro5-4862 and dipyridamole can compete [.sup.3H]diazepam binding to
heart tissue. Diazepam potentiates the actions of adenosine on
isolated cardiac and smooth muscle and the coronary vasodilator
action of adenosine in dogs. There is evidence that diazepam may be
acting in a similar manner to dipyridamole by inhibiting adenosine
uptake. (Davies and Huston, Eur. J. Pharm. 73:209-211 [1981]).
[0014] More recently, PTBR's have been shown to play a role in cell
pathways underlying apoptosis. PTBR's expressed on mitochondria
serve as docking receptors for Bcl.sub.2, a protein that inhibits
apoptosis. The biological pathways in apoptosis modulated by PTBR
ligand interactions are not specifically known.
SUMMARY OF THE INVENTION
[0015] The present invention is based on the recognition that
ligands that interact with the PTBR's are useful in the treatment
of diseases associated with cyst formation, and in particular, slow
down or prevent the progression of polycystic kidney disease (PKD)
to renal failure, and/or slow down or prevent the accompanying
tendency toward hypertension.
[0016] In one aspect, the invention concerns a method for the
treatment of a disease, involving cyst
[0017] formation, comprising administering to a patient having or
at risk of developing such disease an effective amount of a ligand
of a peripheral-type benzodiazepine receptor (PTBR). The patient is
preferably mammal, more preferably human. In a particular
embodiment, the disease to be treated is polycystic kidney disease
(PKD). In a preferred embodiment, the administration of a PTBR
ligand prevents or slows down the progression of PKD. In another
preferred embodiment, the administration of a PTBR ligand prevents
or slows down the development of a symptom of PKD, such as,
hypertension associated with PKD, bleeding into the cyst, or pain
associated with cyst expansion.
[0018] In a further aspect, the invention concerns a method for the
treatment of progressive renal insufficiency associated with the
progression of cystic disease.
[0019] In another aspect, the invention concerns a method for the
treatment of hypertension
[0020] accompanying polycystic kidney disease (PKD) comprising
administering to a patient an effective amount of a ligand of a
peripheral-type benzodiazepine receptor (PTBR).
[0021] In yet another aspect, the invention concerns a
pharmaceutical composition for the treatment of a disease involving
cyst formation or cyst expansion, comprising an effective amount of
a ligand of a peripheral-type benzodiazepine receptor (PTBR) in
admixture with a pharmaceutically acceptable carrier.
[0022] In a further aspect, the invention concerns article of
manufacture comprising
[0023] a container,
[0024] an effective amount of a ligand of a peripheral-type
benzodiazepine receptor (PTBR) within the container,
[0025] and
[0026] a label or package insert with instructions for
administering the ligand for the treatment of a disease involving
cyst formation.
[0027] In all aspects, the disease to be treated preferably
polycystic kidney disease (PKD), including both autosomal dominant
polycystic kidney disease (ADPKD) and autosomal recessive
polycystic kidney disease (ARPKD). Treatment specifically includes
prevention, and slowing down the progression of the disease. If the
objective is to prevent or slow down the progression of PKD,
patients susceptible to the disease can be diagnosed by identifying
mutations in the PKD1, PKD2 or PKD3 genes that are associated with
PKD.
[0028] In all aspects, the PTBR agonist may, for example, be a
native sequence PTBR ligand or a fragment or functional subunit
thereof, an organic small molecule or peptide, a polypeptide
variant of a native sequence ligand, an antibody, a glycopeptide, a
glycolipid, a polysaccharide, an oligosaccharide, a nucleic acid, a
peptidomimetic, a pharmacological agent or a metabolite thereof, a
transcriptional or translational control sequence, and the like.
Similarly, the PTBR antagonist may be a polypeptide, an organic
small molecule or peptide, a polypeptide variant of a native
sequence ligand, an antibody, a glycopeptide, a glycolipid, a
polysaccharide, an oligosaccharide, a nucleic acid, a
peptidomimetic, a pharmacological agent or a metabolite thereof, a
transcriptional or translational control sequence, and the like.
For example, PTBR antagonists include polypeptide variants of a
native sequence PTBR ligand, variants of a native sequence PTBR
that retain the ability to bind an endogenous ligand but are
deficient in their ability to mediate biological activity,
anti-PTBR or anti-PTBR ligand antibodies, and selective inhibitors
of the in vivo production of an endogenous PTBR ligand. The organic
small molecules are preferably selected from the chemical classes
of benzodiazepines, isoquinoline carboxamides, imidazopyridines,
2-aryl-3-indoleacetamides, and pyrolobenzoxazepines. A particularly
preferred agonist is Ro5-4864, while a particularly preferred
antagonist is PK 11195.
[0029] The PTBR ligands can be administered in combination with an
additional therapeutic agent, preferably with an agent known to be
useful to treat the target disease or a related condition. For
example, the PTBR ligands of the present invention can be
administered in combination with one or more therapeutic agents
that inhibit the delivery of membrane proteins to the membrane of a
cell of the patient treated. Such agents include, for example,
taxol, cytochalasin-B, cytochalasin-D, phalloidin and derivatives
of any of the foregoing, and TNF-.alpha.. The PTBR ligands can also
be administered in combination with generic inhibitors or renal
insufficiency progression, such as anti-hypertension therapeutics,
including ACE inhibitors.
[0030] Administration can be performed by various routes known in
the art, including, without limitation, intravenous,
intraperitoneal, intraarterial, subcutaneous, oral or intramuscular
administration.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 shows alignment data comparing the cDNA encoding the
differentially expressed rat peripheral-type benzodiazepine
receptor (P0268) gene with human cDNA corresponding to PTBR (SEQ ID
NOs: 1 and 2).
[0032] FIG. 2 shows the amino acid sequence of human PTBR (SEQ ID
NO: 3).
[0033] FIG. 3 shows alignment data comparing the cDNA encoding the
differentially expressed rat diazepam binding inhibitor (DBI) gene
with human cDNA corresponding to DBI (SEQ ID NOs: 4 and 5).
[0034] FIG. 4 shows the amino acid sequence of human DBI (SEQ ID
NO: 6).
