ILK inhibitors for the treatment of renal disease
United States Patent: 7,678,775
Issued: March 16, 2010
Matthias (Ann Arbor, MI), Logan; Patricia (Vancouver, CA)
Assignee: QLT Inc.
(Vancouver, British Columbia, CA)
Appl. No.: 11/855,884
Filed: September 14, 2007
Master of Science in Law
The invention relates to the treatment of
renal diseases using modulators of integrin linked kinase. Methods of
treatment as well as therapeutic agents including antisense, small
molecules, catalytic peptides and antibodies are disclosed. The agents of
the invention may also be used in combination with traditional therapies
for renal disease including ACE inhibitors. An advantage of the invention
is that it treats one of the causes of renal disease, rather than just
ameliorating symptoms, and can help prevent the progression of renal
disease to the point of acute renal failure.
Description of the
1. Technical Field
The invention relates to the treatment of renal diseases using modulators
of integrin linked kinase.
Progressive renal disease is an increasingly common and economically
burdensome disease. A significant portion of patients progress to acute
renal failure, a life threatening illness whose mortality has remained
high despite the introduction of its only nonsurgical treatment,
hemodialysis, about 25 years ago. Conventional hemodialysis mimics the
filtration function of the kidneys by circulating a patient's blood
through or over a dialysate solution physically separated from the blood
by a porous or permeable wall or membrane. The process results in the
preferential diffusion of small molecules, such as urea, from the
bloodstream into the dialysate solution.
Although dialysis has dramatically changed the prognosis of renal failure,
it is not a complete replacement therapy, since it only provides
filtration function and does not replace the homeostatic, regulatory, and
endocrine functions of the kidney. Patients on dialysis continue to have
major medical problems.
In the United States, kidney failure is experienced by more than 360,000
people who depend on dialysis or a kidney transplant to survive. The
number of people with kidney failure has actually doubled over the past 10
years, and the pool of candidates is large. Conservatively estimated, 10.9
million Americans have kidney disease and face the possibility of a future
on dialysis or with a kidney transplant. Even with these remarkable
treatments, nearly 58,000 people with kidney failure died in 1997. Jones,
C. A., et al., Serum creatinine levels in the US population: third
national health and nutrition examination survey. American Journal of
Kidney Diseases, vol. 32, no. 6, pp. 992-999, December 1998.
Chronic renal failure may result from any major cause of renal
dysfunction. The most common cause of end-stage renal disease is diabetic
nephropathy, followed by hypertensive nephroangiosclerosis and various
primary and secondary glomerulopathies. Plasma concentrations of
creatinine and urea (which are highly dependent on glomerular filtration)
begin a nonlinear rise as the renal function diminishes. Changes in
creatinine and urea concentrations are minimal early on; later levels
increase rapidly and are usually associated with systemic manifestations.
For substances that are excreted mainly through distal nephron secretion,
e.g., K, adaptation usually produces a normal plasma concentration until
advanced failure occurs.
Kidney function depends on an intact glomerular filtration unit, allowing
the excretion of potentially hazardous small molecular substances but
retaining essential macromolecules. The permselectivity of the glomerular
filter is defined by a fenestrated endothelial cell layer, the glomerular
basement membrane (GBM), and podocytes. The podocyte forms the filtration
slit, an ultrastructural membrane bridging the delicate web of
interdigitating podocyte foot processes.
In glomerular disease there is progressive podocyte damage and proteinurea.
Although the mechanisms for the progression of renal impairment remain
fully undetermined, available evidence indicate that renal glomerular
hypertension is responsible in part for the development of renal injury.
In renal disease, afferent arteriolar tone is reported to be reduced,
while the augmented intrarenal angiotensin II serves to act as an efferent
arteriolar constrictor, both of which result in an increase in glomerular
capillary pressure. Angiotensin converting enzyme inhibitors (ACE-I) are
established as the agent possessing both antihypertensive and
renoprotective actions, which exert vasodilator action on efferent
arterioles. Calcium antagonists are also reported to have salutary effect
on renal disease, although their beneficial action varies depending on the
antagonists used and the underlying disease. Chronic progression can be
slowed for 6-12 months using these drugs, but there is no other treatment
at this time besides dialysis and ultimately transplantation of the organ.
