U.S. patent application number 11/101976 was filed with the patent office on 2005-10-13 for compositions and methods for the treatment and prevention of cardiovascular diseases and disorders and for identifying agents therapeutic therefor.
This patent application is currently assigned to Medlyte, Inc.. Invention is credited to Sabbadini, Roger A..
Application Number | 20050226862 11/101976 |
Document ID | / |
Family ID | 22978375 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050226862 |
Kind Code |
A1 |
Sabbadini, Roger A. |
October 13, 2005 |
Compositions and methods for the treatment and prevention of
cardiovascular diseases and disorders and for identifying agents
therapeutic therefor
Abstract
Methods and compositions are disclosed that are useful for the
prevention and/or treatment of cardiovascular and cardiac diseases
and disorders, or damage resulting from surgical or medical
procedures that may cause ischemic or ischemic/reperfusion damage
in humans; and cardiovascular trauma. The beneficial effects of the
compositions and methods are achieved through the use of
pharmaceutical compositions that include agents that interfere with
the production and/or biological activities of sphingolipids and
their metabolites, particularly sphingosine (SPH) and
sphingosine-1-phosphate (S-1-P). Also disclosed are methods for
identifying and isolating therapeutic agents.
Inventors: |
Sabbadini, Roger A.;
(Lakeside, CA) |
Correspondence
Address: |
BIOTECHNOLOGY LAW GROUP
C/O PORTFOLIOIP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Medlyte, Inc.
San Diego
CA
|
Family ID: |
22978375 |
Appl. No.: |
11/101976 |
Filed: |
April 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11101976 |
Apr 7, 2005 |
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10028156 |
Dec 21, 2001 |
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6881546 |
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60257926 |
Dec 22, 2000 |
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Current U.S.
Class: |
424/130.1 ;
514/15.1; 514/15.7; 514/16.4 |
Current CPC
Class: |
A61P 9/00 20180101; G01N
2800/32 20130101; A61P 35/00 20180101; G01N 33/92 20130101; A61K
31/7036 20130101; C12N 9/1288 20130101; C07K 14/705 20130101; G01N
33/6893 20130101; A61K 38/00 20130101; C12N 9/1205 20130101; C07K
16/18 20130101; C07K 16/28 20130101; A61P 29/00 20180101; A61K
2039/505 20130101; C12N 9/80 20130101 |
Class at
Publication: |
424/130.1 ;
514/012 |
International
Class: |
A61K 038/17; A61K
039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2001 |
WO |
PCT/US01/50785 |
Claims
1. A method for treating or preventing cardiovascular or
cerebrovascular disease, comprising administering an agent that
binds a sphingolipid or a sphingolipid metabolite.
2. The method of claim 1 wherein said agent is an antibody or
antibody derivative.
3. The method of claim 1 wherein said agent is a non-catalytic
derivative of an enzyme involved in the sphingolipid metabolic
pathways.
4. The method of claim 1 wherein said agent is a soluble fragment
of a receptor that binds a sphingolipid.
5. The method of claim 1, wherein said sphingolipid or a
sphingolipid metabolite is selected from the group consisting of
sphingomyelin, sphingosine, S-1-P, ceramide, SPC,
3-ketosphinganine, galactosylceramide and dihydroceramide.
6. The method of claim 1 wherein said sphingolipid is selected from
the group consisting of ceramide, sphingosine and S-1-P.
7. The method of claim 4 wherein said sphingolipid is S-1-P.
8. The method of claim 7 wherein said receptor is selected from the
group consisting of Edg-1, Edg-3, Edg-5, Edg-6, Edg-8, the Mil
receptor, AXOR29, NRG1, SCaMPER and homologs and isoforms
thereof.
9. The method of claim 7, wherein said receptor is an Edg
receptor.
10. The method of claim 9, wherein said Edg receptor is rat edg-3
receptor encoded by a nucleic acid having the sequence SEQ ID
NO:7
11. The method of claim 10, wherein said receptor is a SCaMPER.
12. The method of claim 11, wherein said SCaMPER is encoded by a
nucleic acid selected from the group consisting of SEQ ID NO:3 and
SEQ ID NO:4.
13. A method for treating or preventing cardiovascular or
cerebrovascular disease, comprising administering an agent that
binds a receptor of a sphingolipid or a sphingolipid
metabolite.
14. The method of claim 13 wherein said agent is an antibody or
antibody derivative.
15. The method of claim 13 wherein said agent is selected from the
group consisting of a sphingolipid, a sphingolipid metabolite, and
a sphingolipid analog.
16. The method of claim 13 wherein said receptor is selected from
the group consisting of Edg-1, Edg-3, Edg-5, Edg-6, Edg-8, the Mil
receptor, AXOR29, NRG1, SCaMPER and homologs and isoforms
thereof.
17. The method of claim 13, wherein said receptor is an Edg
receptor.
18. The method of claim 17, wherein said Edg receptor is rat edg-3
receptor encoded by a nucleic acid having the sequence SEQ ID
NO:7
19. The method of claim 13, wherein said receptor is a SCaMPER.
20. The method of claim 19, wherein said SCaMPER is encoded by a
nucleic acid selected from the group consisting of SEQ ID NO:3 and
SEQ ID NO:4.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. patent application
Ser. No. 60/257,926 entitled "Compositions and Methods for the
Treatment and Prevention of Cardiac and Myocardial Disorders" by
Sabbadini, Roger A., filed Dec. 22, 2000.
[0002] This application is related to U.S. patent application Ser.
No. ______ (attorney docket No. 078853-0302), Ser. No. ______
(attorney docket No. 078853-0305), and Ser. No. ______ (attorney
docket No. 078853-0306), each entitled "Compositions and Methods
for the Treatment and Prevention of Cardiovascular Diseases and
Disorders, and for Identifying Agents Therapeutic Therefor" by
Sabbadini, Roger A., and filed Dec. 21, 2001.
[0003] All of the preceding applications are hereby incorporated in
their entirety by reference thereto.
FIELD OF THE INVENTION
[0004] The invention relates generally to the area of treatment
and/or prevention of cardiovascular and cerebrovascular diseases,
disorders and physical trauma. The beneficial effect of the
invention is achieved through the use of pharmaceutical
compositions that contain agents that interfere with the production
and/or biological activities of sphingolipids and their
metabolites. The invention is also drawn to methods for isolating,
formulating and using pharmaceutical compositions, and kits and
medical devices comprising such compositions.
BACKGROUND OF THE INVENTION
[0005] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein, or any
publication specifically or implicitly referenced herein, is prior
art, or even particularly relevant, to the presently claimed
invention.
[0006] Cardiovascular Diseases and Disorders
[0007] Ischemic heart disease is the leading cause of death in the
U.S. Each year approximately 1.5 million people suffer heart
attacks (myocardial infarctions), of which 1/3 (i.e. about 500,000)
are fatal. In addition, about 6.75 million Americans suffer from
angina pectoris, the most common manifestation of cardiac ischemia.
Angina pectoris is a painful feeling of pressure in the chest that
results from ischemic heart disease. In total, there are 13.5
million patients living with ischemic heart disease in the U.S.
Americans in the high-risk categories for this disease include
persons having one or more indicators/risk factors therefor,
including but not limited to hypertension, high levels of serum
cholesterol and a family history of heart disease. Many people have
at least one of these indicator/risk factors; for example, there
are 50 million Americans diagnosed with hypertension alone.
[0008] "Ischemia" is a condition associated with an inadequate flow
of oxygenated blood to a part of the body, typically caused by the
constriction or blockage of the blood vessels supplying it.
Ischemia occurs any time that blood flow to a tissue is reduced
below a critical level. This reduction in blood flow can result
from: (i) the blockage of a vessel by an embolus (blood clot); (ii)
the blockage of a vessel due to atherosclerosis; (iii) the breakage
of a blood vessel (a bleeding stroke); (iv) the blockage of a blood
vessel due to acute vasoconstriction; (v) a myocardial infarction
(when the heart stops, the flow of blood to organs is reduced and
ischemia results); (vi) trauma; (vii) surgery, during which blood
flow to a tissue or organ needs to be reduced or stopped to achieve
the aims of surgery (e.g., angioplasty, heart and lung/heart
transplants); (viii) exposure to certain agents, e.g., dobutamine
or adenosine (Lagerqvist et al., Br. Heart J. 68:282-285, 1992) or
(ix) anti-neoplastic and other chemotherapeutic agents, such as
doxorubicin, that are cardiotoxic.
[0009] Even if the flow rate (volume/time) of blood is adequate,
ischemia may nonetheless occur due to hypoxia. "Hypoxia" refers to
conditions in which the oxygen content of blood is insufficient to
satisfy normal cellular oxygen requirements. Hypoxic blood is, by
definition, distinct from normoxic blood, i.e., blood in which the
oxygen content is sufficient to satisfy normal cellular oxygen
requirements. Such conditions include but are not limited to forms
of heart failure that adversely affect cardiac pumping such as
hypertension, arrhythmias, septic shock, trauma, cardiomyopathies
and congestive heart disease.
[0010] Myocardial ischemic disorders occur when cardiac blood flow
is restricted (ischemia) and/or when oxygen supply to the heart
muscle is compromised (hypoxia) such that the heart's demand for
oxygen is not met by the supply. Coronary artery disease (CAD)
arising from arteriosclerosis, particularly atherosclerosis, is the
most common cause of ischemia, and has symptoms such as stable or
unstable angina pectoris. CAD can lead to acute myocardial
infarctions (AMI) and sudden cardiac death. The spectrum of
ischemic conditions which result in heart failure is referred to as
Acute Coronary Syndrome (ACS). Reperfusion injury is often a
consequence of ischemia, in particular when anti-coagulants,
thrombolytic agents, or anti-anginal medications are used or when
the cardiac vasculature is surgically opened by angioplasty or by
coronary artery grafting.
[0011] Cardiotoxic agents are those materials which would cause a
loss of cardiac function, including negative inotropy, arthyTHmias,
heart failure, and cell death (both apoptotic and necrotic).
[0012] Presently, treatments for acute myocardial infarction and
other cardiac diseases include but are not limited to mechanical
devices and associated procedures therewith (e.g., coronary
angioplasty; Grines et al., N. Engl. J. Med. 3298:673-679, 1993);
thrombolytic agents such as streptokinase, tPA, and derivatives
thereof. Adjuvants to these therapies include beta-blockers,
aspirin and heparin, and glycoprotein (GP) IIb/IIIa inhibitors
(Antman et al., Circ. 99:2720-2732, 1999). GP IIb/IIIa inhibitors
decrease platelet aggregation and thrombus formation (for a review,
see Topol, Lancet 353:227-231, 1999). Examples include but are not
limited to monoclonal antibodies (e.g., abciximab), cyclic peptides
(e.g., eptifibatide), and nonpeptide peptidomimetics (e.g.,
tirofibian, lamifiban, xemilofiban, sibrafiban, and
lefradafibian).
[0013] Preventive treatments include but are not limited to those
that reduce a patient's cholesterol levels by, e.g., diet
management and pharmacological intervention. Statins are one type
of agent that have been used to reduce cholesterol levels. Statins
are believed to act by inhibiting the activity of HMG-CoA
reductase, which in turn increases the hepatic production of
cholesterol receptors (Nickenig et al., Circ. 100:2131-2134, 1999).
The hepatic cholesterol receptors bind cholesterol and remove it
from blood. Such agents include but are not limited to lovastatin,
simvastatin, pravastatin, fluvastatin (Lennernas, Clin.
Pharmackinet. 32:403-425, 1997). These and other statins slows the
progression of coronary artery disease, and may induce regression
of atherosclerotic lesions in patients. It is not known, however,
whether other reductases are inhibited by such agents, and what
side effects might occur as a result.
[0014] Cerebrovascular Diseases and Disorders
[0015] Patients experiencing cerebral ischemia often suffer from
disabilities ranging from transient neurological deficit to
irreversible damage (stroke) or death. Cerebral ischemia, i.e.,
reduction or cessation of blood flow to the central nervous system,
can be characterized as either global or focal.
[0016] Focal cerebral ischemia refers to cessation or reduction of
blood flow within the cerebral vasculature resulting from a partial
or complete occlusion in the intracranial or extracranial cerebral
arteries. Such occlusion typically results in stroke, a syndrome
characterized by the acute onset of a neurological deficit that
persists for at least 24 hours, reflecting focal involvement of the
central nervous system and is the result of a disturbance of the
cerebral circulation. Other causes of focal cerebral ischemia
include vasospasm due to subarachnoid hemorrhage or iatrogenic
intervention.
[0017] Global cerebral ischemia refers to reduction of blood flow
within the cerebral vasculature resulting from systemic circulatory
failure. The failure of the circulatory system to maintain adequate
cellular perfusion leads to a in reduction of oxygen and nutrients
to tissues. Thus, global cerebral ischemia results from severe
depression of cardiac performance. The most frequent cause is acute
myocardial infarction with loss of substantial muscle mass. Pump
failure can also result from acute myocarditis or from depression
of myocardial contractility following cardiac arrest or prolonged
cardiopulmonary bypass. Mechanical abnormalities, such as severe
valvular stenosis, massive aortic or mitral regurgitation, acutely
acquired ventricular septal defects, can also reduce cardiac
output. Additional causes include cardiac arrhythmia, such as
ventricular fibrillation, and any cardiac disease described herein.
Further causes include interventional procedures, such as carotid
angioplasty, stenting or endarterectomy, which might otherwise
result in focal cerebral ischemia, and also cardiac procedures
which may result in global cerebral ischemia, such as cardiac
catheterization, electrophysiologic studies, and angioplasty.
[0018] Those skilled in the art are easily able to identify
patients having a stroke or at risk of having a stroke, cerebral
ischemia, head trauma, or epilepsy. For example, patients who are
at risk of having a stroke include, but are not limited to,
patients having hypertension or undergoing major surgery.
[0019] Traditionally, emergent management of acute ischemic stroke
consists of mainly general supportive care, e.g. hydration,
monitoring neurological status, blood pressure control, and/or
anti-platelet or anti-coagulation therapy. Heparin has been
administered to stroke patients with limited and inconsistent
effectiveness. In some circumstances, the ischemia resolves itself
over a period of time due to the fact that some thrombi get
absorbed into the circulation, or fragment and travel distally over
a period of a few days. In 1996, the Food and Drug Administration
approved the use of tissue plasminogen activator (t-PA) or
Activase.RTM., for treating acute stroke. However, treatment with
systemic t-PA is associated with increased risk of intracerebral
hemorrhage and other hemorrhagic complications. Aside from the
administration of thrombolytic agents and heparin, there are no
therapeutic options currently on the market for patients suffering
from occlusion focal cerebral ischemia. Vasospasm may be partially
responsive to vasodilating agents. The newly developing field of
neurovascular surgery, which involves placing minimally invasive
devices within the carotid arteries to physically remove the
offending lesion may provide a therapeutic option for these
patients in the future, although this kind of manipulation may lead
to vasospasm itself.