[0035] FIG. 5 shows the chemical structure of selected PTBR
ligands, including PTBR agonist Ro5-4864, and antagonist PK
11195.
[0036] FIG. 6 is a graphical illustration of the effect of PTBR
agonist, Ro5-4864 on the proliferation of human ADPKD cells. In the
Figure, "A" represents Ro5-4864, and the numbers following
represent the molar concentrations of the agonist. Thus, "-12"
following "A" means that the agonist Ro5-4864 was used in a
concentration of 10.sup.-12 M.
DETAILED DESCRIPTION OF THE INVENTION
[0037] I. Definitions
[0038] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th
ed., John Wiley & Sons (New York, N.Y. 1992), provide one
skilled in the art with a general guide to many of the terms used
in the present application.
[0039] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0040] The terms "peripheral-type benzodiazepine receptor", "PTBR",
and "PTBR polypeptide", whether used in singular or plural, are
used interchangeably, and encompass any native sequence PTBR
polypeptide. Such PTBR polypeptides can be isolated from a variety
of sources, such as from a variety of human or non-human tissue
types, or prepared by recombinant and/or synthetic methods. All
such polypeptides are specifically within the scope of the
definition, regardless of their mode of preparation, and include
variants thereof. Thus, the terms "peripheral-type benzodiazepine
receptor", "PTBR", and "PTBR polypeptide", whether used in singular
or plural, refer to receptor polypeptides which bind to
benzodiazepine molecules but are distinct from those associates
with the central-type benzodiazepine receptors, and which have the
same amino acid sequence as a respective polypeptide derived from
nature. Such PTBR polypeptides can be isolated from nature or can
be produced by recombinant and/or synthetic means. The term "PTBR"
specifically encompasses naturally-occurring truncated or secreted
forms (e.g., an extracellular domain sequence), as well as
naturally occurring variant forms (e.g., alternatively spliced
forms), and naturally occurring allelic variants. PTBR's represent
a subset of the benzodiazepine receptor family that is located
outside the central nervous system. Kruger et al, in: GABA and
Benzodiazepine Receptor Subtypes, Biggio and Costa eds., pp. 1-14
(1990) reported the purification, cloning and expression of a
peripheral-type benzodiazepine receptor. The cDNA of a 18-kDa PTBR
polypeptide, originally identified in heart tissue, has
subsequently been cloned from various sources, such as rat adrenal
(Sprengel et al., J. Biol. Chem. 264:20,415-20,421 [1989]); bovine
adrenal (Parola et al., J. Biol. Chem. 266:14,082-14,087 [1991]); a
human lymphoma cell line (Riond et al., Eur. J. Biochem.
195:305-311 [1991]); and a mouse Leydig tumor cell line (Gamier et
al., Mol. Pharmac. 45:201-211 [1993]). This 169 amino acid protein
has approximately 80% homology between species. Various cells
transfected with these cDNAs displayed binding characteristics for
PTBR ligands Ro5-4864 and PK 11195. It has been suggested that PTBR
is a multimeric complex in which the PK 11195 binding site is on
the 18-kDA subunit, and expression of the benzodiazepine binding
requires another subunit, designated VDAC. Another, 10-kDa protein,
associated with PTBR, has also been tentatively identified as a
further component of the PTBR complex. (See, e.g. Zisterer and
Williams, supra.) All of these polypeptides, alone, or in any
functional combination, are specifically within the definition of
"PTBR". In a particular embodiment, the peripheral-type
benzodiazepine receptor has the amino acid sequence of human PTBR
(SEQ ID NO: 3).
[0041] The terms "ligand" "PTBR ligand" and "ligand of a (native
sequence) PTBR" are interchangeable, and are used in the broadest
sense to include endogenous or exogenous factors that interact with
a PTBR, including native sequence PTBR ligands and their variants,
as well as synthetic polypeptide or small molecule ligands. The
"interaction" is defined as the ability to affect the function of a
PTBR may, but does not need to involve, specific binding to the
native sequence PTBR. The term "PTBR ligand" includes antagonists
and agonists, as defined below.
[0042] The terms "native sequence ligand", "native sequence PTBR
ligand", "native sequence ligand of a PTBR", and grammatical
equivalents thereof, are used interchangeably, and refer to
endogenous ligands of a PTBR, known or hereinafter discovered. Such
native sequence polypeptides can be isolated from nature or can be
produced by recombinant and/or synthetic means. The term "native
sequence" in conjunction with the designation of a particular
polypeptide specifically encompasses naturally-occurring truncated
or secreted forms (e.g. an extracellular domain sequence), as well
as naturally occurring variant forms (e.g., alternatively spliced
forms), and naturally occurring allelic variants of the named
polypeptide.
[0043] The term "antagonist" is used in the broadest sense and
includes any molecule that partially or fully blocks, inhibits or
neutralizes a biological activity mediated by a native sequence
PTBR through preventing the binding of an agonist to the native
sequence PTBR, thereby blocking the biological activity of the
agonist mediated by the PTBR. In a similar manner, the term
"agonist" is used in the broadest sense and includes any molecule
that mimics a biological activity mediated by a PTBR, and
specifically changes the function or expression of a PTBR, or the
efficiency of signalling through a PTBR, thereby altering
(increasing or inhibiting) an already existing biological activity
or triggering a new biological activity.
[0044] The terms "variant" and "amino acid sequence variant" are
used interchangeably and designate polypeptides in which one or
more amino acids are added and/or substituted and/or deleted and/or
inserted at the N- or C-terminus or anywhere within the
corresponding native sequence, and which retain at least one
activity (as defined below) of the corresponding native
polypeptide. In various embodiments, a "variant" polypeptide
usually has at least about 75% amino acid sequence identity, or at
least about 80%amino acid sequence identity, preferably at least
about 85% amino acid sequence identity, even more preferably at
least about 90% amino acid sequence identity, and most preferably
at least about 95% amino acid sequence identity with the amino acid
sequence of the corresponding native sequence polypeptide.
[0045] "Sequence identity", is defined as the percentage of amino
acid residues in a candidate sequence that are identical with the
amino acid residues in a native polypeptide sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence
identity.