A long-term replacement therapy which replaces all of the functions of the
kidney and which is less costly than current dialysis therapies is
2. Description of the Related Art
Integrin-linked kinase (ILK) is a receptor-proximal protein kinase
regulating integrin-mediated signal transduction. The ILK sequence is
described in U.S. Pat. No. 6,013,782, herein incorporated by reference.
The presence of ILK mRNA in proteinurea models and puromycin-induced
podocyte damage is disclosed by Teixeira et al. (2000) Kidney & Blood
Pressure Research 23(3-5):231; Unschuld et al. (1999) Kidney & Blood
Pressure Research 22(4-6):400; Kretzler et al. (1998) Kidney & Blood
Pressure Research 21(2-4):145. The distribution and regulation of ILK in
normal and diabetic kidneys is discussed by Guo et al. (2001) Am J Pathol
The invention provides therapeutic compositions and methods for treatment
of renal disease, specifically for modulating the activity of integrin
linked kinase (ILK) to ameliorate glomerular renal disease states that may
result in proteinuria, or states characterized by tubular or tubulo-interstitial
damage. Treatment includes the administration of agents that interfere
with the ILK signaling pathway, including integrin linked kinase (ILK)
blocking agents; compounds that otherwise prevent the binding of natural
ILK ligands to ILK; or compounds that prevent expression of, or signaling
through, ILK. Such a treatment is used alone as single therapy or in
combination with a second therapy as an adjunct to prevent, to reduce or
to reverse the renal function.
In the subject methods, compounds that modulate the activity of integrin
linked kinase (ILK) are administered systemically or locally to treat
renal diseases, particularly those with underlying glomerular
insufficiency, or tubular damage or insufficiency. Such a treatment is
used alone as single therapy or in combination with a second therapy,
including administration of angiotensin converting enzyme inhibitors as an
adjunct to prevent, to reduce or to reverse the loss of renal function.
Animal and human proteinuric glomerulopathies evolve to terminal renal
failure by a process leading to progressive parenchymal damage, which is
relatively independent of the initial insult. The amount of urinary
proteins, or proteinuria, correlates with the tendency of a given disease
to progress. A constant feature of proteinuric nephritis is also the
concomitant presence of tubulointerstitial inflammation. Biochemical
events associated with tubular cell activation in response to protein
stress include up-regulation of inflammatory and vasoactive genes such as
MCP-1 and endothelins. The corresponding molecules formed in an excessive
amount by renal tubuli are secreted toward the basolateral compartment of
the cell and give rise to an inflammatory reaction that in most forms of
glomerulonephritis consistently precedes renal scarring.
Causes of chronic renal failure include, without limitation,
glomerulopathies, e.g., IgA nephropathy, focal glomerulosclerosis,
membranous nephropathy, membranoproliferative glomerulonephritis,
idiopathic crescentic glomerulonephritis, diabetes mellitus,
postinfectious glomerulonephritis, systemic lupus erythematosus, Wegener's
granulomatosis, hemolytic-uremic syndrome, amyloidosis; chronic
tubulointerstitial nephropathies; hereditary nephropathies, e.g.,
polycistic kidney disease, Alport's syndrome, medullary cystic disease,
Nail-patella syndrome; hypertension, e.g., nephroangiosclerosis, malignant
glomerulosclerosis; renal macrovascular disease; and obstructive uropathy,
e.g., ureteral obstruction, vesicoureteral reflux, benign prostatic
hyperplasia; and the like.
Chronic renal failure (CRF) may result from any major cause of renal
dysfunction. The functional effects of CRF can be categorized as
diminished renal reserve, renal insufficiency (failure), and uremia.
Plasma concentrations of creatinine and urea begin a nonlinear rise as the
renal function diminishes. Na and water balance is well maintained by
increased fractional excretion of Na and a normal response to thirst.