[0020] Documents
[0021] U.S. Pat. No. 6,210,976 B1 and published PCT patent
application WO98/57179 (PCT/US98/10486), both entitled "Methods for
Early Detection of Heart Discase", hereby incorporated by
reference, relate to the use of blood levels of certain
sphingolipids for screening for early ischemic events before
symptoms are presented in persons with high risk for heart disease,
or in a triage setting for patients with acute coronary
syndrome.
[0022] PCT Application PCT/US01/12706, published as WO 01/80903,
entitled "Detection and Treatment of Atherosclerosis Based on
Plasma Sphingomyelin Concentration", relates to enzymatic methods
to measure plasma and tissue sphingomyelin concentrations, and that
human plasma sphingomyelin levels are positively correlated with
atherosclerosis and coronary heart disease.
[0023] U.S. Pat. No. 5,929,039, entitled "Method for Treating
Cardiac Dysfunction and Pharmaceutical Compositions Useful
Therefor", relates to disclose methods methods for the prophylaxis
or treatment of cardiac arrhythmia using an agent capable of
blocking or inhibiting the effect or release of
inositol(1,4,5)trisphosphate in cardiac tissue. The agent may be an
aminoglycoside, including gentamicin.
[0024] U.S. Pat. No. 5,677,288, entitled "Use of Aminoglycosides to
Protect Against Excitotoxic Neuron Damage", relates to the use of
an aminoglycoside, which may be gentamicin, that suppresses the
flow of calcium ions into neurons through N-type calcium channels.
The method relates to reducing excitotoxic damage to neurons, which
can occur as a result of stroke, cerebral ischemia/hypoxia, or
other events or conditions.
[0025] Published U.S. Patent Application 20010041688, entitled
"Methods and Compositions for the Regulation of Vasoconstriction",
relates to modulation of sphingosine kinase and
sphingosine-1-phosphate phosphatase activity and EDG receptor
signaling for the treatment of conditions relating to
vasoconstriction and vasoconstriction, including migraine, stroke,
subarachnoid hemorrhage and vasospasm.
[0026] Ancellin et al., "Extracelluar export of sphingosine
kinase-1 enzyme: Sphingosine 1 phosphate generation and the
induction of angiogenic vascular maturation", JBC Papers in Press.
Published on Dec. 10, 2001 as manuscript M102841200 relates to
events related to angiogenosis that are mediated by a sphingosine
kinase.
SUMMARY OF THE INVENTION
[0027] The invention is drawn to compositions and methods for
treating or preventing cardiovascular, cardiac, myocardial and
other diseases, disorders or physical trauma, and/or cerbrovascular
diseases and disorders, in which therapeutic agents are
administered to a patient that alters the activity or concentration
of an undesirable, toxic and/or cardiotoxic sphingolipids, or
metabolites thereof. The therapeutic methods and compositions of
the invention are said to be "sphingolipid-based" in order to
indicate that they act by changing the absolute, relative and/or
available concentration and/or activities of certain undesirable,
toxic or cardiotoxic sphingolipids. The invention is also drawn to
chemical libraries and screening assays that are used to identify
novel sphingolipid-based therapeutics.
[0028] The compositions of the invention are used in methods of
sphingolipid-based cardiovascular and cardiac therapy. "Cardiac
therapy" refers to the prevention and/or treatment of myocardial
diseases, disorders or physical trauma. Conditions of particular
interest include but not limited to myocardial ischemia; acute
myocardial infarction (AMI); coronary artery disease (CAD); acute
coronary syndrome (ACS); cardiac cell and tissue damage that may
occur during or as a consequence of pericutaneous revascularization
(coronary angioplasty) with or without stenting; coronary bypass
grafting (CABG) or other surgical or medical procedures or
therapies that may cause ischemic or ischemic/reperfusion damage in
humans; and cardiovascular trauma.
[0029] "Cardiovascular therapy" encompasses cardiac therapy as well
as the prevention and/or treatment of other diseases associated
with the cardiovascular system, such as heart disease. The term
"heart disease" encompasses any type of disease, disorder, trauma
or surgical treatment that involves the heart or myocardial tissue.
Of particular interest are heart diseases that relate to hypoxia
and/or ischemia of myocardial tissue and/or heart failure. One type
of heart disease that can result from ischemia is reperfusion
injury, such as can occur when anti-coagulants, thrombolytic
agents, or anti-anginal medications are used in therapy, or when
the cardiac vasculature is surgically opened by angioplasty or by
coronary artery grafting. Another type of heart disease to which
the invention is directed is oronary artery disease (CAD), which
can arise from arteriosclerosis, particularly atherosclerosis, a
common cause of ischemia. CAD has symptoms such as stable or
unstable angina pectoris, and can lead to acute myocardial
infarctions (AMI) and sudden cardiac death. The term "heart
failure" encompasses acute myocardial infarction, myocarditis, a
cardiomyopathy, congestive heart failure, septic shock, cardiac
trauma and idopathic heart failure. The spectrum of ischemic
conditions which result in heart failure is referred to as Acute
Coronary Syndrome (ACS).
[0030] "Cerebrovascular therapy" refers to therapy directed to the
prevention and/or treatment of diseases and disorders associated
with cerebral ischemia and/or hypoxia. Of particular interest is
cerebral ischemia and/or hypoxia resulting from global ischemia
resulting from a heart disease, including without limitation heart
failure.
[0031] "Toxic sphingolipids" are those sphingolipids that can cause
or enhance the necrosis and/or apoptosis of cells, including, in
some instances, particular cell types that are found in specific
tissues or organs. "Cardiotoxic sphingolipids" are toxic
sphingolipids that directly or indirectly cause or enhance cardiac
arrythmias, the negative inotropy (loss of contractile function) of
the heart and the necrosis and/or apoptosis of cells found in or
associated with the heart, including but not limited to
cardiomyocytes, cardiac neurons and the like. "Undesirable
sphingolipids" include toxic and cardiotoxic sphingolipids, as well
as metabolites, particularly metabolic precursors, of toxic and
cardiotoxic sphingolipids. Undesirable, cardiotoxic and/or toxic
sphingolipids of particular interest include but are not limited to
ceramide (CER), sphingosine-1-phosphate (S-1-P) and sphingosine
(SPH; D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol, or
sphinganine).
[0032] The term "metabolites" refers to compounds from which
sphingolipids are made, as well as those that result from the
degradation of sphingolipids; that is compounds that are involved
in the sphingolipid metabolic pathways (FIGS. 1 and 2). Metabolites
include metabolic precursors and metabolic products. The term
"metabolic precursors" refers to compounds from which sphingolipids
are made. Metabolic precursors of particular interest include but
are not limited to SPC, sphingomyelin, dihydrosphingosine,
dihydroceramide, and 3-ketosphiganine. The term "metabolic
products" refers to compounds that result from the degradation of
sphingolipids, such as phosphorylcholine (a.k.a. phosphocholine,
choline phosphate), fatty acids, including free fatty acids, and
hexadecanal (a.k.a. palmitaldehyde).
[0033] As used herein, the term "therapeutic" encompasses the full
spectrum of treatments for a disease or disorder. A "therapeutic"
agent of the invention may act in a manner that is prophylactic or
preventive, including those that incorporate procedures designed to
target individuals that can be identified as being at risk
(pharmacogenetics); or in a manner that is ameliorative or curative
in nature; or may act to slow the rate or extent of the progression
of a disease or disorder; or may act to minimize the time required,
the occurrence or extent of any discomfort or pain, or physical
limitations associated with recuperation from a disease, disorder
or physical trauma; or may be used as an adjuvant to other
therapies and treatments. The term "cardiotherapeutic agent" refers
to an agent that is therapeutic to diseases and diseases caused by
or associated with cardiac and mycardial diseases and
disorders.
[0034] Without wishing to be bound by any particular theory, it is
believed that the level of undesirable sphingolipids such as SPH or
S-1-P, and/or one or more of their metabolites, cause or contribute
to the development of cardiac and myocardial diseases and
disorders. Because sphingolipids are also involved in fibrogenesis
and wound healing of liver tissue (Davaille et al., J. Biol. Chem.
275:34268-34633, 2000; Ikeda et al., Am J. Physiol. Gastrointest.
Liver Physiol 279:G304-G310, 2000), healing of wounded vasculatures
(Lee et al., Am. J. Physiol. Cell Physiol. 278:C612-C618, 2000),
and other disease states or disorders, or events associated with
such diseases or disorders, such as cancer, angiogenesis and
inflammation (Pyne et al., Biochem. J. 349:385-402, 2000), the
compositions and methods of the present disclosure may be applied
to treat these diseases and disorders as well as cardiac and
myocardial diseases and disorders.
[0035] One form of sphingolipid-based therapy involves manipulating
the metabolic pathways of sphingolipids in order to decrease the
actual, relative and/or available in vivo concentrations of
undesirable, toxic and/or cardiotoxic sphingolipids. The invention
provides compositions and methods for treating or preventing
cardiac and myocardial diseases, disorders or physical trauma, in
which therapeutic agents are administered to a patient that alters
the activity or concentration of an enzyme, wherein the enzyme
catalyzes a reaction that produces or degrades undesirable, toxic
and/or cardiotoxic sphingolipids, or metabolites thereof. An
"enzyme" is a protein or polypeptide that catalyzes (causes,
accelerates or enhances) a chemical reaction. The term "metabolism"
is used to describe the biological construction or destruction of a
compound. Metabolism comprises the synthesis (constructive
metabolism, a.k.a. anabolism) of compounds and the degradation
(destructive metabolism, a.k.a. catabolism) thereof. Enzymes of
particular interest, and preferred modulating agents thereof
(inhibitors/activators or stimulators/blocking agents), are
described in the Detailed Description (see also Examples 7 through
10).
[0036] In one version of this form of sphingolipid-based therapy,
metabolic steps that involve the production of sphingolipids are
inhibited or blocked. Therapeutic agents and methods are used to
decrease the amount or activity of enzymes that catalyze chemical
reactions that degrade undesirable sphingolipids and/or metabolic
precursors thereof. Thus, net sphingolipid catabolism is
increased.
[0037] In another version of this form of sphingolipid-based
therapy, metabolic steps that involve the destruction of
sphingolipids are activated or stimulated. Therapeutic agents and
methods are used to increase the amount or activity of enzymes that
catalyze chemical reactions that degrade undesirable sphingolipids
and/or metabolic precursors thereof. Thus, net sphingolipid
anabolism is decreased.
[0038] One form of sphingolipid-based therapy involves the use of
agents that bind undesirable, toxic and/or cardiotoxic
sphingolipids, or metabolites thereof. Such sphingolipid-binding
agents include but are not limited to proteins and polypeptide
derivatives thereof that bind undesirable, toxic and/or cardiotoxic
sphingolipids or metabolites thereof. Such a protein and
polypeptide may, by way of non-limiting example, be a non-catalytic
derivative of an enzyme involved in the sphingolipid metabolic
pathways, a derivative of proteins that participate in the
sphingomyelin signaling pathway, a derivative of a receptor that
binds an undesirable, toxic and/or cardiotoxic sphingolipid, an
antibody or antibody derivative that is directed to (specifically
binds) an undesirable, toxic and/or cardiotoxic sphingolipid. Such
derivatives are preferably water soluble. (Sphingolipid-binding
agents are described in the Detailed Description of the Invention;
see also Examples 6 and 14).
[0039] One form of sphingolipid-based therapy involves the use of
agents that bind sphingolipid receptors that initiate and stimulate
the sphingomyelin signaling pathway. This pathway ultimately
results in increased ceramide production. An increased level of
ceramide would, in turn, be expected to result in elevated
concentrations of undesirable sphingolipids such as, e.g., S-1-P
and SPH. Thus, inhibiting or blocking such receptors decreases, or
at least prevents an increase due to the sphingomyelin signaling
pathway, the intracellular production of ceramide and metabolites
thereof (see the Detailed Description and Example 9). Another form
of sphingolipid-based therapy involves the use of molecular
genetics to generate therapeutic agents (see the Detailed
Description and Example 18).
[0040] In one version of this form Of sphingolipid-based therapy,
the therapeutic agent is a protein (including, without limitation,
polypeptides, oligopeptides, and peptidomimetics). A "protein" is a
molecule having a sequence of amino acids that are linked to each
other in a linear molecule by peptide bonds. The term protein
refers to a polypeptide that is isolated from a natural source, or
produced from an isolated cDNA using recombinant DNA technology;
and has a sequence of amino acids having a length of at least about
200 amino acids. As used herein, the term "polypeptide" includes
proteins, fusion proteins, oligopeptides and polypeptide
derivatives, with the exception that peptidomimetics are considered
to be small molecules herein. An "oligopeptide" is a polypeptide
having a short amino acid sequence (i.e., 2 to about 200 amino
acids). An oligopeptide is generally prepared by chemical
synthesis. Although oligopeptides and protein fragments may be
otherwise prepared, it is possible to use recombinant DNA
technology and/or in vitro biochemical manipulations. For example,
a nucleic acid encoding an amino acid sequence may be prepared and
used as a template for in vitro transcription/translation
reactions.
[0041] A "protein fragment" is a proteolytic fragment of a larger
polypeptide, which may be a protein or a fusion protein. A
proteolytic fragment may be prepared by in vivo or in vitro
proteolytic cleavage of a larger polypeptide, and is generally too
large to be prepared by chemical synthesis. Preferably, proteolytic
fragments have amino acid sequences having a length from about 10
to about 5,000 amino acids; more preferably about 200 to 1000 amino
acids; most preferably 200 to about 1,000 amino acids.
[0042] A therapeutic protein may be a dominant negative mutant of
an enzyme that catalyzes a reaction that results in the production
of an undesirable, toxic and/or cardiotoxic sphingolipid or a
metabolite thereof, of a receptor for such a sphingolipid, or of a
protein that participates in the sphingomyelin signaling pathway. A
"dominant negative mutant protein" is one that, when expressed, (I)
docs not itself provide the activity of the wildtype protein and
(ii) inhibits the action of the wildtype form of the protein. The
therapeutic protein may be an enzyme, produced by recombinant DNA
technology or any other appropriate method, that catalyzes a
reaction that results in the degradation of a undesirable, toxic
and/or cardiotoxic sphingolipid, or a metabolite thereof; and such
an enzyme may be one that has been altered via molecular genetics
to have improved desirable properties such as enhanced catalytic
activity, tighter substrate binding, etc.