[0046] The local homology algorithm of Smith and Waterman (Smith et
al., Adv. Appl. Math. 2:482 (1981)) can conduct optimal alignment
of sequences for comparison, e.g., by the homology alignment
algorithm of Needleman and Wunsch (Needleman et al., J. Mol. Biol.
48:443 (1970)), by the search for similarity method of Pearson and
Lipman (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988)),
by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection.
[0047] In a preferred embodiment, the homology alignment algorithms
employed in the BLAST program (Altschul et al., Nucleic Acids Res.
25:3389-3402 (1997)) may be used. The BLAST family of programs
allows all combinations of DNA or protein query sequences with
searches against DNA or protein databases. Within the context of
the present invention, the specific BLAST programs that may be
utilized include: blastp, which compares an amino acid query
sequence against a protein sequence database; blasin, which
compares a nucleotide query sequence against a nucleotide sequence
database; blastx, which compares the six-frame conceptual
translation products of a nucleotide query sequences (both strands)
against a protein sequence database; tblastn, which compares a
protein query sequence against a nucleotide sequence database
dynamically translated in all six reading frames (both strands);
and tblastx, which compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database. For the blastn program, the following
parameters and their default values are utilized: -G: cost to open
a gap, default =5; -E: cost to extend a gap, default =2; -q:
penalty for a mismatch in the blast portion of run, default =-3;
-r: rewared for a match in the blast portion of run, default =1;
-e: expectation value (E), default =10.0; -W: word size, default is
11 for blastn, 3 for other programs; -v number of one-line
descriptions (V), default =100; and -b: number of alignments to
show (B), default =100.
[0048] Most preferably, the % sequence identity values are
generated by the NCBI BLAST2.0 software as defined by Altschul et
al., (1997), "Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs", Nucleic Acids Res.,
25:3389-3402. The parameters are set to default values, with the
exception of the Penalty for mismatch, which is set to -1.
[0049] "Active" or "activity" means a qualitative biological and/or
immunological property. In the context of the present invention, a
preferred biological activity of a PTBR antagonist is the ability
to prevent, slow down the progression of or eliminate cyst
formation or cyst expansion, or treat a disease dependent upon or
mediating cyst formation. Even more preferably, a PTBR antagonist
is biologically active, if it is effective in the treatment of
polycystic kidney disease (PKD).
[0050] The phrase "immunological property" means immunological
cross-reactivity with at least one epitope of the reference (native
sequence) polypeptide molecule, wherein, "immunological
cross-reactivity" means that the candidate polypeptide is capable
of competitively inhibiting the qualitative biological activity of
the reference (native sequence) polypeptide. The immunological
cross-reactivity is preferably "specific", which means that the
binding affinity of the immunologically cross-reactive molecule
identified to the corresponding polypeptide is significantly higher
(preferably at least about 2-times, more preferably at least about
4-times, most preferably at least about 6-times higher) than the
binding affinity of that molecule to any other known native
polypeptide.
[0051] The phrases "polycystic kidney disease" "PKD" and
"polycystic renal disease" are used interchangeably, and refer to a
group of disorders characterized by a large number of cysts
distributed throughout dramatically enlarged kidneys. The resultant
cyst development leads to impairment of kidney function and can
eventually cause kidney failure. "PKD" specifically includes
autosomal dominant polycystic kidney disease (ADPKD) and recessive
autosomal recessive polycystic kidney disease (ARPKD), in all
stages of development, regardless of the underlying cause.
[0052] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, or the expansion of such
disorder, such as the development of polycystic kidney disease. For
purposes of this invention, beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0053] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the desired effect for an extended period of time.
[0054] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0055] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0056] An "individual" is a vertebrate, preferably a mammal, more
preferably a human.
[0057] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal herein is human.
[0058] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an effective amount of a PTBR ligand is an amount that
is sufficient to effect the desired treatment, as hereinabove
defined.
[0059] The term "recombinant" when used with reference to a cell,
animal, or virus indicates that the cell, animal, or virus encodes
a foreign DNA or RNA. For example, recombinant cells optionally
express nucleic acids (e.g., RNA) not found within the native
(non-recombinant) form of the cell.
[0060] The term "antibody" is used in the broadest sense and
specifically covers anti-PTBR monoclonal antibodies (including
agonist, antagonist, and neutralizing antibodies), polyclonal
antibodies, multi-specific antibodies (e.g., bispecific
antibodies), as well as antibody fragments. The monoclonal
antibodies specifically includes "chimeric" antibodies in which a
portion of the heavy and/or light chain is identical with or
homologous to corresponding sequences in antibodies derived from a
particular species or belonging to a particular antibody class or
subclass, while the remainder of the chain(s) is identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). The
monoclonal antibodies further include "humanized" antibodies or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a CDR of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity, and
capacity. In some instances, Fv FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues which are
found neither in the recipient antibody nor in the imported CDR or
framework sequences. These modifications are made to further refine
and maximize antibody performance. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al, Nature,
321:522-525 (1986); and Reichmann et al., Nature, 332:323-329
(1988). The humanized antibody includes a PRIMATIZED.RTM. antibody
wherein the antigen-binding region of the antibody is derived from
an antibody produced by immunizing macaque monkeys with the antigen
of interest.
[0061] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0062] II. Modes of Carrying Out the Invention
[0063] A. Ligands of PTBR
[0064] There are several native polypeptides that have been
putatively identified as endogenous
[0065] ligands for PTBR or as components of such ligands. One
possible endogenous ligand is the diazepam-binding inhibitor (DBI)
(Berkovich et al., Mol. Pharmac. 37:164-172 [1990]; Guidotti et
al., Nature 257:533-535 [1978]), an endogenous 11-kDa polypeptide
of 86 amino acids (Besman et al., Proc. Natl. Acad. Sci. USA
86:4897-4901 [1989]). The same ligand is also referred to in the
literature as acyl coenzyme A-binding protein (Knudsen et al.,
Biochem. J. 26:513-519 [1989]). This ligand is not selective as it
has the same affinity (.mu.M range) for both the
GABA.sub.A/benzodiazepine receptor and PTBR. A shorter fragment of
DBI (fragment 17-50, also referred to as trikontetraneuropeptide)
is more selective for PTBR.