Thus, the plasma Na concentration is typically normal and hypervolemia is
infrequent despite unmodified dietary intake of Na. However, imbalances
may occur if Na and water intakes are very restricted or excessive.
Patients with mildly diminished renal reserve are asymptomatic, and renal
dysfunction can be detected only by laboratory testing. A patient with
mild to moderate renal insufficiency may have only vague symptoms despite
elevated BUN and creatinine; nocturia is noted, principally due to a
failure to concentrate the urine during the night. Lassitude, fatigue, and
decreased mental acuity often are the first manifestations of uremia.
Neuromuscular features include coarse muscular twitches, peripheral
neuropathies with sensory and motor phenomena, muscle cramps, and
convulsions. Anorexia, nausea, vomiting, stomatitis, and an unpleasant
taste in the mouth are almost uniformly present. In advanced CRP, GI
ulceration and bleeding are common. Hypertension is present in >80% of
patients with advanced renal insufficiency and is usually related to
hypervolemia and occasionally to activation of the
renin-angiotensin-aldosterone system. Cardiomyopathy and renal retention
of Na and water may lead to congestive heart failure or dependent edema.
Pericarditis, usually seen in chronic uremia, may occur in acute,
potentially reversible, uremia. Abnormalities with lipid metabolism also
occur with CRF, on dialysis, and after renal transplantation. The primary
finding in CRF and dialysis is hypertriglyceridemia; the total cholesterol
level is usually normal.
Treatments useful as an adjunct to the present methods include diet for
controlling hyperglycemia in diabetic nephropathy and hypertension, and
protein restriction. Administration of ACE inhibitors, and angiotensin
receptor blockers is also of interest.
ILK Modulating Agents
ILK is a 59 kDa serine/threonine kinase that associates with the
cytoplasmic tails of .beta.1 and .beta.3 integrins. The enzymatic activity
for ILK is modulated by the interaction of cells with the extracellular
matrix component fibronectin, integrin clustering and a number of growth
factors. Because of its intimate association with a wide variety of
signaling pathways that have been directly or indirectly implicated in
various pathological processes, ILK may represent a therapeutic target for
a variety of clinical conditions including angiogenesis, cancer,
inflammation and autoimmunity. The genetic sequence of human ILK is
disclosed in U.S. Pat. Nos. 6,013,782; and 6,001,622, herein incorporated
Agents that block ILK activity are used in the treatment of renal disease
relating to neovascularization. Numerous agents are useful in reducing ILK
activity, including agents that directly modulate ILK expression, e.g.,
anti-sense specific for ILK, ILK specific antibodies and analogs thereof,
small organic molecules that block ILK catalytic or binding activity,
etc.; and agents that affect ILK activity through direct or indirect
modulation of [PtdIns(3,4,5)P.sub.3] levels in a cell. For example, small
molecule inhibitors of integrin linked kinase are described in U.S. Pat.
No. 6,214,813. Antisense inhibitors of ILK are described in U.S. Pat. No.
6,177,273, each herein incorporated by reference.
Agents of interest for down-regulating ILK activity include direct
blocking of [PtdIns(3,4,5)P.sub.3] binding sites through competitive
binding, steric hindrance, etc. Of particular interest are antibodies that
bind to the PH domains, thereby blocking the site. Antibodies include
fragments, e.g., F(Ab), F(Ab)', and other mimetics of the binding site.
Such antibodies can be raised by immunization with the protein or the
specific domain. Mimetics are identified by screening methods. Analogs of
[PtdIns(3,4,5)P.sub.3] that compete for binding sites but do not result in
activation of ILK are also of interest.
Because ILK activity is upregulated by the presence of the lipid
[PtdIns(3,4,5)P.sub.3], the activity of ILK can be manipulated by agents
that affect cellular levels of [PtdIns(3,4,5)P.sub.3], or that block the
binding of [Ptdins(3,4,5)P.sub.3] to ILK. The amino acid sequence of ILK
contains a sequence motif found in pleckstrin homology (PH) domains, which
are involved in the binding of phosphatidylinositol phosphates. The
activity of ILK is also down-regulated by inhibiting the activity of PI(3)
kinase, thereby decreasing cellular levels of [PtdIns(3,4,5)P.sub.3].