[0043] In another version of this form of sphingolipid-based
therapy, the therapeutic agent is a nucleic acid (including,
without limitation, DNA, RNA, and oligonucleotides). A therapeutic
nucleic acid may have a sequence that is antisense to a nucleotide
sequence found within an mRNA that encodes an enzyme that catalyzes
a reaction that results in the production of a undesirable, toxic
and/or cardiotoxic sphingolipid, or a metabolite thereof, or a
receptor thereof. Such nucleic molecules include antisense
oligonucleotides. Such antisense nucleic acids bind to a specific
target mRNA due to their complementary sequences, and prevent the
mRNA from being processed or translated, or enhance or cause the
degradation of the mRNA. A therapeutic nucleic acid may be a gene
therapy construct that comprises and expresses, over-expresses or
constitutively expresses (i) nucleic acids that are antisense to
those that encode an enzyme that catalyzes a reaction that results
in the production of a undesirable, toxic and/or cardiotoxic
sphingolipid or a metabolite thereof; (ii) therapeutic proteins,
such as enzyme that degrades a sphingolipid, or a dominant negative
mutant that inhibits such an enzyme, or a sphingolipid-binding
protein.
[0044] Any composition and method of the invention that may be used
in sphingolipid-based therapy may be used in combination with any
other compositions and methods for sphingolipid-based therapy, as
well as in conjunction with therapeutic agents and compositions
that are not sphingolipid-based. Useful adjuvant treatments for the
sphingolipid-based treatments of the invention modulate the
sphingomyelin signaling pathway and/or inhibit cytokines (see the
Detailed Description and Example 14). An "adjuvant" is any agent
that is added to a composition or therapeutic regimen to aid the
therapeutic effect of the active agent(s) thereof.
[0045] An agent for sphingolipid-based therapy is formulated in a
pharmaceutical composition. The pharmaceutical compositions of the
invention may be formulated for rapid cardiac delivery. By "rapid
cardiac delivery" it is meant that the therapeutic agent reaches a
therapeutically effective concentration in the blood, serum, or
specified tissue within about 30 to 60 minutes, preferably within
about 15 to 20 minutes, more preferably within about 5 to 10
minutes, and most preferably within about 5 seconds to about 5
minutes, after its administration. The pharmaceutical compositions
are used to treat cardiac, myocardial and other diseases, disorders
or physical trauma.
[0046] Pharmaceutical and pharmaceutical compositions comprising
one or more therapeutic agents of the invention are incorporated
into kits and medical devices for such treatments. Medical devices
are used to administer the pharmaceutical compositions of the
invention to a patient in need thereof, and kits that include such
devices. Such devices and kits may be designed for the routine
administration, including self-administration, of the
pharmaceutical compositions of the invention. Such devices and kits
may also be designed for emergency use, i.e., in ambulances or
emergency rooms, or during surgery, or in activities where injury
is possible but where full medical attention may not be immediately
forthcoming (i.e., hiking and camping, or combat situations). The
invention thus provides cardiac and myocardial therapies based on
the role of sphingolipids in cardiac and myocardial diseases,
disorders and physical trauma.
[0047] The invention also provides screening assays, including
high-throughput screening (HTS) assays, that are useful for
identifying novel sphingolipid-based therapeutics. Chemical
libraries are screened using these assays, preferably in a high
throughput manner, to identify lead compounds and therapeutic
agents.
[0048] In a related aspect, the invention provides a method of
identifying molecules that specifically bind to, and/or otherwise
interfere with the action of a sphingolipid target. A "sphingolipid
target" is any molecule or moiety that is desired to obtain novel
compounds that bind thereto or otherwise inhibit the activity
thereof. Sphingolipid targets of the inventions include, but are
not limited to, sphingolipids per se; sphingolipid receptors; and
molecules involved in sphingolipid metabolism, including but not
limited to enzymes that act on sphingolipids and sphingolipid
metabolites.
[0049] In another embodiment, sphingolipids that are cardiotoxic at
relatively high concentrations are used to preconditon hearts.
Preconditioning hearts with short cycles of ischemia and
reperfusion is known to have a cardioprotective effect in rodents
(Yellon et al., Cardiovasc Res 26.983-987, 1992; Napoli et al., J
Clin Bas Cardiol 1:37-42, 1998). In the preconditioning methods of
the invention, sphingolipids that are cardiotoxic are administered
in small doses. In the methods of the invention, sphingolipids,
including but not limited to ceramide, sphingosine and
sphingosine-1-phosphate given in low, intermittent doses may
protect cardiac tissue from ischemia.
[0050] The invention provides benefits not previously obtainable in
cardiovascular, cardiac and myocardial treatments. By way of
non-limiting example, the consequences of acute cardiac or
myocardial events may result from the end result of a cascade of
molecular events that evolve rapidly after symptoms become
apparent. Treatments that address early events in the cascade may
not be able to "catch up" with such events, i.e., may not achieve
an effective level until after some undesirable molecules that lead
to cardiac or myocardial damage have been produced.
Sphingolipid-based therapies act on undesirable events and
molecules that occur or are present at the later stages of these
cascades, they can act before such undesirable events occur or
undesirable molecules are produced, and thus can prevent the
occurrence of such events and/or production of such compounds to a
greater degree than can be realized by therapies that act earlier
in the cascade. Sphingolipid-based therapies addresses events that
lead directly (rather than indirectly) to myocardial ischemia and
other cardiac disorders, and undesirable side-effects of indirect
treatments are thus reduced, minimized or eliminated.
Sphingolipid-based therapies provide for preventative treatments
that achieve an effective state relatively quickly and
non-intrusive as compared to other preventative measures, e.g.,
changes in diet or surgery.
[0051] The summary of the invention described above is not limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the preferred
embodiments, as well as from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] FIG. 1 shows a set of biochemical reactions that are a
central part of the sphingolipid metabolic pathways.
[0053] FIG. 2 is a more expansive view of sphingolipid metabolism
and includes the biochemical reactions and enzymes shown in FIG. 1.
Abbreviations: DAG, diacylglycerol); PtdCho,
phosphatidylcholine.
[0054] FIG. 3 shows the fate of .sup.3H-labeled SPH in whole blood.
After labeled sphingosine (SPH) is added to human whole blood, the
concentration of SPH drops while the concentration of labeled
sphingosine-1-phosphate (S1P) increases, suggesting that SPH is
converted into S-1-P in blood; in contrast, little of the label is
detected as labeled hexadecanal (HD). Symbols: solid line, S1P;
line with long dashes, HD; line with short dashes, SPH.
[0055] FIG. 4 shows results of experiments that demonstrate that
L-carnitine blocks the hypoxia-induced production of sphingosine in
a cellular model.
[0056] FIG. 5 shows results of experiments in which rat hearts are
subject to ischemia with (grey lines) or without (black lines) an
inhibitor of sphingomyelinase.
[0057] FIG. 6 shows the general chemical structure of
aminoglycosides. "R1" through "R13" are substituent groups.
ABBREVIATIONS
[0058] Unless otherwise indicated, the following abbreviations are
used herein.
1 Sphingolipids DHSPH Dihydrosphingosine CER ceramide
(N-acylsphingosine) SPC Sphingosylphosphorylcholinc SPH Sphingosine
S-1-P sphingosine-1-phosphate (a.k.a. S1P or SPP) SM
Sphingomyelin
[0059]
2 Enzymes CER kinase ceramide kinase SMase Sphingomyelinase
SM-deacylase Sphingomyelin deacylase SPH kinase Sphingosine kinase
S-1-P Lyase Sphingosine-1-phosphate lyase S-1-P
Sphingosine-1-phosphate lyase phosphatase Phosphatase
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention provides methods and compositions
useful for the treatment of cardiovascular and cerebrovascular
diseases and disorders, as well as other disease states that relate
to sphingolipids and sphingolipid metabolites. The methods and
compositions act by interfering with the metabolism of various
sphingolipids and/or their metabolites; by binding sphingolipids,
thereby reducing their effective concentration; by modulating the
sphingomyelin signaling pathway; via modalities based on molecular
genetics (including but not limited to the use of dominant negative
proteins, antisense, gene therapy, and the like). All the above
modalities of cardiovascular therapy may be used alone, in
combination with each other, and/or in combination with other
methods and compositions useful for cardiovascular therapy
(including but not limited to those that interfere with the action
of certain cytokines). The therapeutic methods and compositions of
the invention are said to be "sphingolipid-based" in order to
indicate that these therapies act by changing the relative,
absolute or available concentration(s) of certain undesirable,
toxic or cardiotoxic sphingolipids. Therapeutic administration of
exogenous sphingolipids may have therapeutic benefit if give in a
preconditiong regimen (i.e., low doses given intermittently).
[0061] Applicants believe, without wishing to be bound by any
particular theory, that the level of undesirable sphingolipids such
as CER, SPH or S-1-P, and/or one or more of their metabolites, may
be directly responsible for cardiac dysfunction, during or
immediately after cardiac ischemia such as during reperfusion
injury. For example, sphingosine has negative inotropic effects on
the heart (Oral H, Dom GW, Mann DL. Sphingosine mediates the
immediate negative inotropic effects of tumor necrosis factor-a in
the adult mammalian cardiac myocyte. J. Biol. Chem.
1997;272:4836-4842; Krown K, Yasui K, Brooker M, et al. TNF.alpha.
receptor expression in rat cardiac myocytes: TNF.alpha. inhibition
of L-type Ca2+ current and Ca2.div. transients. FEBS Letters
1995;376:24-30; Smith G W, Constable P D, Eppley R M, Tumbleson M
E, Gumprecht L A, Haschek-Hock W M. Purified fumonisin B1 decreases
cardiovascular function but does not alter pulmonary capillary
permeability in swine. Toxicological Sciences 2000;56:240-249,
blocks Na/Ca exchangers Condrescu M, Reeves J P. Inhibition of
sodium-calcium exchange by ceramide and sphingosine. J. Biol. Chem.
2001;276:4046-4054, and the L-type calcium channel in heart cells
Krown K, Yasui K, Brooker M, et al. TNFa receptor expression in rat
cardiac myocytes: TNTa inhibition of L-type Ca2+ current and Ca2+
transients. FEBS Letters 1995;376:24-30, and modulates the
ryanodine receptor McDonough PM, Yasui K, Betto R, et al. Control
of cardiac Ca2+ levels: inhibitory actions of sphingosine on Ca2+
transients and L-channel conductance. Circ. Res. 1994;75:981-989,
all of which can cause calcium deregulation that is observed when
cardiac cells are treated with sphingosine (McDonough P M, Yasui K,
Betto R, et al. Control of cardiac Ca2+ levels: inhibitory actions
of sphingosine on Ca2+ transients and L-channel conductance. Circ.
Res. 1994;75:981-989; Krown K, Yasui K, Brooker M, et al. TNF(L
receptor expression in rat cardiac myocytes: TNF.alpha. inhibition
of L-type Ca2+ current and Ca2+ transients. FEBS Letters
1995;376:24-30). In addition, SPH inhibits the Na/H exchanger (Lowe
J H N, Huang C-L, Ives HE. Sphingosine differentially inhibits
activation of the Na+/H+exchange by phorbol esters and growth
factors. J. Biol. Chem. 1990;265:7188-7194), that is responsible
for pH regulation. Sphingosine has also been shown to produce cell
death in heart cells Krown K A, Page M T, Nguyen C, et al.
TNF.alpha.-induced apoptosis in cardiac myocytes: Involvement of
the sphingolipid signalling cascade in cardiac cell death. J. Clin.
Invest. 1996;98:2854-2865; Zechner D, Craig R, Hanford D, McDonough
P M, Sabbadini R A, Glembotski C C. MKK6 inhibits myocardial cell
apoptosis via a p38 MAP kinase-dependent pathway. J. Biol. Chem.
1998;273:8232-8239), and can also produce free radica damage during
reperfusion Hernandez 0, Discher, D., Bishorpric, N., Webster, K.
Rapid Activation of Neutral Sphingomyelinase by
Hypoxia-Reoxygenation of Cardiac Myocytes. Circ. Res 2000:198-204.
Sphingosine-1-phosphate has been shown to produe cell death Zechner
D, Craig R, Hanford D, McDonough .mu.M, Sabbadini R A, Glembotski C
C. MKK6 inhibits myocardial cell apoptosis via a p38 MAP
kinase-dependent pathway. J. Biol. Chem. 1998;273:8232-8239, and
promote arrthymias and coronary vasoconstriction Sugiyama A, Yatomi
Y, Ozaki Y, Hashimoto K. Sphingosine 1-phosphate induces sinus
tachycardia and coronary vasoconstriction in the canine heart.
Cardiovasc. Res. 2000;46:119-125; MacDonnell K, Severson D, Giles
W. Depression of excitability by sphingosine 1-phosphate in rat
ventricular myocytes. Am. J. Physiol. 1998;44:H2291-H2299; and
Liliom K, Sun G, Bunemanns M, et al. Sphingosylphosphocholine is a
naturally occuring lipid mediator in blood plasma: a possible role
in regulating cardiac function via sphingolipid receptors. Biochem
J. 2001;355:189-197).
[0062] Because sphingolipids such as S-1-P are involved in
fibrogenesis and wound healing of liver tissue (Davaille et al., J.
Biol. Chem. 275:34268-34633, 2000; Ikeda et al., Am J. Physiol.
Gastrointest. Liver Physiol 279:G304-G310, 2000), healing of
wounded vasculatures (Lee et al., Am. J. Physiol. Cell Physiol.
278:C612-C618, 2000), and other disease states, or events
associated with such diseases, such as cancer, angiogenesis and
inflammation (Pyne et al., Biochem. J. 349:385-402, 2000), the
compositions and methods of the disclosure may be applied to treat
these diseases as well as cardiac diseases. For example, S-1-P may
be used therapeutically as a promoter of cardiac angiogenesis. The
ability of S-1-P to stimulate angiogenesis in cell culture and in
non-cardiac tissue has been reported (Lee et al., Sphingosine
1-Phosphate induces angiogenesis: its angiogenic action and
signaling mechanism in human umbilical endothelial cells. Biochem
Biophys Res Commun 1999;264:743-325; Lee et al., Am J Physiol Cell
Physiol 278:C612-C618, 2000). Recent evidence suggests that
exogenously administered S-1-P crosses the blood-brain barrier and
promotes cerebral vasoconstriction (Tosaka et al., Stroke 32:
2913-2919. 2001). This suggests that sphingolipids derived from
cardiac or other non-cerebral sources could contribute to stroke.
Consequently, interfering with sphingolipid production and/or
action may be beneficial in mitigating stroke, particularly in
stroke casued by peripherical vascular disease, atherosclerosis and
cardiac disorders. s.For example, S1P may be used therapeutically
as a promoter of cardiac angiogenesis. The ability of S1P to
stimulate angiogenesis in cell culture and in non-cardiac tissue
has been reported [Lee, 1999 #1508]. Recent evidence suggests that
exogenously administered S1P crosses the blood-brain barrier and
promotes cerebral vasoconstriction (Tosaka et al., Stroke 32:
2913-2919. 2001). This suggests that sphingolipids derived from
cardiac or other non-cerebral sources could contribute to stroke.
Consequently, interfering with sphingolipid production and/or
action may be beneficial in mitigating stroke, particularly in
stroke casued by peripherical vascular disease, atherosclerosis and
cardiac disorders.