[0066] Another set of putative endogenous ligands are naturally
occurring porphyrins which have been reported to have high affinity
for the PTBR. (Taketani et al, J. Biochem. 117:875-880 [1995] and
Zisterer and Williams, supra.)
[0067] Synthetic ligands of the PTBR are also known and well
characterized. Such synthetic ligands include benzodiazepines, such
as, for example, Ro5-4864 and Clonazepam; isoquinoline
carboxamides, e.g. PK 11195 [1
-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline
carboxamide] and PK 14105
[(2-fluoro-5-nitro-phenyl)-N-methyl-N-(1-methyl-
propyl)-3-isoquinoline carboxamide]; imidazopuridines, e.g. Alpidem
and Zolpidem; and 2-amyl-3-indoleacetamides, e.g. FGIN-1-27; and
pyrolobenoxapines, e.g. NF 182. The chemical structures of some
selected synthetic PTBR ligands are shown in FIG. 5. Further
synthetic PTBR ligands are also well known in the art, and are
discussed, for example, in Zister and Williams, supra; Anzini et
al., J. Med. Chem. 4275 (1996); Cappelli et al., J. Med. Chem. 2910
(1997) (confornationally constrained analogues of Ro5-4864); WO
96/32383 [(2-phenylpyrimidin-4-yl) (oxy or amino) acetamide
derivatives]; FR 2,678,269 [1-(4-chlorophenyl)-2-(1-pipe-
ridinyl)ethanol derivatives]; EP 524,846
[2-(1-piperidinyl)-2-(6(3,4-quino- lin-2-(1H)-one))-ethanol
derivatives]; FR 2,669,926 (phenylurea derivatives); U.S. Pat. No.
5,128,338 and EP 446,141 [imidazo(1,2-c)quinazoline derivatives];
U.S. Pat. No. 5,026,711 (4substituted amino-quinoline or
naphtyridine-3-carboxylic acid derivatives); U.S. Pat. No.
4,808,599 and EP 248,734 (benzothiphene or benzofuran carboxamide
derivatives); and EP 210,084 (amide or carbamate derivatives of
(iso)quinoline and quinazoline), the disclosures of which are
hereby expressly incorporated by reference.
[0068] The use of these and similar ligands, native or synthetic,
known or hereinafter discovered, is specifically within the scope
of the present invention. Preferred ligands show high selectivity
for the PTBR, relative to the benzodizepine receptors present in
the brain (CBR) or GABA. In competitive binding experiments, the
difference in binding affinity is preferably at least 10-fold, more
preferably at least 100-fold, most preferably at least
1000-fold.
[0069] PTBR ligands include agonist and antagonist of PTBR.
Representative PTBR agonists include benzodiazepines, e.g. Ro5-4864
and its derivatives, while representatives PTBR antagonists include
isoquinoline carboxamides, e.g. PK 11195 and PK 14105 and
derivatives.
[0070] B. Screening for New Antagonists and Agonists of PTBR.
[0071] The first step in identifying new ligands of the PTBR
(whether agonists or antagonists), is
[0072] in vitro screening to identify compounds that selectively
bind the peripheral-type receptor. Receptor-binding can be tested
using peripheral-type and brain-derived receptors isolated from
their respective native sources, or produced by recombinant DNA
technology and/or chemical synthesis. The binding affinity of the
candidate compounds can be tested by direct binding (see, e.g.
Schoemaker et al., J. Pharmacol. Exp. Ther., 285:61-69 [1983]) or
by indirect, e.g. competitive, binding. In competitive binding
experiments, the concentration of a compound necessary to displace
50% of another compound bound to the receptor (IC.sub.50) is
usually used as a measure of binding affinity. The other ligand can
be any compound known to bind to PTBR with high affinity and
selectivity, e.g. PK11195 or Ro5-4864.
[0073] In a specific embodiment, in order to identify novel
ligands, DNA encoding the full length sequence of the human
peripheral benzodiazepine receptor (GenBAnk M36035) is cloned into
an expression vector containing a selectable marker. The vector is
used to transfect recombinant host cells, for example mammalian
cells, e.g., the human embryonic kidney cell line (HEK-293).
Following several rounds of selection stable lines which express
PTBRs are identified by Western blot using immunoreactivity toward
an epitope tag that is genetically engineered into the PTBR gene.
Membrane fractions are prepared from the stably expressing cell
lines in bulk and stored frozen for HTP screening. Authentification
of the PTBR containing membrane fractions is achieved by
reproducing binding coefficients of known radiolabelled ligands
(such as [3H]Ro5-4864). Screening for novel ligands is performed by
virtue of their ability to compete effectively with [3H]Ro5-4864 in
competitive binding assays. Binding coefficients can be determined
by any known manner, e.g. by Scatchard analysis.
[0074] It might also be necessary to distinguish between PTBR
agonists and antagonists. This can be done in in vitro or in vivo
experiments, by monitoring the response of a cell following the
binding of the ligand to the receptor. An agonist will produce a
cellular response, which results in increased or new activity or in
the inhibiting of an already occurring cellular activity. In
contrast, an antagonist will have no effect on cellular response,
rather will have the effect of preventing binding of agonists to
the same receptor sites. It may be desirable to screen for
antagonists in a fashion that the readout is functional to find
molecules that activate the receptor without affecting the binding
site(s) of the native ligand(s). Antagonists can be screened in a
similar fashion.
[0075] For example, the following methods are suitable for
identifying antagonists and agonists of the PTBR that are useful in
the methods of the present invention:
[0076] 1. The proliferation of renal epithelial, fibroblast, or
smooth muscle cells derived from normal or polycystic mammalian
(including human) kidneys.
[0077] 2. The regulation of induced apoptosis in renal epithelial
or fibroblast cells derived from normal or polycystic mammalian
(including human) kidneys.
[0078] 3. Monitoring the in vitro formation of cysts by cells
derived from human or nonhuman mammals with polycystic kidney
disease.
[0079] 4. The expression of a phenotype indicating loss of
differentiation by renal epithelial cells.
[0080] 5. Alterations in ion conductance or in electrical phenomena
dependent on ion conductance in renal epithelial cells or
insterstitial cells derived from normal or polycystic kidneys.