Agents of interest include inhibitors of PI(3) kinase, e.g., wortmannin,
LY294002, etc. Physiologically effective levels of wortmannin range from
about 10 to 1000 nM, usually from about 100 to 500 nM, and optimally at
about 200 nM. Physiologically effective levels of LY294002 range from
about 1 to 500 .mu.M, usually from about 25 to 100 gM, and optimally at
about 50 .mu.M. The inhibitors are administered in vivo or in vitro at a
dose sufficient to provide for these concentrations in the target tissue.
Drug screening can be used to identify agents that modulate ILK function.
One can identify ligands or substrates that inhibit the action of ILK. A
wide variety of assays may be used for this purpose, including labeled in
vitro protein-protein binding assays, electrophoretic mobility shift
assays, immunoassays for protein binding, and the like. Knowledge of the
3-dimensional structure of ILK, derived from crystallization of purified
recombinant ILK protein, leads to the rational design of small drugs that
specifically inhibit ILK activity. These drugs may be directed at specific
domains of ILK, e.g., the kinase catalytic domain, ankyrin repeat domains,
pleckstrin homology domains, etc. Among the agents of interest for drug
screening are those that interfere with the binding of cytoplasmic
integrin tails to ILK; the kinase activity of ILK; binding of
[Ptdins(3,4,5)P.sub.3] to the PH domains of ILK and agents that inhibit
the production of [PtdIns(3,4,5)P.sub.3] by P1 (3) kinase.
The term "agent" as used herein describes any molecule, e.g., protein or
pharmaceutical, with the capability of altering the physiological function
of ILK. Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic compounds
having a molecular weight of more than 50 and less than about 2,500
daltons. Candidate agents comprise functional groups necessary for
structural Interaction with proteins, particularly hydrogen bonding, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl group,
preferably at least two of the functional chemical groups. The candidate
agents often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of the
above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or combinations
Assays of interest may detect agents that block ILK function, such as
integrin binding, kinase activity, down regulation of E-cadherin, up
regulation of LEF-1, binding properties, etc. For example, an expression
construct comprising an ILK gene may be introduced into a cell line under
conditions that allow expression. The level of ILK activity is determined
by a functional assay, as previously described. In one screening assay,
candidate agents are added, and the formation of fibronectin matrix is
detected. In another assay, the ability of candidate agents to enhance ILK
function is determined.
Methods of Treatment
The subject methods are used for prophylactic or therapeutic purposes to
treat renal diseases, particularly to prevent, reduce or reverse the loss
of glomerular function. As used herein, the term "treating" is used to
refer to both prevention of disease, and treatment of pre-existing
conditions. While treatment during early stages is desirable, the adverse
symptoms of the disease may be at least partially alleviated by treatment
during later stages.
Suitable animal models exist for determination of an effective dose of an
ILK inhibitor, for example as set forth in the examples. However the
efficacy of a therapeutic effect for different mammals varies widely; for
example, doses typically are 20, 30 or even 40 times smaller (per unit
body weight) in man than in the rat. Similarly the mode of administration
can have a large effect on dosage. Formulation or treatment with an
adjunct therapeutic agent or regimen may also affect the effective dose
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of an ILK inhibitor is administered to a
subject afflicted with a disease or disorder related to loss of renal
function, particularly to glomerular insufficiency relating to progressive
podocyte damage and proteinurea. The inhibitor may be administered in
accordance with the method of the invention either alone or in combination
with other known therapies. When co-administered with one or more other
therapies, the inhibitor may be administered either simultaneously with
the other treatment(s), or sequentially. If administered sequentially, the
attending physician will decide on the appropriate sequence of
administration, which may be before or after a second therapy.