[0063] It has been suggested that an early event in the course of
cardiac ischemia (i.e., lack of blood supply to the heart) is an
excess production by the heart muscle of the naturally occurring
compound sphingosine, and that other metabolites, particularly
sphingosine-1-phosphate (S-1-P), are also produced either by the
heart tissue itself or by components of blood as a consequence of
cardiac sphingolipid production and subsequent conversion in the
blood. The present invention provides methods and the compositions
thereof to inhibit and/or activate sphingolipid production and/or
metabolism. More specifically, the present invention provides
methods and the compositions that may block production of SPH,
S-1-P and other metabolites by inhibiting and/or activating
metabolic enzymes and/or sphingolipid receptors involving in the
sphingolipid metabolic pathways. Since either hypoxia per se and/or
cardiac-derived TNF.alpha. and/or other cytokines may trigger the
sphingomyelin signal transduction cascade in the heart to increase
the production of SPH, S-1-P and other metabolites, the present
invention also provides methods and compositions to block cytokine
release and/or its action.
[0064] The present invention thus provides methods and compositions
thereof to reduce blood and tissue levels of key sphingolipids,
e.g., SPH and S-1-P. Such methods and compositions include, but are
not limited to, monoclonal and/or polyclonal antibodies directed to
sphingolipids, which may be used, for example, to bind and thus
lower the effective concentration of, undesirable sphingolipids in
whole blood. The present invention also provides methods and the
compositions thereof to indirectly reduce the absolute or effective
(available) blood and tissue levels of key sphingolipids, e.g. SPH
and S-1-P, including but not limited to methods and compositions
for inhibiting and/or activating enzymes involving in the
sphingolipid metabolic pathways; for the use of soluble fragments
containing the sphingolipid binding domain of enzymes involved in
sphingolipid metabolism, or the binding domain of sphingolipid
binding proteins, to bind and reduce the effective concentration of
undesirable sphingolipids; for the use of negative dominant (a.k.a.
"transdominant") mutants of sphingolipid receptors and enzymes
involved in sphingolipid metabolism; for genetic therapy to provide
or alter a function of a sphingolipid enzyme or receptor; and for
the use of anti sense oligonucleotides or transcripts against mRNAs
of the sphingolipid metabolic enzymes, and/or sphingolipid
receptors, to reduce or eliminate the genetic expression of these
enzymes. For a review of sphingolipid metabolism, see Liu et al.,
Crit Rev. Clin. Lab. Sci. 36:511-573, 1999.
[0065] The present invention also provides compositions for
inhibiting the action or expression of cytokines, interferons,
chemokines and the like, that may modulate events that occur during
the sphingomyelin signaling pathway. This pathway, which it has
been suggested is activated during cardiac ischemia/hypoxia
(Bielawska et al., Am. J. Pathol. 151:1257-1263, 1997; Meldrum, Am.
J. Physiol. 274:R577-R595, 1998; and Cain et al., J. Mol. Cell.
Cardiol. 31:931-947, 1999), and which is stimulated by cytokines,
interferons, chemokines and the like, ultimately results in
increased ceramide production. An increased level of ceramide
would, in turn, be expected to result in elevated concentrations of
undesirable sphingolipids such as, e.g., S-1-P and SPH. For reviews
of the sphingomyelin signaling pathway, see Hannun et al., Adv.
Lipid Res. 25:27-41, 1993; Liu et al., Crit. Rev. Clin. Lab. Sci.
36:511-573, 1999; Igarashi, J. Biochem. 122:1080-1087, 1997; Oral
et al., J. Biol. Chem. 272:4836-4842, 1997; and Spiegel et al.,
Biochemistry (Moscow) 63:69-83, 1998.
[0066] Sphingolipids
[0067] The therapeutic methods and compositions of the invention
are said to be "sphingolipid-based" in order to indicate that these
therapies can change the relative, absolute or available
concentration(s) of certain undesirable, toxic or cardiotoxic
sphingolipids. "Toxic sphingolipids" are those that can, under
certain circumstances, disturb the normal function of cells such as
ones that cause or enhance the necrosis and/or apoptosis of cells,
including, in some instances, particular cell types that are found
in specific tissues or organs. "Cardiotoxic sphingolipids" are
toxic sphingolipids that directly or indirectly cause a negative
inotropic state or cause or enhance the necrosis and/or apoptosis
of cells found in or associated with the heart, including but not
limited to cardiomyocytes, cardiac neurons and the like, and/or can
cause loss of cardiac function due to the negative inotropic,
arrhythmic coronary vasoconstriction/spasm effects of the
sphingolipids and/or their metabolites. "Undesirable sphingolipids"
include toxic and cardiotoxic sphingolipids, as well as
metabolites, particularly metabolic precursors, of toxic and
cardiotoxic sphingolipids. Undesirable sphingolipids of particular
interest include but are not limited to ceramide (CER),
sphingosine-1-phosphate (S-1-P), and sphingosine (SPH).
[0068] Sphingolipids are a unique class of lipids that were named,
due to their initially mysterious nature, after the Sphinx.
Sphingolipids were initially characterized as primary structural
components of cell membranes, but recent studies indicate that
sphingolipids also serve as cellular signaling and regulatory
molecules (Hannun et al., Adv. Lipid Res. 25:27-41, 1993; Speigel
et al., FASEB J. 10:1388-1397, 1996; Igarashi, J. Biochem
127:1080-1087, 1997). The metabolic pathways for sphingolipids are
shown in FIGS. 1 and 2.
[0069] One group of sphingolipids of particular interest is the set
of sphingolipids involved in the sphingomyelin signal transduction
pathway (Hannun et al., Adv. Lipid Res. 25:27-41, 1993; Liu et al.,
Crit. Rev. Clin. Lab. Sci. 36:511-573, 1999). In this regard,
ceramide, sphingosine and sphingosine-1-phosphate have been most
widely studied (Hannun et al., Science 243:500-507, 1989).
Sphingolipid signaling molecules are derived from sphingomyelin and
include but are not limited to sphingosine [(SPH;
D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol or sphingenine)],
sphingosine-1-phosphate (S-1-P), ceramide (CER), as well as
sphingosylphosphorylcholine (SPC) (see FIG. 1).
[0070] Ceramide and sphingomyelin (SPH) are intracellular second
messengers activated by the sphingomyelin signal transduction
cascade that occurs in response to inflammatory cytokines such as
TNF.alpha., .gamma.IFN, and IL-1.beta., and in response to
ischemia/reperfusion (Bielawska et al., Am. J. Pathol.
151:1257-1263, 1997; Zager et al., Kidney Int. 54:60-70, 1997).
[0071] Accumulations of ceramide in ischemia of human and rat
brains, and in renal ischemia have been alleged to occur (Kubota et
al., Japan J. Exp. Med. 59:59-64, 1989; Kubota et al., Neuro. Res.
18:337-341, 1996; and Zager et al., Kidney Int. 54:60-70, 1997).
Further, S1P causes cerebral vasoconstriction (Tosaka et al.,
Stroke 32: 2913-2919. 2001). Taken together, it is reasoned that
either brain-derived or non-brain derived sphingolipids may
contribute to stroke and that interfering with sphingolipid
production and/or action may mitigate stroke.
[0072] Hernandez et al. (Circ. Res. 86:198-204, 2000) is stated to
show that one of the earliest responses of cardiac myocytes to
hypoxia and reoxygenation is the activation of neutral
sphingomyelinase and the accumulation of ceramide. SPH has been
allegedly implicated as mediating an early signaling event in
apoptotic cell death in a variety of cell types (Ohta et al., FEBS
Letters 355:267-270, 1994; Ohta et al., Cancer Res. 55:691-697,
1995; Cuvlilier et al., Nature 381:800-803, 1996). It is postulated
that the cardiotoxic effects of hypoxia may result in part from
sphingolipid production and/or from the inappropriate production of
other metabolites (e.g. protons, calcium, certain free radicals) or
signaling molecules (e.g. MAP kinases, caspases) that adversely
affect cardiac function.
[0073] S-1-P is stored in platelets and is a normal constituent of
human plasma and serum (Yatomi et al., J. Biochem. 121:969-973,
1997). Sugiyama et al. (Cardiovascular Res. 46:119-125, 2000) is
stated to demonstrate that S-1-P is a coronary vasoconstrictor and
has other biological effects on canine hearts. Siess et al. have
proposed a role for S-1-P in artherosclerosis (IUBMB Life
49:161-171, 2000). This has been supported by other data, including
evidence that the protective effect of HDL is due to blocking S1P
production (Xia et al., PNAS 95:14196-14201, 1988; Xia et al., J
Biol Chem 274:33143-33147, 1999).
[0074] Treatment of neonatal and adult cardiac cells in culture
with physiologically relevant levels of SPH and its immediate
metabolite, S-1-P, has been related to the activation of
cardiomyocyte cell death by apoptosis, a form of programmed cell
death that may contribute to the size of the size of myocardial
infarct (Krown et al., J. Clin. Invest. 98:2854-2865, 1996; Zechner
et al., J. Biol. Chem. 273:8232-8239, 1998; Kajstura et al., Lab.
Invest. 74:86-107, 1996).
[0075] Cordis et al. (J. Pharm. and Biomed. Analysis 16:1189-1193,
1998) states that levels of sphingosine are reduced in
ischemia/reperfused rat hearts. In contrast, however, Bielawska et
al., Am. J. Pathol. 151:1257-1263, 1997) is stated to present
evidence that the levels of the immediate metabolic precursor of
SPH, ceramide, are increased in rat neonatal cardiomyocytes
perfused under ischemic conditions.
[0076] Sphingomyelin, the metabolic precursor of ceramide, has been
stated to be increased in experimental animals subjected to hypoxia
(Sergeev et al., Kosm. Biol. Aviakosm. Ailed. (Russian) 15:71-74,
1981). Other studies have been stated to show that internal
membranes of muscle cells contain high amounts of SPH and
sphingomyelin (Sumnicht et al., Arch. Biochem. Biophys.
215:628-637, 1982; Sabbadini et al., Biochem. Biophys. Res. Comm.
193752-758, 1993). Treatment of experimental animals with
fumonisinB fungal toxins result in increase serum levels of SPH and
DHSPH(S1P was not measured) with coincident negative inotropic
effects on the heart (Smithe et al., Toxicological Sciences
56:240-249, 2000).
[0077] Modulation of the Metabolism of Sphingolipids for
Therapeutic Benefit
[0078] One way to control the amount of undesirable sphingolipids
in a patient is to alter the activity of an enzyme that catalyzes a
reaction that is part of sphingolipid metabolism (see FIGS. 1 and
2). Specifically, to lower the amount of undesirable sphingolipids,
one can inhibit or block enzymes involved in sphingolipid anabolism
(constructive metabolism, i.e., reactions that lead to the
production of undesirable sphingolipids). Additionally or
alternatively, one can stimulate or activate enzymes involved in
sphingolipid catabolism (destructive metabolism, i.e., reactions
that lead to the breakdown of undesirable sphingolipids). For
further details, see Examples 7-10.
[0079] There are several enzymes involved in the sphingolipid
metabolic pathway that can be inhibited in order to reduce the
amount of undesirable sphingolipids. As is explained herein, due to
their deleterious effects on cardiac cells and tissues, two
particularly undesirable sphingolipids are SPH and S-1-P. Enzymes
that are inhibited for the purpose of lowering levels of SPH, S-1-P
and/or other undesirable sphingolipids are assigned to different
classes based on the product(s) of the reaction that they
catalyze.
[0080] Similarly, there are several enzymes involved in
sphingolipid metabolism that can be stimulated in order to reduce
the amount of undesirable sphingolipids including but not limited
to SPH and S-1-P. Stimulation of these enzymes leads to a more
rapid degradation of undesirable sphingolipids. Enzymes that are
stimulated for the purpose of lowering levels of SPH, S-1-P and
other undesirable sphingolipids are assigned to different classes
based on whether they promote the production or degradation of a
selected undesirable, toxic and/or cardiotoxic sphingolipid or a
precursor thereof. In general, enzymes that catalyze the production
of a undesirable, toxic and/or cardiotoxic sphingolipid or its
precursor are inhibited, whereas enzymes that catalyze the
degradation of the undesirable, toxic and/or cardiotoxic
sphingolipid are stimulated.
[0081] Binding Sphingolipids, and Receptors Thereof, for
Therapeutic Benefit
[0082] One way to control the amount of undesirable sphingolipids
in a patient is by providing a composition that binds one or more
sphingolipids, or receptors thereof.
[0083] Antibodies and other compounds that bind to undesirable
sphingolipids may be used as therapeutic "sponges" that reduce the
level of free undesirable sphingolipids. When a compound is stated
to be "free," the compound is not in any way restricted from
reaching the site or sites where it exerts its undesirable effects.
Typically, a free compound is present in the cardiovascular system,
which either is or contains the site(s) of action of the free
compound, or from which a compound can freely migrate to its
site(s) of action. A free compound may also be available to be
acted upon by any enzyme that converts the compound into an
undesirable compound.
[0084] Antibodies and other compounds that bind to cellular
receptors of undesirable sphingolipids may be used to compete with
and/or prevent sphingolipids from binding to receptors and thereby
causing or enhancing undesirable cellular or biochemical events.
Such events include, but are not limited to, the entry of
undesirable sphingolipids into cells, initiation of a signal
cascade pathway that has an undesirable outcome, and a reaction,
which may be catalyzed by an enzyme, that produces an undesirable
product. Receptors of interest include but are not limited to Edg
receptors, SCaMPER. and other receptors that bind sphingolipids,
and receptors for cytokines, including but not limited to the
TNF.alpha. receptor.
[0085] Antibodies
[0086] Several antibodies have recently been approved for
therapeutic use in humans by the Federal Drug Administration
(Kling, Mod. Drug Disc. 2:33-45, 1999). In one aspect of
sphingolipid-based cardiovascular therapy, antibodies that bind
sphingolipids can be delivered to a patient, e.g., incorporation
into pharmaceutical compositions, medical devices, and the like,
for use in sphingolipid-based cardiovascular therapy. Such methods
may, by way of non-limiting example, (1) modulate the effective
concentration of a undesirable, toxic and/or cardiotoxic
sphingolipid or a metabolic precursor thereof; (2) sterically
inhibit the binding of a sphingolipid to a cellular receptor
therefor, or to lower the concentration of a sphingolipid that is
available for binding to such a receptor; (3) sterically inhibit
the enzymatic conversion of a metabolic precursor of a undesirable,
toxic and/or cardiotoxic sphingolipid, or lower the concentration
of such a precursor that is available for enzymatic conversion into
a undesirable, toxic and/or cardiotoxic sphingolipid; and (4)
remove undesirable, toxic and/or cardiotoxic sphingolipids and
their metabolic precursors from blood in vivo or ex vivo.