[0081] 6. Modulation in the secretion by renal or circulating cells
of protein or lipid or carbohydrate factors associated with the
expression or progression of polycystic kidney disease, including
cytokines and lipid factors produced in or around cysts.
[0082] C. Other PTBR Antagonists
[0083] The PTBR antagonists of the present invention are not
limited to PTBR ligands. Other PTBR
[0084] antagonists include (1) variants of a native PTBR that
retain the ability to bind an endogenous PTBR ligand but are
deficient in their ability to mediate a biological response, (2)
soluble receptors, (3) antibodies specifically binding an
endogenous PTBR ligand at or around its receptor binding site so
that they block the binding of the ligand to its native receptor,
and (4) selective inhibitors of the in vivo production of an
endogenous PTBR ligand, such as transcriptional regulators of the
expression of an endogenous PTBR ligand in vivo. Another preferred
PTBR antagonist is a bioorganic molecule, usually an orally active
compound that is based on synthetic and/or molecular modeling
studies, that is capable of preventing the interaction between a
native PTBR receptor and its endogeneous ligand. Such PTBR
antagonists can be identifying using the same type of assays as
those discussed above.
[0085] D. Availability of PTBR Antagonists and Agonists
[0086] The PTBR antagonist and agonists of the present invention
can be small molecules, e.g. organic
[0087] compounds or peptides that can be synthesized by known
techniques of chemical synthesis. Some PTBR antagonists or agonists
will be polypeptides, e.g. native sequence PTBR ligands, or
fragments, variants or derivatives thereof, and may be produced by
recombinant DNA technology, chemical synthesis or a combination of
these or similar techniques. Some PTBR agonist or antagonists are
commercially available, e.g. from Hoffmann-La Roche AG (Nutley,
N.J.), and Synthelabo (France).
[0088] The PTBR ligands, either agonists or antagonist, of the
present invention may also be agonist or antagonist antibodies to a
PTBR. Methods of preparing polyclonal antibodies are known in the
art. Polyclonal antibodies can be raised in a mammal, for example,
by one or more injections of an immunizing agent and, if desired,
an adjuvant. Typically, the immunizing agent and/or adjuvant will
be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal
being immunized, such as serum albumin, or soybean trypsin
inhibitor. Examples of adjuvants which may be employed include
Freund's complete adjuvant and MPL-TDM.
[0089] According to one approach, monoclonal antibodies may be
prepared using hybridoma methods, such as those described by Kohler
and Milstein, Nature, 256:495 (1975). In a hybridoma method, a
mouse, hamster, or other appropriate host animal, is typically
immunized with an immunizing agent to elicit lymphocytes that
produce or are capable of producing antibodies that will
specifically bind to the immunizing agent. Alternatively, the
lymphocytes may be immunized in vitro. Generally, either peripheral
blood lymphocytes ("PBLs") are used if cells of human origin are
desired, or spleen cells or lymph node cells are used if non-human
mammalian sources are desired. The lymphocytes are then fused with
an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986) pp.
59-103]. Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. Preferred
immortalized cell lines are those that fuse efficiently, support
stable high level expression of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium.
[0090] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the particular PTBR used. Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma
cells is determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA). Such techniques and assays are known
in the art. 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).
[0091] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0092] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0093] Alternatively, monoclonal antibodies may be made by
recombinant DNA methods, such as those described in U.S. Pat. No.
4,816,567. DNA encoding the monoclonal antibodies of the invention
can be 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). The hybridoma cells discussed above serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as 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.
[0094] The antibodies, including antibody fragments, such as Fv,
Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of
antibodies, may be humanized. Humanized antibodies contain minimal
sequence derived from a non-human immunoglobulin. More
specifically, in humanized antibodies residues from a complementary
determining region (CDR) of a human immunoglobulin (the recipient)
are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are also replaced by
corresponding non-human residues. Humanized antibodies may
additionally comprise residues that are found neither in the
recipient antibody nor in the imported CDR or framework sequences
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,
Nature, 332:323-329 (1988)].
[0095] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a non-human source. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers [Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
In addition, human antibodies can be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0096] The antibodies may be bispecific, in which one specificity
is for a PTBR, and the other specificity for another protein, such
as, a second, different PTBR, or a different epitope of the same
PTBR, or a PTBR ligand.
[0097] D. Compositions Comprising PTBR Agonists and Antagonists
[0098] The PTBR agonists and antagonists (ligands and others) can
be administered to a patient at therapeutically effective doses to
treat (including prevention) a specific cystic disease, e.g.
polycystic kidney disease. A therapeutically effective dose refers
to that amount of the compound sufficient to result in desired
treatment.
[0099] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
exhibiting large therapeutic indices are preferred. While compounds
that exhibit toxic side effects can be used, care should be taken
to design a delivery system that targets such compounds to the site
of affected tissue in order to minimize potential damage to
uninfected cells and, thereby, reduce side effects.
[0100] Data obtained from cell culture assays and animal studies
can be used in formulating a range of dosage for use in humans. The
dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED.sub.50 with little
or no toxicity. The dosage can vary within this range depending
upon the dosage form employed and the route of administration
utilized. For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose can be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound, which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to accurately determine
useful doses in humans. Levels in plasma can be measured, for
example, by high performance liquid chromatography. A typical daily
dose for a PTBR agonist or antagonist of the present invention
might range from about 1 .mu.g/kg to about 100 mg/kg of patient
body weight or more per day, depending on the factors mentioned
above, preferably about 10 .mu.g/kg/day to 10 mg/kg/day.
[0101] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates can be formulated for administration by inhalation or
insufflation (either through the mouth or the nose) or oral,
buccal, parenteral or rectal administration.
[0102] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate. talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0103] Preparations for oral administration can be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions can take the form of tablets
or lozenges formulated in conventional manner.
[0104] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
can be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0105] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
or dispersing agents. Alternatively, the active ingredient can be
in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use. The compounds can also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides.
[0106] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example, subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0107] The compositions can, if desired, be presented in a pack or
dispenser device that can contain one or more unit dosage forms
containing the active ingredient. The pack can for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device can be accompanied by instructions for
administration.