In addition to dietary management, administration of ACE inhibitors is
useful as adjunct therapy. ACE inhibitor include, but are not limited to,
captopril, benazeprile, enalapril, fosinopril, lisinopril, quinapril,
Ramipril, imidapril, perindopril, erbumine, and trandolapril. ACE receptor
blockers may also be used in place of or as well as ACE inhibitors, and
these include losartan, irbesartan, candesartan, cilexetil, and valsartan.
The dose of ILK inhibitor in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition being
treated, and on the nature of prior treatments which the patent has
undergone. Ultimately, the attending physician will decide the dose with
which to treat each individual patient. Initially, the attending physician
may administer low doses and observe the patient's response. Larger doses
may be administered until the optimal therapeutic effect Is obtained for
the patient, and at that point the dosage is not increased further.
The compounds of this invention can be incorporated into a variety of
formulations for therapeutic administration. Administration of an ILK
inhibitor may be by delivery using any appropriate means including, but
not limited to, systemic, local, or even direct application to the target
tissue. Local delivery of an ILK inhibitor provides a high local
concentration while reducing the likelihood of non-specific anti-angiogenic
or other undesirable side effects that may follow systemic administration
of an ILK inhibitor.
The compounds of the present invention are formulated into pharmaceutical
compositions by combination with appropriate, pharmaceutically acceptable
carriers or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules, powders,
granules, ointments, solutions, suppositories, injections, inhalants,
gels, microspheres, and aerosols. As such, administration of the compounds
can be achieved in various ways, including oral, buccal, rectal,
parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration. The ILK may be systemic after administration or may be
localized by the use of an implant that acts to retain the active dose at
the site of implantation.
The compounds of the present invention can be administered alone, in
combination with each other, or they can be used in combination with other
known compounds and therapies. In pharmaceutical dosage forms, the
compounds may be administered in the form of their pharmaceutically
acceptable salts, or they may also be used alone or in appropriate
association, as well as in combination with other pharmaceutically active
For oral preparations, the compounds can be used alone or in combination
with appropriate additives to make tablets, powders, granules or capsules,
for example, with conventional additives, such as lactose, mannitol, corn
starch or potato starch; with binders, such as crystalline cellulose,
cellulose derivatives, acacia, corn starch or gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium
stearate; and if desired, with diluents, buffering agents, moistening
agents, preservatives and flavoring agents.
The compounds can be formulated into preparations for injections by
dissolving, suspending or emulsifying them in an aqueous or nonaqueous
solvent, such as vegetable or other similar oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol; and if
desired, with conventional additives such as solubilizers, isotonic
agents, suspending agents, emulsifying agents, stabilizers and
Implants for sustained release formulations are well-known in the art.
Implants are formulated as microspheres, slabs, etc. with biodegradable or
non-biodegradable polymers. For example, polymers of lactic acid and/or
glycolic acid form an erodible polymer that is well-tolerated by the host.
The implant is placed in proximity to the site of Infection, so that the
local concentration of active agent is increased relative to the rest of
The term "unit dosage form," as used herein, refers to physically discrete
units suitable as unitary dosages for human and animal subjects, each unit
containing a predetermined quantity of compounds of the present invention
calculated in an amount sufficient to produce the desired effect in
association with a pharmaceutically acceptable diluent, carrier or
vehicle. The specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect to be
achieved, and the pharmacodynamics associated with each compound in the
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or diluents, are readily available to the public. Moreover,
pharmaceutically acceptable auxiliary substances, such as pH adjusting and
buffering agents, tonicity adjusting agents, stabilizers, wetting agents
and the like, are readily available to the public.
Those of skill will readily appreciate that dose levels can vary as a
function of the specific compound, the severity of the symptoms and the
susceptibility of the subject to side effects. Some of the specific
compounds are more potent than others. Preferred dosages for a given
compound are readily determinable by those of skill in the art by a
variety of means. A preferred means is to measure the physiological
potency of a given compound.
Claim 1 of 24 Claims
1. A method for treating a patient with
renal dysfunction, the method comprising: administering an effective dose
of an integrin linked kinase (ILK) inhibitor to said patient.
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