[0087] The term "antibody" is meant to encompass an immunoglobulin
molecule obtained by in vitro or in vivo generation of an
immunogenic response, and includes polyclonal, monospecific and
monoclonal antibodies, as well as T cell receptors, and fragments
and derivatives thereof. An "immunogenic response" is one that
results in the production of antibodies directed to one or more
proteins after the appropriate cells have been contacted with such
proteins, or polypeptide derivatives thereof, in a manner such that
one or more portions of the protein function as epitopes. An
epitope is a single antigenic determinant in a molecule. In
proteins, particularly denatured proteins, an epitope is typically
defined and represented by a contiguous amino acid sequence.
However, in the case of nondenatured proteins, epitopes also
include structures, such as active sites, that are formed by the
three-dimensional folding of a protein in a manner such that amino
acids from separate portions of the amino acid sequence of the
protein are brought into close physical contact with each
other.
[0088] Polyclonal antibodies are generated in a immunogenic
response to a protein having many epitopes, and thus include a
variety of different antibodies directed to different epitopes
within the protein. Methods for producing polyclonal antibodies are
known in the art (see, e.g., Cooper et al., Section III of Chapter
11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et
al., eds., John Wiley and Sons, New York, 1992, pages 11-37 to
1-41).
[0089] Monospecific antibodies (a.k.a. antipeptide antibodies) are
generated in a humoral response to a short (typically, 5 to 20
amino acids) immunogenic polypeptide that corresponds to a few
(preferably one) isolated epitopes of the protein from which it is
derived. A plurality of monospecific antibodies includes a variety
of different antibodies directed to a specific portion of the
protein, i.e., to an amino acid sequence that contains at least
one, preferably only one, epitope. Methods for producing
monospecific antibodies are known in the art (see, e.g., Id., pages
11-42 to 11-46).
[0090] A monoclonal antibody is a specific antibody that recognizes
a single specific epitope of an immunogenic protein. In a plurality
of a monoclonal antibody, each antibody molecule is identical to
the others in the plurality. In order to isolate a monoclonal
antibody, a clonal cell line that expresses, displays and/or
secretes a particular monoclonal antibody is first identified; this
clonal cell line can be used in one method of producing the
antibodies of the invention. Methods for the preparation of clonal
cell lines and of monoclonal antibodies expressed thereby are known
in the art (see, for example, Fuller et al., Section II of Chapter
11 in: Short Protocols int Molecular Biology, 2nd Ed., Ausubel et
al., eds., John Wiley and Sons, New York, 1992, pages 1'-22 to
11-11-36).
[0091] T cell receptors (TCR) are a distinct class of proteins that
are genetically and structurally related to antibodies. TCR
proteins belong to the immunoglobulin superfamily of proteins and
have molecular structures similar to those of antibodies and, like
antibodies, specifically recognize (i.e., specifically and bind)
specific ligands. Complexes of TCR are displayed on T cells and
bind specific antigens for the purpose of triggering molecular
events associated with T cell differentiation and activation. Like
antibodies, TCR proteins recognize particular antigens. However,
because of differences in the precise structures of the portions of
TCR proteins that bind ligands and the amino acid sequences
associated with those structures, as well as different mechanisms
by which genes encoding a protein are diversified by rearrangement
and mutation. Thus, the "molecular rules" for specific binding of
TCR molecules to their ligands are different from those of
antibodies, and the use of TCR proteins expands the population of
potential sphingolipid-binding proteins.
[0092] Antibody fragments and derivatives are proteins that are
derived from antibodies and T-cell receptors and which retain the
ability to specifically recognize the ligand recognized by the
"parent" antibody or TCR (see Gavilondo et al., BioTechniques
29:128-145, 2000, and Morrow, Amer. Lab. 32:15-19, 2000). Preferred
fragments include Fab fragments (i.e., an antibody fragment that
contains the antigen-binding domain and comprises a light chain and
part of a heavy chain bridged by a disulfide bond); Fab' (an
antibody fragment containing a single anti-binding domain
comprising an Fab and an additional portion of the heavy chain
through the hinge region); F(ab')2 (two Fab' molecules joined by
interchain disulfide bonds in the hinge regions of the heavy
chains; the Fab' molecules may be directed toward the same or
different epitopes); a bispecific Fab (an Fab molecule having two
antigen binding domains, each of which may be directed to a
different epitope); and camelized VH domains (the variable,
antigen-binding determinative region of a single heavy chain of an
antibody in which some amino acids at the VH interface are those
found in the heavy chain of naturally occurring camel
antibodies).
[0093] Single chain antibodies (scFv) comprise a variable region,
a.k.a., a scFv (the variable, antigen-binding determinative region
of a single light and heavy chain of an antibody linked together by
a chain of 10-25 amino acids). For reviews, see Raag et al.,
Single-chain Fvs. FASEB J. 9:73-80, 1995, and Hudson, Recombinant
antibody fragments. Curr. Op. Biotechnol. 9, 395-402, 1999. See
also Bird et al., Single-chain antigen-binding proteins. Science
242, 423-426, 1988, and U.S. Pat. Nos. 5,260,203; 5,869,620;
5,455,030; 5,518,889; 5,534,621; 4,946,778; 6,025,165; and
6,027,725.
[0094] The well-known technique of phage display is used to prepare
scFv molecules. For reviews, see Winter et al., Making antibodies
by phage display technology. Annu. Rev. Immunol. 12:433-455, 1994;
Little et al., Surface display of antibodies. Biotechn. Adv.
12:539-555, 1994; and Burton et al., Human antibodies from
combinatorial libraries. Adv. Immunol. 57:191-280, 1994. See also
U.S. Pat. Nos. 5,821,047; 5,702,892; 6,031,071; and 6,310,191.
[0095] Complexes of single chain antibodies are also within the
scope of the invention and include, but are not limited to, a
disulfide-linked Fv, or dsFv (the variable, antigen-binding
determinative region of a single light and heavy chain of an
antibody linked together by a disulfide bond; a bispecific sFv (a
scFv or a dsFv molecule having two antigen-binding domains, each of
which may be directed to a different epitope); a diabody (a
dimerized scFv formed when the VH domain of a first scFv assembles
with the VL domain of a second scFv and the VL domain of the first
scFv assembles with the VH domain of the second scFv; the two
antigen-binding regions of the diabody may be directed towards the
same or different epitopes); and a triabody (a trimerized sFv,
formed in a manner similar to a diabody, but in which three
antigen-binding domains are created in a single complex; the three
antigen binding domains may be directed towards the same or
different epitopes).
[0096] The term "antibody" also includes genetically engineered
antibodies and/or antibodies produced by recombinant DNA techniques
and "humanized" antibodies. Humanized antibodies have been
modified, by genetic manipulation and/or in vitro treatment to be
more human, in terms of amino acid sequence, glycosylation pattern,
etc., in order to reduce the antigenicity of the antibody or
antibody fragment in an animal to which the antibody is intended to
be administered (Gussow et al., Methods Enz. 203:99-121, 1991).
[0097] Methods of Preparing Antibodies and Antibody Variants
[0098] The antibodies and antibody fragments of the invention may
be produced by any suitable method, for example, in vivo (in the
case of polyclonal and monospecific antibodies), in cell culture
(as is typically the case for monoclonal antibodies, wherein
hybridoma cells expressing the desired antibody are cultured under
appropriate conditions), in in vitro translation reactions, and in
recombinant DNA expression systems (Johnson et al., Methods Enz.
203:88-98, 1991). Antibodies and antibody variants can be produced
from a variety of animal cells, preferably from mammalian cells,
with murine and human cells being particularly preferred.
Antibodies that include non-naturally occurring antibody and T-cell
receptor variants that retain only the desired antigen targeting
capability conferred by an antigen binding site(s) of an antibody
can be produced by known cell culture techniques and recombinant
DNA expression systems (see, e.g., Johnson et al., Methods in
Enzymol. 203:88-98, 1991; Molloy et al., Mol. Immunol. 32:73-81,
1998; Schodin et al., J. Immunol. Methods 200:69-77, 1997).
Recombinant DNA expression systems are typically used in the
production of antibody variants such as, e.g., bispecific
antibodies and sFv molecules. Preferred recombinant DNA expression
systems include those that utilize host cells and expression
constructs that have been engineered to produce high levels of a
particular protein. Preferred host cells and expression constructs
include Escherichia coli; harboring expression constructs derived
from plasmids or viruses (bacteriophage); yeast such as
Sacharomyces cerevisieae or Fichia pastoras harboring episomal or
chromosomally integrated expression constructs; insect cells and
viruses such as Sf9 cells and baculovirus; and mammalian cells
harboring episomal or chromosomally integrated (e.g., retroviral)
expression constructs (for a review, see Verma et al., J. Immunol.
Methods 216:165-181, 1998). Antibodies can also be produced in
plants (U.S. Pat. No. 6,046,037; Ma et al., Science 268:716-719,
1995) or by phage display technology (Winter et al., Annu. Rev.
Immunol. 12:433-455, 1994).
[0099] XenoMouse strains are genetically engineered mice in which
the murine IgH and Igk loci have been functionally replaced by
their Ig counterparts on yeast artificial YAC transgenes. These
human Ig transgenes can carry the majority of the human variable
repertoire and can undergo class switching from IgM to IgG
isotypes. The immune system of the xenomouse recognizes
administered human antigens as foreign and produces a strong
humoral response. The use of XenoMouse in conjunction with
well-established hybridomas techniques, results in fully human IgG
mAbs with sub-nanomolar affinities for human antigens (see U.S.
Pat. No. 5,770,429, entitled "Transgenic non-human animals capable
of producing heterologous antibodies"; U.S. Pat. No. 6,162,963,
entitled "Generation of Xenogenetic antibodies"; U.S. Pat. No.
6,150,584, entitled "Human antibodies derived from immunized
xenomice"; U.S. Pat. No. 6,114,598, entitled Generation of
xenogeneic antibodies; and U.S. Pat. No. 6,075,181, entitled "Human
antibodies derived from immunized xenomice"; for reviews, see
Green, Antibody engineering via genetic engineering of the mouse:
XenoMouse strains are a vehicle for the facile generation of
therapeutic human monoclonal antibodies, J. Immunol. Methods
231:11-23, 1999; Wells, Eek, a XenoMouse: Abgenix, Inc., Chem Biol
2000 August;7(8):R185-6; and Davis et al., Transgenic mice as a
source of fully human antibodies for the treatment of cancer Cancer
Metastasis Rev 1999;18(4):421-5).
[0100] Soluble Receptor Fragments
[0101] Soluble polypeptides derived from membrane bound, typically
hydrophobic, sphingolipid receptors that retain the receptor's
ability to bind sphingolipids may also be used to bind
sphingolipids and sphingolipid metabolites. In the case of Edg
receptors, in some instances, particualr amino acid residues may be
involved in the specificity of sphingolipid binding, i.e., the
amino acids that determine which sphingolipid is bound by a
specific receptor (Parrill et al., "Identification of Edg1 Receptor
Residues That Recognize Sphingosine 1-Phosphate", J. Biol. Chem.
275:39379-39384, 2000; and Wang et al., "A Single Amino Acid
Determines Lysophospholipid Specificity of the S1P1 (EDG1) and LPA1
(EDG2) Phospholipid Growth Factor Receptors", JBC Papers in Press
Published Oct. 16, 2001 as Manuscript M107301200). Such information
may be used to provide soluble receptor fragments comprising
receptor residues of interest, i.e., the stretches of amino acids
that bind the sphingolipid. Soluble receptor fragments derived from
the naturally soluble TNF.alpha. receptor have been prepared and at
least one of these, ENBREL.RTM. (Etanercept) is in development as a
therapeutic agent for arthritis. In addition, modification of such
residues may permit the skilled artisan to tailor the binding
specificities and/or affinity of soluble receptor fragments.
[0102] Soluble receptor fragments of particular interest include
Edg-1, Edg-3, Edg-5, Edg-6 and Edg-8, all of which bind the
undesirable sphingolipid sphingosine-1-phosphate (S-1-P). The
Edg-1, Edg-3, Edg-5 receptors are of particular interest because
binding of S-1-P thereto seems to stimulate the production of
intracellular S-1-P (Heringdorf et al., Stimulation of
intracellular sphingosine-1-phosphate production by
G-protein-coupled sphingosine-1-phosphate receptors, Eur J.
Pharmacol. 414:145-54, 2001). The P2Y(2) receptor is of interest as
it also increases intracellular production of S-1-P (Alemany et
al., Stimulation of sphingosine-1-phosphate formation by the P2Y(2)
receptor in HL-60 cells: Ca(2+) requirement and implication in
receptor-mediated Ca(2+) mobilization, but not MAP kinase
activation, Mot Pharmacol. 58:491-7, 2000).
[0103] Soluble receptor fragments may be prepared in various ways
including but not limited to proteolytic digestion of cells or
cellular membrane preparations comprising the receptor (Bartfeld et
al., Active acetylcholine receptor fragment obtained by tryptic
digestion of acetylcholine receptor from Torpedo californica,
Biochem Biophys Res Commun. 89:512-9, 1979; Borhani et al.,
Crystallization and X-ray diffraction studies of a soluble form of
the human transferrin receptor, J Mol. Biol. 218:685-9, 1991),
recombinant DNA technologies (Marlovits et al., Recombinant soluble
low-density lipoprotein receptor fragment inhibits common cold
infection, J Mol Recognit. 11:49-51, 1998; Huang et al., Expression
of a human thyrotrophin receptor fragment in Escherichia coli and
its interaction with the hormone and autoantibodies from patients
with Graves' disease, J Mol Endocrinol. 8:137-44, 1992), or by in
vitro synthesis of oligopeptides.
[0104] Nucleic Acids
[0105] Traditionally, techniques for detecting and purifying target
molecules have used polypeptides, such as antibodies, that
specifically bind such targets. While nucleic acids have long been
known to specifically bind other nucleic acids (e.g., ones having
complementary sequences), aptamers (i.e., nucleic acids that bind
non-nucleic target molecules) have been disclosed. See, e.g.,
Blackwell et al., Science (1990) 250:1104-1110; Blackwell et al.,
Science (1990) 250:1149-1152; Tuerk et al., Science (1990)
249:505-510; Joyce, Gene (1989) 82:83-87; and U.S. Pat. No.
5,840,867 entitled "Aptamer analogs specific for biomolecules".
[0106] As applied to aptamers, the term "binding" specifically
excludes the "Watson-Crick"-type binding interactions (i.e., A:T
and G:C base-pairing) traditionally associated with the DNA double
helix. The term "aptamer" thus refers to a nucleic acid or a
nucleic acid derivative that specifically binds to a target
molecule, wherein the target molecule is either (i) not a nucleic
acid, or (ii) a nucleic acid or structural element thereof that is
bound through mechanisms other than duplex- or triplex-type base
pairing. Such a molecule is called a "non-nucleic molecule"
herein.