[0108] If an agonist or an antagonist is coadministered with
another agonist or antagonist, or with another agent having similar
biological activity, the different active ingredients may be
formulated together in an appropriate carrier vehicle to form a
pharmaceutical composition. The PTBR agonists of the present
invention may, for example, be combined or otherwise coadministered
with other therapeutics used in the treatment of the target
disease, for example, polycystic kidney disease, including taxol
and taxol derivatives, TNF-.alpha., anti-hypertensives, including
ACE inhibitors, therapeutics targeting protein factors or their
receptors, etc.
[0109] E. Gene therapy
[0110] The cystic diseases, e.g. polycystic kidney disease, can be
treated in accordance with the present
[0111] invention also by gene-based therapies, using either ex vivo
or in vivo transfer of a gene encoding a PTBR ligand polypeptide or
other polypeptide PTBR agonist or antagonist. In the ex vivo form
of gene delivery, cells derived either from the patient or from
other sources, are first modified outside the body by introduction
of a particular gene or genes. These modified cells are then
reintroduced into the patient's body, so as to achieve local,
regional or widespread distribution.
[0112] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11:205-210
(1993)). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, and proteins
that target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87:3410-3414
(1990). For review of the currently known gene marking and gene
therapy protocols see Anderson et al., Science 256:808-813
(1992).
[0113] The most advanced technologies for using nucleic acids in
the course of gene therapy use retroviral systems for delivering
genes into the cell (Wilson et al.,
[0114] Proc. Natl. Acad. Sci. U.S.A. 87, 439-443 (1990), and Kasid
et al, Proc. Natl. Acad. Sci. U.S.A. 87 473-477 (1990).
[0115] Efficient gene transfer into the kidney is relatively
difficult, particularly since the kidney is a structurally complex
organ that has a relatively low mitotic index. Moreover, the
architectural organization of the kidney is critical for proper
organ function.
[0116] Therefore, efficient gene delivery to one particular cell
type within the kidney is relatively difficult to achieve. A
specific method for gene transfer into the kidney is disclosed, for
example, in U.S. Pat. No. 5,869,230 issued on Feb. 9, 1999.
According to this approach, a vector carrying the genetic material
of interest is introduced into the vasculature of the kidney under
conditions that allow infection of kidney but protects it from
ischemic damage, for example, by maintaining the kidney at a low
temperature (e.g. on ice) during incubation with the gene transfer
vector.
[0117] The following examples illustrate, but do not limit, the
invention. All references cited throughout the specification,
including the examples, are hereby expressly incorporated by
reference.
EXAMPLE 1
[0118] Differential Expression of PTBR in an in vivo Model of
Kidney Disease as Determined by Microarray Analysis
[0119] The expression profile of representative genes was assessed
in normal tissues and tissues
[0120] obtained from subjects suffering from a disease,
specifically cardiac, kidney or inflammatory disease.
Identification of the differentially expressed genes involved the
following steps: (1) construction of normalized and subtracted cDNA
libraries from mRNA extracted from the cells or tissue of healthy
animals and an animal model of disease; (2) purification of DNA;
(3) microarraying the purified DNA for expression analysis; and (4)
probing microarrays to identify the genes from the clones that are
differentially expressed using labeled cDNA from healthy and
diseased cells or tissues.
[0121] (1) In Vivo Model of Kidney Disease
[0122] As an in vivo model of kidney disease, a rat model of an
inherited form of autosomal dominant polycystic kidney disease
(ADPKD) was used, based on the observation that ADPKD develops in
Han:SPRD rats (Kaspareit-Rittinghaus et al., Transplant Proc. 6:
2582-3 (1990); Cowley et al., Kidney Int. 43:522-34 (1993)). Renal
cysts and renal failure were evident in six months old male
heterozygous rats (Cy/+), whereas control rats (+/+) showed no sign
of cysts or renal failure. Five diseased animals (Cy/+) and one
normal (+/+) were sacrificed and the kidneys removed.
[0123] (2) Preparation of Normalized and Subtracted cDNA
Libraries
[0124] Poly A+mRNA was isolated from the kidneys of normal and
diseased animals, following techniques known in the art, such as
those described in Ausubel et al., eds., Current Protocols in
Molecular Biology, J. Wiley and Sons (New York, N.Y. 1993). Large
numbers of tissue samples can be readily processed using techniques
well known in the art, such as, for example, the single-step RNA
isolation process of Chomczynski (U.S. Pat. No., 4,843,155) which
is hereby expressly incorporated by reference in its entirety.
Methods for making normalized cDNA libraries are also well known in
the art, see, e.g. Soares et al., Proc. Natl. Acad. Sci. USA 91
(20):9228-32 (1994); and Bonaldo et al., Genome Res. 6 (9):791-806
(1996). Following the method of Bonaldo et al., supra, a normalized
version of a cDNA library was generated from normal and diseased
tissue. In particular, poly A+RNA was purified from the normal and
diseased tissue samples provided by the in vivo model of kidney
disease described above. A directionally cloned cDNA library was
first generated by conventional methods. Briefly, double stranded
cDNA was generated by priming first strand synthesis for reverse
transcription using oligo dT primers which contain a Not I
restriction site. After second strand synthesis, Xba I adapters are
added to the 5' end of the cDNA, and the cDNA size was selected for
>500 bp and ligated into the corresponding restriction sites of
phagemid vector pCR2. 1 (Invitrogen, San Diego Calif.).
[0125] From the total cDNA library, a normalized library was
generated as detailed elsewhere (Bonaldo et al, supra), and
described here briefly. Phagemid vector pCR2.1 contains an F1
origin of replication. Thus, the cDNA library can be propagated as
single stranded phage with appropriate helper virus. Single
stranded, circular DNA was extracted from the phage library and
serves as "tester" DNA in the hybridization step of normalization.
The other component of the hybridization, "driver" DNA, was
generated from the library by PCR amplification using a set of
primers specific for the region of the vector, which flanks the
cloned inserts. Purified tester DNA (50 ng) and driver DNA (0.5
.mu.g) was combined in 120 mM NaCI, 50% formamide, 10 mM Tris (pH
8.0), 5 mM EDTA, and 1% SDS. A pair of oligonucleotides (10 .mu.g
each), corresponding to polylinker sequence (same strand as tester
DNA) which is present in the PCR product, was included in the
hybridization reaction to block annealing of vector-specific
sequences which are in common between tester and driver DNA.
[0126] The reaction mixture, under oil, was heated 3 min. at
80.degree. C., and hybridization performed at 30.degree. C. for 24
hr (calculated C.sub.ot.about.5). Single stranded circles were
purified from the reaction mixture by hydroxylapatite (HAP)
chromatography, converted to double strand DNA, and electroporated
into bacteria to yield a normalized cDNA library representative of
genes expressed in the left ventricle of rat. To evaluate the
effectiveness of the normalization protocol, the frequency of a few
clones (ANP, BNP, actin, and myosin) was assessed in both in the
starting library and the normalized library. The frequency of
abundant cDNAs (actin and myosin) was reduced and roughly
equivalent to rarer cDNA clones (ANP and BNP). Clone frequency in
the two libraries was determined with standard screening techniques
by immobilizing colonies onto nylon membranes and hybridizing with
radiolabeled DNA probes.
[0127] Certain genes, unexpressed in a normal tissue and turned on
in diseased tissue, may be absent from the normalized cDNA library
generated from normal tissue. To obtain disease-specific clones to
include on the microarray, one can repeat the normalization
strategy outlined above using diseased tissue obtained from the
appropriate disease model. However, since most genes are expressed
commonly between normal and diseased tissue, microarraying
normalized libraries from diseased and nonnal tissue may introduce
significant redundancy. In a preferred embodiment, clone redundancy
is reduced, yet cDNAs are obtained which are expressed
specifically, as well as substantially elevated, in diseased
tissue. To obtain disease-specific cDNAs, a subtracted library can
be made using protocols similar to those used to generate
normalized libraries. Again, the method of Bonaldo et al, supra,
described here briefly is used.
[0128] To make a subtracted library, a total cDNA library is
generated from the tissue obtained from the disease model. The cDNA
library is directionally cloned in pCR2. 1 vector and single
stranded tester DNA derived as described above for library
normalization. The driver DNA is generated by PCR amplification of
cloned inserts from the total cDNA library prepared from the normal
kidney. Hybridization occurs between sequences, which are in common
to normal and diseased kidneys. For this subtracted library, the
reaction is driven more thoroughly (calculated C.sub.ot.about.27)
than normalization by using more driver (1.5 .mu.g vs. 0.5 .mu.g)
and longer hybridization time (48 hr vs. 24 hr). Purification of
nonhybridized, single stranded circles by HAP chromatography,
conversion to double strand DNA, and electroporation into bacteria
yields a subtracted cDNA library enriched for genes which are
expressed in diseased rat kidneys.
[0129] (3) Microarray Analysis
[0130] A microtiter plate protocol for PCR amplification of DNA and
its subsequent purification was established that provides
acceptable quality and quantity of DNA for printing on microarrays
for use in a preferred embodiment of the present invention.
Specifically, PCR primers were synthesized that amplify insert DNA
from the vector pCR2.1, which was used for library construction.
After 30 cycles of amplification each PCR product is passed over a
gel filtration column to remove unincorporated primers and salts.
To maintain robustness, the columns are packed in 96-well filter
plates and liquid handling is performed robotically. The yield, per
PCR reaction, is generally 2-5 .mu.g, enough DNA for printing
several hundred chips.
[0131] To test the quality of DNA that was prepared by this PCR
method, 96 purified samples from a single microtiter plate were
produced as a microarray. Using a robotic liquid handler (Biomek
2000, Beckman), 85 .mu.l of PCR reaction mixture was aliquoted into
each well of a thin walled, 0.2 ml 96-well plate. The reaction
mixture contained 0.2 mM each dNTP, 1.25 units of Taq polymerase,
and 1.times.Taq buffer (Boehringer Mannheim). Primers, 1 .mu.m
each, are from vector regions, which flank the cloning site of
pCR2.1 and include a 5' primary amine with a 6 carbon linker to
facilitate attachment of DNA product to the glass surface of the
microarray chip. 1.0 .mu.l of bacterial culture of individual cDNA
clones was added to each well. PCR conditions are: 2 min.,
95.degree. C. to denature, then 30 cycles of 95.degree., 30 sec.
/65.degree. C., 40 sec. /72.degree. C., 1 min. 30 sec., and a final
extension of 72.degree. C., 5 min. using a MJResearch PTC 100
thermocycler.
[0132] PCR products were purified by gel filtration over Sepliacryl
400 (Sigma). Briefly, 400 .mu.l of pre-swollen Sephacryl 400 was
loaded into each well of a 96-well filter plate (PallBiosupport)
and spun into a collection plate at 800 g for 1 min. Wells were
washed 5 times with 0.2.times.SSC. PCR reaction mixtures were
loaded onto the column and purified DNA (flow-thru) was collected
at 800 g for 1 min. Samples are dried down at 50.degree. C.
overnight and arrayed.
[0133] Fluorescent probe pairs were synthesized by reverse
transcription of poly A+RNA using, separately, Cy3 dCTP and Cy5
dCTP (Amersham). In 16.5 .mu.l, 1 .mu.g poly A+RNA and 2 .mu.g of
oligo dT 21mer, were denatured at 65.degree. C., 5 min. and
annealed at 25.degree. C., 10 min. Reverse transcription was
performed for 2 hours at 37.degree. C. with Superscript RT (Life
Technologies, Gaithersburg, Md.) in 1.times.buffer, 10 units RNase
block, 500.mu.M each dATP/dGTP/dTTP, 280 .mu.M dCTP, 40 .mu.M Cy5
or Cy3 dCTP, and 200 units RT. RNA is degraded in 0.1 M NaOH,
65.degree. C. for 10 min. Labeled cDNA was purified by successive
filtration with Chroma Spin 30 spin columns (Clontech) following
manufacturer's instructions. Samples were dried at room temperature
in the dark using a covered Speed-Vac. Probes were applied to the
test chip for hybridization and the data collected essentially as
described in Schena et al., Proc. Natl. Acad. Sci. USA
93(20):106-49 (1996). The intensity of hybridization signal at each
element reflected the level of expression of the mRNA for each gene
in the rat ventricle. Digitized signal data was stored and prepared
for analysis. A series of control DNA elements were included on
each chip to ensure consistency in labeling and hybridization
between experiments and to aid in balancing the signal when two
fluorescence channels are used. For each element hybridized with
dual labeled probes, absolute and relative intensity of signal was
determined. The results from these and other experiments indicate
that these methods for production of template DNA and labeled cDNA
probes are suitable for generating high quality microarrays within
a preferred embodiment of the methods of the present invention. The
evaluation of tens of thousands of genes for expression generates a
large amount of data that can be manipulated by commercially
available software packages that facilitate handling this type and
quantity of data. The expression data can be stored, analyzed, and
sorted from each experiment using this software. In addition,
expression of each clone can be tracked from experiment to
experiment using known methodologies.