[0107] Structures of Nucleic Acids
[0108] "Nucleic acids," as used herein, refers to nucleic acids
that are isolated a natural source; prepared in vitro, using
techniques such as PCR amplification or chemical synthesis;
prepared in vivo, e.g., via recombinant DNA technology; or by any
appropriate method. Nucleic acids may be of any shape (linear,
circular, etc.) or topology (single-stranded, double-stranded,
supercoiled, etc.). The term "nucleic acids" also includes without
limitation nucleic acid derivatives such as peptide nucleic acids
(PNA's) and polypeptide-nucleic acid conjugates; nucleic acids
having at least one chemically modified sugar residue, backbone,
internucleotide linkage, base, nucleoside, or nucleotide analog; as
well as nucleic acids having chemically modified 5' or 3' ends; and
nucleic acids having two or more of such modifications. Not all
linkages in a nucleic acid need to be identical.
[0109] Nucleic acids that are aptamers are often, but need not be,
prepared as oligonucleotides. Oligonucleotides include without
limitation RNA, DNA and mixed RNA-DNA molecules having sequences of
lengths that have minimum lengths of 2, 4, 6, 8, 10, 11, 12, 13,
14, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and
maximum lengths of about 100, 75, 50, 40, 25, 20 or 15 or more
nucleotides, irrespectively. In general, a minimum of 6
nucleotides, preferably 10 nucelotides, more preferably 14 to 20
nucleotides, is necessary to effect specific binding.
[0110] In general, the oligonucleotides may be single-stranded (ss)
or double-stranded (ds) DNA or RNA, or conjugates (e.g., RNA
molecules having 5' and 3' DNA "clamps") or hybrids (e.g., RNA:DNA
paired molecules), or derivatives (chemically modified forms
thereof). However, single-stranded DNA is preferred, as DNA is
often less labile than RNA. Similarly, chemical modifications that
enhance an aptamer's specificity or stability are preferred.
[0111] Chemical Modifications of Nucleic Acids
[0112] Chemical modifications that may be incorporated into
aptamers and other nucleic acids include, with neither limitation
nor exclusivity, base modifications, sugar modifications, and
backbone modifications.
[0113] Base modifications: The base residues in aptamers may be
other than naturally occurring bases (e.g., A, G, C, T, U, 5MC, and
the like). Derivatives of purines and pyrimidines are known in the
art; an exemplary but not exhaustive list includes
aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine (5MC),
N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5-methoxyuracil, 2-methylthio-N-6-isopentenylade- nine,
uracil-5-oxyacetic acid methylester, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine. In
addition to nucleic acids that incorporate one or more of such base
derivatives, nucleic acids having nucleotide residues that are
devoid of a purine or a pyrimidine base may also be included in
aptamers.
[0114] Sugar modifications: The sugar residues in aptamers may be
other than conventional ribose and deoxyribose residues. By way of
non-limiting example, substitution at the 2'-position of the
furanose residue enhances nuclease stability. An exemplary, but not
exhaustive list, of modified sugar residues includes 2' substituted
sugars such as 2'-O-methyl-, 2'-O-alkyl, 2'-O-allyl, 2'-S-alkyl,
2'-S-allyl, 2'-fluoro-, 2'-halo, or 2'-azido-ribose, carbocyclic
sugar analogs, alpha-anomeric sugars, epimeric sugars such as
arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,
sedoheptuloses, acyclic analogs and abasic nucleoside analogs such
as methyl riboside, ethyl riboside or propylriboside.
[0115] Backbone modifications: Chemically modified backbones
include, by way of non-limiting example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Chemically modified backbones that do not contain a
phosphorus atom have backbones that are formed by short chain alkyl
or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages, including
without limitation morpholino linkages; siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; and amide backbones.
[0116] Preparation and Identification of Aptamers
[0117] In general, techniques for identifying aptamers involve
incubating a preselected non-nucleic target molecule with mixtures
(2 to 50 members), pools (50 to 5,000 members) or libraries (50 or
more members) of different nucleic acids that are potential
aptamers under conditions that allow complexes of target molecules
and aptamers to form. By "different nucleic acids" it is meant that
the nucleotide sequence of each potential aptamer may be different
from that of any other member, that is, the sequences of the
potential aptamers are random with respect to each other.
Randomness can be introduced in a variety of manners such as, e.g.,
mutagenesis, which can be carried out in vivo by exposing cells
harboring a nucleic acid with mutagenic agents, in vitro by
chemical treatment of a nucleic acid, or in vitro by biochemical
replication (e.g., PCR) that is deliberately allowed to proceed
under conditions that reduce fidelity of replication process;
randomized chemical synthesis, i.e., by synthesizing a plurality of
nucleic acids having a preselected sequence that, with regards to
at least one position in the sequence, is random. By "random at a
position in a preselected sequence" it is meant that a position in
a sequence that is normally synthesized as, e.g., as close to 100%
A as possible (e.g., 5'-C-T-T-A-G-T-3') is allowed to be randomly
synthesized at that position (C-T-T-N-G-T, wherein N indicates a
randomized position where, for example, the synthesizing reaction
contains 25% each of A,T,C and G; or x % A, w % T, y % C and z % G,
wherein x+w+y+z=100. In later stages of the process, the sequences
are increasingly less randomized and consensus sequences may
appear; in any event, it is preferred to ultimately obtain an
aptamer having a unique nucleotide sequence.
[0118] Aptamers and pools of aptamers are prepared, identified,
characterized and/or purified by any appropriate technique,
including those utilizing in vitro synthesis, recombinant DNA
techniques, PCR amplification, and the like. After their formation,
target:aptamer complexes are then separated from the uncomplexed
members of the nucleic acid mixture, and the nucleic acids that can
be prepared from the complexes are candidate aptamers (at early
stages of the technique, the aptamers generally being a population
of a multiplicity of nucleotide sequences having varying degrees of
specificity for the target). The resulting aptamer (mixture or
pool) is then substituted for the starting apatamer (library or
pool) in repeated iterations of this series of steps. When a
limited number (e.g., a pool or mixture, preferably a mixture with
less than 10 members, most preferably 1) of nucleic acids having
satisfactory specificity is obtained, the aptamer is sequenced and
characterized. Pure preparations of a given aptamer are generated
by any appropriate technique (e.g., PCR amplification, in vitro
chemical synthesis, and the like).
[0119] For example, Tuerk and Gold (Science (1990) 249:505-510)
disclose the use of a procedure termed "systematic evolution of
ligands by exponential enrichment" (SELEX). In this method, pools
of nucleic acid molecules that are randomized at specific positions
are subjected to selection for binding to a nucleic acid-binding
protein (see, e.g., PCT International Publication No. WO 91/19813
and U.S. Pat. No. 5,270,163). The oligonucleotides so obtained are
sequenced and otherwise characterization. Kinzler, K. W., et al.
(Nucleic Acids Res. (1989) 17:3645-3653) used a similar technique
to identify synthetic double-stranded DNA molecules that are
specifically bound by DNA-binding polypeptides. Ellington, A. D.,
et al. (Nature (1990) 346: 818-822) disclose the production of a
large number of random sequence RNA molecules and the selection and
identification of those that bind specifically to specific dyes
such as Cibacron blue.
[0120] Another technique for identifying nucleic acids that bind
non-nucleic target molecules is the oligonucleotide combinatorial
technique disclosed by Ecker, D. J. et al. (Nuc. Acids Res. 21,
1853 (1993)) known as "synthetic unrandomization of randomized
fragments" (SURF), which is based on repetitive synthesis and
screening of increasingly simplified sets of oligonucleotide
analogue libraries, pools and mixtures (Tuerk, C. and Gold, L.
(Science 249, 505 (1990)). The starting library consists of
oligonucleotide analogues of defined length with one position in
each pool containing a known analogue and the remaining positions
containing equimolar mixtures of all other analogues. With each
round of synthesis and selection, the identity of at least one
position of the oligomer is determined until the sequences of
optimized nucleic acid ligand aptamers are discovered.
[0121] Once a particular candidate aptamer has been identified
through a SURF, SELEX or any other technique, its nucleotide
sequence can be determined (as is known in the art), and its
three-dimensional molecular structure can be examined by nuclear
magnetic resonance (NMR). These techniques are explained in
relation to the determination of the three-dimensional structure of
a nucleic acid ligand that binds thrombin in Padmanabhan, K. et
al., J. Biol. Chem. 24, 17651 (1993); Wang, K. Y. et al.,
Biochemistry 32, 1899 (1993); and Macaya, R. F. et al., Proc.
Nat'l. Acad. Sci. USA 90, 3745 (1993). Selected aptamers may be
resynthesized using one or more modified bases, sugars or backbone
linkages. Aptamers consist essentially of the minimum sequence of
nucleic acid needed to confer binding specificity, but may be
extended on the 5' end, the 3' end, or both, or may be otherwise
derivatized or conjugated.
[0122] Small Molecules
[0123] The term "small molecule" includes any chemical or other
moiety, other than polypeptides and nucleic acids, that can act to
affect biological processes. Small molecules can include any number
of therapeutic agents presently known and used, or can be small
molecules synthesized in a library of such molecules for the
purpose of screening for biological function(s). Small molecules
are distinguished from macromolecules by size. The small molecules
of this invention usually have molecular weight less than about
5,000 daltons (Da), preferably less than about 2,500 Da, more
preferably less than 1,000 Da, most preferably less than about 500
Da.
[0124] Small molecules include without limitation organic
compounds, peptidomimetics and conjugates thereof. As used herein,
the term "organic compound" refers to any carbon-based compound
other than macromolecules such nucleic acids and polypeptides. In
addition to carbon, organic compounds may contain calcium,
chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen,
oxygen, sulfur and other elements. An organic compound may be in an
aromatic or aliphatic form. Non-limiting examples of organic
compounds include acetones, alcohols, anilines, carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
nucleosides, nucleotides, lipids, retinoids, steroids,
proteoglycans, ketones, aldehydes, saturated, unsaturated and
polyunsaturated fats, oils and waxes, alkenes, esters, ethers,
thiols, sulfides, cyclic compounds, heterocylcic compounds,
imidizoles and phenols. An organic compound as used herein also
includes nitrated organic compounds and halogenated (e.g.,
chlorinated) organic compounds. Methods for preparing
peptidomimetics are described below. Collections of small
molecules, and small molecules identified according to the
invention are characterized by techniques such as accelerator mass
spectrometry (ALMS; see Turteltaub et al., Curr Pharm Des 2000
6:991-1007, Bioanalytical applications of accelerator mass
spectrometry for pharmaceutical research; and Enjalbal et al., Mass
Spectrom Rev 2000 19:139-61, Mass spectrometry in combinatorial
chemistry.)
[0125] Preferred small molecules are relatively easier and less
expensively manufactured, formulated or otherwise prepared.
Preferred small molecules are stable under a variety of storage
conditions. Preferred small molecules may be placed in tight
association with macromolecules to form molecules that are
biologically active and that have improved pharmaceutical
properties. Improved pharmaceutical properties include changes in
circulation time, distribution, metabolism, modification,
excretion, secretion, elimination, and stability that are favorable
to the desired biological activity. Improved pharmaceutical
properties include changes in the toxicological and efficacy
characteristics of the chemical entity.
[0126] Peptidomimetics
[0127] In general, a polypeptide mimetic ("peptidomimetic") is a
molecule that mimics the biological activity of a polypeptide, but
that is not peptidic in chemical nature. While, in certain
embodiments, a peptidomimetic is a molecule that contains no
peptide bonds (that is, amide bonds between amino acids), the term
peptidomimetic may include molecules that are not completely
peptidic in character, such as pseudo-peptides, semi-peptides and
peptoids. Examples of some peptidomimetics by the broader
definition (e.g., where part of a polypeptide is replaced by a
structure lacking peptide bonds) are described below. Whether
completely or partially non-peptide in character, peptidomimetics
according to this invention may provide a spatial arrangement of
reactive chemical moieties that closely resembles the
three-dimensional arrangement of active groups in a polypeptide. As
a result of this similar active-site geometry, the peptidomimetic
may exhibit biological effects that are similar to the biological
activity of a polypeptide.
[0128] There are several potential advantages for using a mimetic
of a given polypeptide rather than the polypeptide itself. For
example, polypeptides may exhibit two undesirable attributes, i.e.,
poor bioavailability and short duration of action. Peptidomimetics
are often small enough to be both orally active and to have a long
duration of action. There are also problems associated with
stability, storage and immunoreactivity for polypeptides that may
be obviated with peptidomimetics.
[0129] Candidate, lead and other polypeptides having a desired
biological activity can be used in the development of
peptidomimetics with similar biological activities. Techniques of
developing peptidomimetics from polypeptides are known. Peptide
bonds can be replaced by non-peptide bonds that allow the
peptidomimetic to adopt a similar structure, and therefore
biological activity, to the original polypeptide. Further
modifications can also be made by replacing chemical groups of the
amino acids with other chemical groups of similar structure, shape
or reactivity. The development of peptidomimetics can be aided by
determining the tertiary structure of the original polypeptide,
either free or bound to a ligand, by NMR spectroscopy,
crystallography and/or computer-aided molecular modeling. These
techniques aid in the development of novel compositions of higher
potency and/or greater bioavailability and/or greater stability
than the original polypeptide (Dean (1994), BioEssays, 16: 683-687;
Cohen and Shatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley
and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends
Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33:
1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98, all
incorporated herein by reference].
[0130] Specific examples of peptidomimetics are set forth below.
These examples are illustrative and not limiting in terms of the
other or additional modifications.
[0131] Peptides With A Reduced Isostere Pseudopeptide Bond
[0132] Proteases act on peptide bonds. Substitution of peptide
bonds by pseudopeptide bonds may confer resistance to proteolysis
or otherwise make a compound less labile. A number of pseudopeptide
bonds have been described that in general do not affect polypeptide
structure and biological activity. The reduced isostere
pseudopeptide bond is a suitable pseudopeptide bond that is known
to enhance stability to enzymatic cleavage with no or little loss
of biological activity (Couder, et al., (1993), Int. J. Polypeptide
Protein Res. 41:181-184, incorporated herein by reference). Thus,
the amino acid sequences of these compounds may be identical to the
sequences of their parent L-amino acid polypeptides, except that
one or more of the peptide bonds are replaced by an isostere
pseudopeptide bond. Preferably the most N-terminal peptide bond is
substituted, since such a substitution would confer resistance to
proteolysis by exopeptidases acting on the N-terminus.
[0133] Peptides With A Retro-Inverso Pseudopeptide Bond
[0134] To confer resistance to proteolysis, peptide bonds may also
be substituted by retro-inverso pseudopeptide bonds (Dalpozzo, et
al. (1993), Int. J. Polypeptide Protein Res. 41:561-566,
incorporated herein by reference). According to this modification,
the amino acid sequences of the compounds may be identical to the
sequences of their L-amino acid parent polypeptides, except that
one or more of the peptide bonds are replaced by a retro-inverso
pseudopeptide bond. Preferably the most N-terminal peptide bond is
substituted, since such a substitution will confer resistance to
proteolysis by exopeptidases acting on the N-terminus.