[0134] (4) Detection of Differentially Expressed Genes using
Microarray Analysis
[0135] As disclosed in detail above, probes were applied to the
microarrays for hybridization and the data collected essentially as
described in Schena et al., supra. The intensity of hybridization
signal at each element reflected the level of expression of the
mRNA for each gene. For each element hybridized with dual labeled
probes, absolute and relative intensity of signal is determined,
which translates into the relative expression levels of the subject
genes. The numeric data generated reflects the relative expression
level of the gene in the disease state as compared to the
expression level of the gene in the normal, or non-disease state,
in the five PKD disease model delineated above and as determined by
microarray analysis. Data are reported as differential expression
values with positive numbers indicative of genes expressed at
higher levels in the diseased tissue relative to normal tissue, and
negative values indicative of lower expression in disease. While in
general microarray data are the average values from multiple
experiments performed with separate DNA arrays, in the present case
one experiment was performed and the RNA was obtained from one
animal (n=1).
[0136] In a preferred embodiment, clones that reproducibly scored
in microarray analysis to be at least about two-fold elevated or
decreased were microarrayed on separate secondary chips and their
expression levels determined. It is understood, however, that
differentially expressed genes exhibiting less than about a
two-fold change in expression, e.g., less than one, one-half, or
one-quarter, or greater than about a two-fold change in expression,
e.g., greater than three, five, ten, twenty, one hundred-fold, or
one thousand-fold, are also of interest.
[0137] Using cDNA obtained from the in vivo kidney disease model,
microarrays were constructed and probed as described above. It has
been found that a gene, originally assigned clone ID No: P0242_B03
and later identified as the rat equivalent of human peripheral type
benzodiazepine receptor (PTBR) gene, is overexpressed by a factor
of 2 in the rat model of polycystic kidney disease (PKD) described
above, as compared to normal kidney tissue. It has further been
found that the gene for a native ligand of PTBR, DBI is
underexpressed by a factor of -1.9 in diseased kidney tissue, as
compared to normal tissue.
[0138] (5) Identification of Differentially Expressed Human
Genes
[0139] Differentially expressed clones obtained from the microarray
analysis of DNA obtained from the disease model described above
were sequenced and compared to known human gene sequence databases
for matches to known human genes. FIG. 1 shows alignment data
comparing the nucleotide sequence of the cDNA encoding the
differentially expressed rat P0268 gene with the sequence of human
cDNA corresponding to PTBR (SEQ ID NO: 1 and 2). FIG. 2 shows the
amino acid sequence of human PTBR (SEQ ID NO: 3). FIG. 3 shows
alignment data comparing the nucleotide of the cDNA encoding the
differentially expressed rat DBI protein (native PTBR ligand) with
the sequence of human cDNA corresponding to DBI (SEQ ID NO: 4 and
5). FIG. 4 shows the amino acid sequence of human DBI.
EXAMPLE 2
[0140] Effect of a PTBR agonist on Proliferation of Human ADPKD
Cells
[0141] In this experiment, the effect of concentrations from
10.sup.-12 to 10.sup.-6 M of the known PTBR agonist, Ro5-4864 on
the rate of proliferation of human autosomal dominant polycystic
kidnay disease (ADPKD) cells was examined.
[0142] Epithelial cells from human ADPKD kidneys have been cultured
from the domes of cysts and maintained in primary culture through
several passages. These cells exhibit several phenotypic properties
of intact cysts, including epithelial orientation, fluid secretion
in response to cAMP stimulation and cell proliferation), and have
been used for studies of cellular mechanisms of fluid secretion and
cell proliferation.
[0143] The rate of cell proliferation was determined using the
Promega Cell titer 96 MTT Assay method. This assay is a
modification of that described by Mosmann T 1983 J Immunol Meth.
65, 55. This method, which measures the optical density (O.D.) of a
proliferation-dependent reaction product (MTT), was found to
correlate directly with direct determinations of cell number using
the classical hemacytometer technique. The relation between cell
number and O.D. was linear, and r.sup.2 was >0.98. To determine
the effect of agonists on the rate of proliferation, approximately
4000 cells were seeded into individual chambers of a 96-well plate.
The cells were incubated initially in DME/F12 medium supplemented
only with penicillin, streptomycin, ITS and 1% FBS. After 24 hours,
the FBS was reduced to 0.002% (ITS deleted) to arrest growth. This
small amount of serum was needed to potentiate cell attachment to
the plastic surface. The human ADPKD cells were then cultured for
48 hours, after adding the cAMP (a stimulator of cell
proliferation) at a concentration of 100 .mu.M. A control and
forskolin group were examined as well. Preliminary studies
determined that growth was sustained over this entire interval.
[0144] Thereafter the effect of a range of concentrations from
10.sup.-12 to 10.sup.-6 M of the known PTBR agonist, Ro5-4864 on
the rate of proliferation of PTBR cells pretreated with cAMP or not
treated (baseline proliferation) was examined. The results are
graphically illustrated in FIG. 6. Each data set shown in FIG. 6 is
mean, and was obtained from from six determinations. Accordingly,
the results are highly statistically significant.
[0145] The data demonstrate that in all concentrations examined,
Ro5-4864 acted as a potent inhibitor of cell proliferation, which
blocked both basal cell proliferation and cAMP stimulated cell
proliferation. Visual observation of the cells supports the finding
that the PTBR agonist, Ro5-4864 acts by arresting cell growth and
not be killing the cells.
* * * * *