[0135] Peptoid Derivatives
[0136] Peptoid derivatives of polypeptides represent another form
of modified polypeptides that retain the important structural
determinants for biological activity, yet eliminate the peptide
bonds, thereby conferring resistance to proteolysis (Simon, et al.,
1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371 and incorporated
herein by reference). Peptoids are oligomers of N-substituted
glycines. A number of N-alkyl groups have been described, each
corresponding to the side chain of a natural amino acid.
[0137] Polypeptides
[0138] The polypeptides of this invention, including the analogs
and other modified variants, may generally be prepared following
known techniques. Preferably, synthetic production of the
polypeptide of the invention may be according to the solid phase
synthetic method. For example, the solid phase synthesis is well
understood and is a common method for preparation of polypeptides,
as are a variety of modifications of that technique [Merrifield
(1964), J. Am. Chem. Soc., 85: 2149; Stewart and Young (1984),
Solid Phase polypeptide Synthesis, Pierce Chemical Company,
Rockford, Ill.; Bodansky and Bodanszky (1984), The Practice of
polypeptide Synthesis, Springer-Verlag, New York; Atherton and
Sheppard (1989), Solid Phase polypeptide Synthesis: A Practical
Approach, IRL Press, New York].
[0139] Alternatively, polypeptides of this invention may be
prepared in recombinant systems using polynucleotide sequences
encoding the polypeptides. For example, fusion proteins are
typically prepared using recombinant DNA technology.
[0140] Polypeptide Derivatives
[0141] A "derivative" of a polypeptide is a compound that is not,
by definition, a polypeptide, i.e., it contains at least one
chemical linkage that is not a peptide bond. Thus, polypeptide
derivatives include without limitation proteins that naturally
undergo post-translational modifications such as, e.g.,
glycosylation. It is understood that a polypeptide of the invention
may contain more than one of the following modifications within the
same polypeptide. Preferred polypeptide derivatives retain a
desirable attribute, which may be biological activity; more
preferably, a polypeptide derivative is enhanced with regard to one
or more desirable attributes, or has one or more desirable
attributes not found in the parent polypeptide.
[0142] Mutant Polypeptides: A polypeptide having an amino acid
sequence identical to that found in a protein prepared from a
natural source is a "wildtype" polypeptide. Mutant oligopeptides
can be prepared by chemical synthesis, including without limitation
combinatorial synthesis.
[0143] Mutant polypeptides larger than oligopeptides can be
prepared using recombinant DNA technology by altering the
nucleotide sequence of a nucleic acid encoding a polypeptide.
Although some alterations in the nucleotide sequence will not alter
the amino acid sequence of the polypeptide encoded thereby
("silent" mutations), many will result in a polypeptide having an
altered amino acid sequence that is altered relative to the parent
sequence. Such altered amino acid sequences may comprise
substitutions, deletions and additions of amino acids, with the
proviso that such amino acids are naturally occurring amino
acids.
[0144] Thus, subjecting a nucleic acid that encodes a polypeptide
to mutagenesis is one technique that can bc used to prepare mutant
polypeptides, particularly ones having substitutions of amino acids
but no deletions or insertions thereof. A variety of mutagenic
techniques are known that can be used in vitro or in vivo including
without limitation chemical mutagenesis and PCR-mediated
mutagenesis. Such mutagenesis may be randomly targeted (i.e.,
mutations may occur anywhere within the nucleic acid) or directed
to a section of the nucleic acid that encodes a stretch of amino
acids of particular interest. Using such techniques, it is possible
to prepare randomized, combinatorial or focused compound libraries,
pools and mixtures.
[0145] Polypeptides having deletions or insertions of naturally
occurring amino acids may be synthetic oligopeptides that result
from the chemical synthesis of amino acid sequences that are based
on the amino acid sequence of a parent polypeptide but which have
one or more amino acids inserted or deleted relative to the
sequence of the parent polypeptide. Insertions and deletions of
amino acid residues in polypeptides having longer amino acid
sequences may be prepared by directed mutagenesis.
[0146] Chemically Modified Polypeptides: As contemplated by this
invention, the term "polypeptide" includes those having one or more
chemical modification relative to another polypeptide, i.e.,
chemically modified polypeptides. The polypeptide from which a
chemically modified polypeptide is derived may be a wildtype
protein, a mutant protein or a mutant polypeptide, or polypeptide
fragments thereof, an antibody or other polypeptide ligand
according to the invention including without limitation
single-chain antibodies, bacterial proteins and polypeptide
derivatives thereof, or polypeptide ligands prepared according to
the disclosure. Preferably, the chemical modification(s) confer(s)
or improve(s) desirable attributes of the polypeptide but does not
substantially alter or compromise the biological activity thereof.
Desirable attributes include but are limited to increased
shelf-life; enhanced serum or other in vivo stability; resistance
to proteases; and the like. Such modifications include by way of
non-limiting example N-terminal acetylation, glycosylation, and
biotinylation.
[0147] Polypeptides with N-Terminal or C-Terminal Chemical Groups:
An effective approach to confer resistance to peptidases acting on
the N-terminal or C-terminal residues of a polypeptide is to add
chemical groups at the polypeptide termini, such that the modified
polypeptide is no longer a substrate for the peptidase. One such
chemical modification is glycosylation of the polypeptides at
either or both termini. Certain chemical modifications, in
particular N-terminal glycosylation, have been shown to increase
the stability of polypeptides in human serum (Powell et al. (1993),
Pharma. Res. 10: 1268-1273). Other chemical modifications which
enhance serum stability include, but are not limited to, the
addition of an N-terminal alkyl group, consisting of a lower alkyl
of from 1 to 20 carbons, such as an acetyl group, and/or the
addition of a C-terminal amide or substituted amide group.
[0148] Polypeptides with a Terminal D-Amino Acid: The presence of
an N-terminal D-amino acid increases the serum stability of a
polypeptide that otherwise contains L-amino acids, because
exopeptidases acting on the N-terminal residue cannot utilize a
D-amino acid as a substrate. Similarly, the presence of a
C-terminal D-amino acid also stabilizes a polypeptide, because
serum exopeptidases acting on the C-terminal residue cannot utilize
a D-amino acid as a substrate. With the exception of these terminal
modifications, the amino acid sequences of polypeptides with
N-terminal and/or C-terminal D-amino acids are usually identical to
the sequences of the parent L-amino acid polypeptide.
[0149] Polypeptides With Substitution of Natural Amino Acids By
Unnatural Amino Acids: Substitution of unnatural amino acids for
natural amino acids in a subsequence of a polypeptide can confer or
enhance desirable attributes including biological activity. Such a
substitution can, for example, confer resistance to proteolysis by
exopeptidases acting on the N-terminus. The synthesis of
polypeptides with unnatural amino acids is routine and known in the
art (see, for example, Coller, et al. (1993), cited above).
[0150] Post-Translational Chemical Modifications: Different host
cells will contain different post-translational modification
mechanisms that may provide particular types of post-translational
modification of a fusion protein if the amino acid sequences
required for such modifications is present in the fusion protein. A
large number (.about.100) of post-translational modifications have
been described, a few of which are discussed herein. One skilled in
the art will be able to choose appropriate host cells, and design
chimeric genes that encode protein members comprising the amino
acid sequence needed for a particular type of modification.
[0151] Glycosylation is one type of post-translational chemical
modification that occurs in many eukaryotic systems, and may
influence the activity, stability, pharmacogenetics, immunogenicity
and/or antigenicity of proteins. However, specific amino acids must
be present at such sites to recruit the appropriate glycosylation
machinery, and not all host cells have the appropriate molecular
machinery. Saccharomyces cerevisieae and Pichia pastoris provide
for the production of glycosylated proteins, as do expression
systems that utilize insect cells, although the pattern of
glyscoylation may vary depending on which host cells are used to
produce the fusion protein.
[0152] Another type of post-translation modification is the
phosphorylation of a free hydroxyl group of the side chain of one
or more Ser, Thr or Tyr residues. Protein kinases catalyze such
reactions. Phosphorylation is often reversible due to the action of
a protein phosphatase, an enzyme that catalyzes the
dephosphorylation of amino acid residues.
[0153] Differences in the chemical structure of amino terminal
residues result from different host cells, each of which may have a
different chemical version of the methionine residue encoded by a
start codon, and these will result in amino termini with different
chemical modifications.
[0154] For example, many or most bacterial proteins are synthesized
with an amino terminal amino acid that is a modified form of
methionine, i.e., N-formyl-methionine (fMct). Although the
statement is often made that all bacterial proteins are synthesized
with an fMet initiator amino acid; although this may be true for E.
coli, recent studies have shown that it is not true in the case of
other bacteria such as Pseudomonas aeruginosa (Newton et al., J.
Biol. Chem. 274:22143-22146, 1999). In any event, in E. coli, the
formyl group of fMet is usually enzymatically removed after
translation to yield an amino terminal methionine residue, although
the entire fMet residue is sometimes removed (see Hershey, Chapter
40, "Protein Synthesis" in: Escherichia Coli and Salmonella
Typhimurium: Cellular and Molecular Biology, Neidhardt, Frederick
C., Editor in Chief, American Society for Microbiology, Washington,
D.C., 1987, Volume 1, pages 613-647, and references cited therein.)
E. coli mutants that lack the enzymes (such as, e.g., formylase)
that catalyze such post-translational modifications will produce
proteins having an amino terminal fMet residue (Guillon et al., J.
Bacteriol. 174:4294-4301, 1992).
[0155] In eukaryotes, acetylation of the initiator methionine
residue, or the penultimate residue if the initiator methionine has
been removed, typically occurs co- or post-translationally. The
acetylation reactions are catalyzed by N-terminal
acetyltransferases (NATs, a.k.a. N-alpha-acetyltransferases),
whereas removal of the initiator methionine residue is catalyzed by
methionine aminopeptidases (for reviews, see Bradshaw et al.,
Trends Biochem. Sci. 23:263-267, 1998; and Driessen et al., CRC
Crit. Rev. Biochem. 18:281-325, 1985). Amino terminally acetylated
proteins are said to be "N-acetylated," "N alpha acetylated" or
simply "acetylated."
[0156] Another post-translational process that occurs in eukaryotes
is the alpha-amidation of the carboxy terminus. For reviews, see
Eipper et al. Annu. Rev. Physiol. 50:333-344, 1988, and Bradbury et
al. Lung Cancer 14:239-251, 1996. About 50% of known endocrine and
neuroendocrine peptide hormones are alpha-amidated (Treston et al.,
Cell Growth Differ. 4:911-920, 1993). In most cases, carboxy
alpha-amidation is required to activate these peptide hormones.
[0157] Aminoglycosides
[0158] A class of small molecules of particular interest are known
as aminoglycosides, particularly those that inhibit a
sphingomyelinase (SMase), particularly a neutral SMase. Example 13
describes the use of the screening methods of the invention to
prepare novel therapeutic agents using chemical libraries based on
the aminoglycoside structure.
[0159] Aminoglycosides were first identified as antibiotics
produced by microorganisms of the genus Micromonospora. The
antibiotics, recovered from the Micromonospora culture media,
included Gentamicin (Weinstein et at., Antimicrobial Agents and
Chemotherapy, 1963, page 1; Cooper et al., J. Infect. Dis. 119:342,
1969; Waitz: Antimicrobial Agents and Chemotherapy 2:464, 1972),
Antibiotic No. 460 (Japanese Pat. No. 16153/71), Sisomicin
(Weinstein et al., J. Antibiotics 23:551, 555, 559, 1970),
Kanamycin, Neomycin, and many others as described below. For a
review, see Edson et al., The Aminoglycosides, Mayo Clin Proc
74:519-528, 1999.
[0160] Aminoglycosides are a group of antibiotics that exert their
bactericidal activity primarily by inhibition of protein synthesis.
Aminoglycoside molecules bind to the bacterial 30S ribosomal
subunit rendering the ribosomes unavailable for translation, which
results in cell death.
[0161] The first aminoglycoside, streptonmycin, was isolated from
Streptomyces griseus in 1943. Neomycin, isolated from Streptomyces
fradiae, had better activity than streptomycin against aerobic
gram-negative bacilli but, because of its formidable toxicity,
could not safely be used systemically. Gentamicin, isolated from
Micromonospora in 1963, was a breakthrough in the treatment of
gram-negative bacillary infections, including those caused by
Pseudomonas acruginosa. Other aminoglycosides were subsequently
developed, including amikacin (Amikin), netilmicin (Netromycin) and
tobramycin (Nebcin), which are all currently available for systemic
use in the United States.
[0162] Within the aminoglycoside family, the suffix "-mycin" is
used in the name when the antibiotic is produced by Streptomyces
species and "micin," when produced by Micromonospora species.
[0163] Structure of Aminoglycosides
[0164] Aminoglycosides are water soluble weak bases that are
polycations at body pH. They are chemically similar in that they
have one of two bases to which is attached two or three
aminosugars. The aminosugars are linked to the by glycosidic bonds,
hence the group name. The base in streptomycin is streptidine, but
all of the others have 2-deoxystreptamine so that most of the
members differ in the number and nature of the aminosugars attached
to the 2-deoxystreptamine.
[0165] The terms "aminoglycoside" is used herein according to its
conventional chemical meanings, as described in various standard
texts on organic chemistry. The following is a condensed summary of
these terms.
[0166] As implied by the term, an aminosaccharide is a saccharide
molecule (the term saccharide is used interchangeably with sugar)
having at least one amine group coupled to it, either directly or
indirectly. Saccharide molecules (i.e., polyhydroxylated aldehydes
or ketones) exist as both straight chains and ring structures,
which spontaneously convert back and forth between straight and
ring forms in an equilibrium-type "tautomeric" mode. Since the
equilibria between straight and ring structures tends to generate
more ring structures than straight chains at any given moment when
dissolved in an aqueous solvent, most saccharides are usually drawn
and discussed as ring structures.
[0167] Saccharide rings are called furanose rings if the ring
structure itself (excluding any pendant groups) contains five
atoms, and pyranose rings if the ring contains six atoms. Most
furanose molecules are derived from pentose sugars (i.e., sugars
which contain five carbon atoms, such as ribose, arabinose, or
xylose). In a pentose molecule, one of the atoms in the ring form
of the molecule is an oxygen atom; the fifth carbon atom is
attached to the ring in a pendant structure, usually as a
hydroxylated methyl group. In the same manner, most pyranose
molecules (with six-membered rings) are hexose sugars such as
glucose, galactose, and mannose, or derivatives thereof. Hexose
sugars contain six carbon atoms; in the most common pyranose ring,
five carbons are in the ring along with an oxygen atom; the sixth
carbon atom is attached to the ring in a pendant group.
[0168] A glycoside molecule contains at least one saccharide
component (usually drawn as a ring) attached through an oxygen atom
(which can be regarded as an ether linkage) to a second molecular
group having at least one carbon atom. If a glycoside molecule is
chemically hydrolyzed to break the ether linkage(s), it will
release at least one saccharide molecule. Usually, the glycoside
linkage is between adjacent saccharide rings, to form
disaccharides, trisaccharides, polysaccharides, etc.
[0169] As implied by the name, an aminoglycoside is a glycoside
with one or more amino groups. Because of their biological
properties, aminoglycosides are an important class of
aminosaccharides. Neomycin A (neamine), Neomycin B and C,
Gentamicin, sisomycin, streptomycin, and tobramycin are all
aminoglycosides, since they have the requisite amine groups,
saccharide rings, and oxygen linkages. Most aminoglycosides were
initially identified due to the anti-bacterial activities of
various microbes that synthesize such compounds in nature. Many of
these aminoglycoside antibiotics can be altered or derivatized in
various ways that do not destroy their antibiotic activity; for
example, if Neomycin B or Neomycin C is cleaved between the
disaccharide structure and the pentose ring, the two cleavage
products are Neomycin A (a disaccharide, also known as neamine) and
either Neobiosamine B or Neobiosamine C.
[0170] The aminoglycosides and derivatives thereof that are of
particular interest to the present invention have the structure:
1
[0171] Wherein each of R1-R13 is independently hydrogen, alkyl,
optionally substituted alkyl, alkenyl, optionally substituted
alkenyl, alkynyl, optionally substituted alkynyl, aryl, optionally
substituted aryl, cycloalkyl, optionally substituted cycloalkyl,
alkoxy, optionally substituted alkoxy, heterocyclic, optionally
substituted heterocyclic, heteroaryl, optionally substituted
heteroaryl, hydroxyl, halogen, nitro, carboxyl, thioalkyl, amino,
alkylamino, arylamino, amido, ammonium, alkylammonium, sulfonyl,
aminosulfonyl, alkylsulfonyl, alkoxycarbonyl, acetyl, or acyl.
[0172] In one embodiment, each of R1-R13 is independently hydrogen,
alkyl, optionally substituted alkyl, alkoxy, optionally substituted
alkoxy, cycloalkyl, optionally substituted cycloalkyl,
cyclooxyalkyl, optionally substituted cyclooxyalkyl, hydroxyl,
halogen, amino, alkylamino, amido, ammonium, alkoxycarbonyl,
acetyl, or acyl.
[0173] In another embodiment, each of R1-R13 is independently
hydrogen, alkyl, optionally substituted alkyl, hydroxyl, alkoxy,
optionally substituted alkoxy, halogen, amino, acetyl, or acyl. In
a related embodiment, at least one of R1-R13 is independently
acetyl.
[0174] In another embodiment, at least one of R2, R3, R6, R12, or
R13 is halogen.
[0175] In a related embodiment, the halogen is fluorine.
[0176] In another embodiment, at at least one of R4, R5, or R6 is
acyl.
[0177] In another embodiment, R7 is alkyl or optionally substituted
alkyl. In a related embodiment, the optionally substituted alkyl is
a C6 to C12 alkyl.
[0178] In another embodiment, each of R1-R13 is independently
hydrogen, alkyl, optionally substituted alkyl, alkenyl, optionally
substituted alkenyl, alkynyl, optionally substituted alkynyl,
alkoxy, optionally substituted alkoxy, aryl, optionally substituted
aryl, cycloalkyl, optionally substituted cycloalkyl, heterocyclic,
optionally substituted heterocyclic, heteroaryl, optionally
substituted heteroaryl, hydroxyl, halogen, nitro, carboxyl,
thioalkyl, amino, alkylamino, arylamino, amido, ammonium,
alkylammonium, sulfonyl, aminosulfonyl, alkylsulfonyl,
alkoxycarbonyl, acetyl, or acyl, with the proviso that when R6=H,
R7=H, R8=CH3, R9=OH, R10=CH3,R11=H, R12=OH, and R13=OH; if R1=NH2,
R2=H, R3=H, and R4=CH3, then R5 is not NH2 or NHCH3; and if R1=OH,
R2=OH, R3=OH, and R4 is H, then R5 is not NH2.
[0179] In another embodiment, R1=NH2, R2=H, R3=H, R4=CH3, R5=NH2 or
NHCH3, R6=H, R7=H, R8=CH3, R9=OH, R10=CH3, R11=H, R2=OH, and
R13=OH.
[0180] In another embodiment, R1=OH, R2=OH, R3=OH, R4=H, R5=NH2,
R6=H, R7=H, R8=CH3,R9=OH, R10=CH3,R11 H, R120H, and R13=OH.
[0181] In addition to the above, some compounds are also called
aminoglycosides even though they do not have a glycosidic oxygen
linkage, since they are components of larger molecules which are
true aminoglycosides, and they are commonly synthesized using
bacterial aminoglycosides as starting reagents. Examples include
2,6-diamino-2,6-dideoxy-D-glucose (which can be obtained by
hydrolyzing Neomycin) and streptidine (which can be obtained by
hydrolyzing streptomycin). As used herein, the term
"aminoglycoside" encompasses these compounds as well.
[0182] Gentamicin and derivatives thereof are one type of
aminoglycoside of interest, as gentamicin is known to inhibit SMase
(Ghosh et al., J. Biol. Chem. 262:12550-12556, 1987). Several
isoforms of gentamicin are known, including Gentamicin C,
Gentamicin C1a, Gentamicin C2, Gentamicin C26 and Gentamicin B (see
Example 13 for the structures of these isoforms). Methods of
preparing gentamicin isoforms and derivatives are disclosed in U.S.
Pat. No. 3,984,395 (Method of isolating gentamicin C2a); U.S. Pat.
No. 4,288,547 (Fermentative process for preparing antibiotics of
the gentamicin class); and U.S. Pat. No. 5,814,488 (Semisynthetic
1-N-ethylgentamicin C1a and method for its preparation). Gentamicin
derivatives are disclosed in U.S. Pat. No. 4,387,219 (2-Hydroxy
gentamicin compounds); U.S. Pat. No. 4,283,528
(1-N-aminohydroxyacyl derivatives of gentamicin B); and U.S. Pat.
No. 4,223,024 (4"-O-Alkylgentamicins and sagamicins). U.S. Pat. No.
4,150,949, (Immunoassay for gentamicin), discloses
fluorescein-labelled gentamicins, including fluoresceinthiocarbanyl
gentamicin.
[0183] Kanamycin and derivatives thereof are of interest. Many
chemically modified derivatives of kanamycin are known; for a
review, see Mingeot-Leclercq et al., Antimicrobial Agents and
Chemoctherapy 43:727-737, 1999. Kanamycin derivatives are described
in U.S. Pat. No. 4,873,225
(1-n-(4-amino-3-fluoro-2-hydroxybutyryl)-kanamycins); U.S. Pat. No.
4,455,419 (2'-Modified kanamycins and production thereof); U.S.
Pat. No. 4,424,343 (Preparation of
1-N->.omega.-amino-.alpha.-hydroxyalkano- ylkanamycin
polysilylates and products); U.S. Pat. No. 4,337,336 (Derivative of
kanamycin A and a process for the preparation thereof); U.S. Pat.
No. 4,195,170 (3',4'-Episulfido kanamycin B compounds); U.S. Pat.
No. 4,178,437 (1-N-Kanamycin derivatives); U.S. Pat. No. 4,170,642
(Derivatives of kanamycin A); U.S. Pat. No. 4,140,849 (Kanamycin C
derivatives); U.S. Pat. No. 4,120,955 (Method for production of
kanamycin C and its derivatives); U.S. Pat. No. 3,974,137 (Process
for the preparation of
1-[L-(-)-gamma-amino-alpha-hydroxybutyryl]-kanamycin A (RD-1341A));
U.S. Pat. No. 3,940,382 (1,2'-Di-N-substituted kanamycin B
compounds); U.S. Pat. No. 4,178,437 (1-N-Kanamycin derivatives);
U.S. Pat. No. 4,140,849 (Kanamycin C derivatives); U.S. Pat. No.
4,181,797 (1-N-(omega-amino-alpha-hydroxyalkanoyl) derivatives of
4'-deoxy-6'-N-methylkanamycin A); and U.S. Pat. No. 4,051,315
(6"-Deoxykanamycin B and 6"-deoxytobramycin).
[0184] Fortimicin and derivatives thereof are of interest.
Fortimicin is a naturally occurring aminoglycoside antibiotic,
first produced by fermentation of a microorganism belonging to the
genus Micromonospora. Studies of fortimicin showed that blocking
the 2-hydroxy group by inactivates the antibiotic. As a result,
much attention was focused on developing chemical modifications to
the aminoglycoside that position in order to develop more stable
fortimicin derivatives. Fortimicin and chemical derivatives of
Fortimicin are described in U.S. Pat. No. 4,214,079 (4-N, 2'-N and
4,2'-Di-N-fortimicin AL derivatives); U.S. Pat. No. 4,214,078
(Fortimicin AL); U.S. Pat. No. 4,214,076 (2'-N-Substituted
fortimicin B and derivatives); U.S. Pat. No. 4,220,756 (Method of
producing 3-O-demethylfortimicin B,4-N-alkylfortimicin B
derivatives and related aminoglycoside antibiotics); U.S. Pat. No.
4,219,644 (Fortimicins AH and AI); U.S. Pat. No. 4,219,643
(Fortimicin AN); U.S. Pat. No. 4,219,642 (Fortimicin AO); U.S. Pat.
No. 4,214,080 (Fortimicins AM and AP); U.S. Pat. No. 4,214,075
(6'-Epi-fortimicin A and B derivatives); U.S. Pat. No. 4,213,974
(4-N,2'-N and 4,2'-Di-N-fortimicin AO derivatives); U.S. Pat. No.
4,213,972 (4-N, 2'-N and 4,2'Di-N-fortimicins AH and AI); U.S. Pat.
No. 4,213,971 (4-N, 2'-N and 4,2'-Di-N-fortimicin AD derivatives);
U.S. Pat. No. 4,207,415 (Method of producing 2-deoxyfortimicin A);
U.S. Pat. No. 4,205,070 (6'N-Alkyl- and 6',6'-di-N-alkyl
derivatives of fortimicins A and B); U.S. Pat. No. 4,176,178
(2-Deoxy-2'3-N-acyl and alkyl fortimicins A and B); U.S. Pat. No.
4,169,198 (2-Deoxyfortimicin B); U.S. Pat. No. 4,183,920 (4-N-Acyl,
2'-N-acyl and 4,2'-N,N'-diacylfortimicin E derivatives); U.S. Pat.
No. 4,192,867 (2-Deoxyfortimicin A, 4-N-alkyl and
4-N-acyl-2-deoxyfortimicin B derivatives); U.S. Pat. No. 4,220,756
(Method of producing 3-O-demethylfortimicin B,4-N-alkylfortimicin B
derivatives and related aminoglycoside antibiotics); U.S. Pat. No.
4,196,197 (2'N-Acyl and alkyl-6'-N-alkyl- and 6',6'-di-N-alkyl
derivatives of fortimicins A and B); U.S. Pat. No. 4,187,299
(Fortimicin E); U.S. Pat. No. 4,187,298 (2N-acyl and alkyl
fortimicin B and derivatives, 4,2'-N,N'diacyl and dialkyl
fortimicin B derivatives 4-N-acyl-2'-N-alkyl and
4-N-alkyl-2'-N-acyl fortimicin B derivatives); U.S. Pat. No.
4,187,297 (3-De-O-methyl-2-N-acyl and alkyl fortimicins A and B);
U.S. Pat. No. 4,187,296 (2-N-acyl and alkyl 6-epi-fortimicin B and
derivatives); U.S. Pat. No. 4,208,407 (5-Deoxyfortimicin A,
2,5-dideoxyfortimicin A and the corresponding 4-N-acyl and alkyl
fortimicin B derivatives thereof and intermediates therefor); U.S.
Pat. No. 4,216,210 (Fortimicins AM and AP derivatives); U.S. Pat.
No. 4,319,022 (2-O-Substituted sulfonyl derivatives of fortimicin
B); U.S. Pat. No. 4,251,516 (2-Deoxy-3-O-Demethylfortimicins); U.S.
Pat. No. 4,251,511 (Antibiotic and fermentation process of
preparing); U.S. Pat. No. 4,250,304 (2-Deoxy-2-substituted
fortimicin A and B and derivatives); U.S. Pat. No. 4,317,904
(1,2-Epiminofortimicin B); U.S. Pat. No. 4,255,421 (Fortimicin
aminoglycosides, process for production thereof, and use thereof);
U.S. Pat. No. 4,252,972 (Fortimicin B-1,2:4,5-bis-carbamates); U.S.
Pat. No. 4,418,193 (Method of producing 2-epi-fortimicin A); and
U.S. Pat. No. 4,207,415 (Method of producing 2-deoxyfortimicin
A).
[0185] Sisomicin and derivatives thereof are disclosed in U.S. Pat.
No. 4,438,260 (Sisomicin compounds); U.S. Pat. No. 4,369,251
(Method for the production of sisomicin); U.S. Pat. No. 4,365,020
(Method for the preparation of antibiotic sisomicin); U.S. Pat. No.
4,336,369 (Selectively protected
1-N-(omega-aminoalkoxycarbonyl)-sisomicin derivatives); U.S. Pat.
No. 4,312,859 (Sisomicin derivatives, processes for their
production and their medicinal use); U.S. Pat. No. 3,997,524
(Process for the manufacture of 6'-N-alkyl derivatives of sisomicin
and verdamicin; novel intermediates useful therein, and novel
6'-N-alkylverdamicins prepared thereby); and U.S. Pat. No.
3,988,316 (Antibiotics sisomicin and verdamicin I and complex
containing same).
[0186] Amikacin and derivatives thereof are disclosed in U.S. Pat.
No. 5,763,587 (Process for the synthesis of amikacin); U.S. Pat.
No. 5,656,735 (Process for the preparation of amikacin precursors);
U.S. Pat. No. 5,621,085 (Process for the preparation of amikacin
precursors); U.S. Pat. No. 4,985,549 (Process for preparing
amikacin); and U.S. Pat. No. 4,902,790 (Novel process for the
synthesis of amikacin).
[0187] Dibekacin and derivatives thereof are disclosed in U.S. Pat.
No. 5,618,795 (Dibekacin derivatives and arbekacin derivatives
active against resistant bacteria, and the production thereof); and
U.S. Pat. No. 5,488,038 (Dibekacin derivatives and arbekacin
derivatives active against resistant bacteria).
[0188] Other aminoglycosides are disclosed in U.S. Pat. No.
4,855,287 (Aminoglycoside compounds, processes for production
thereof, and pharamaceutical composition containing the same); U.S.
Pat. No. 5,442,047 (Process for preparing isepamicin); U.S. Pat.
No. 4,208,531 (Synthetic aminoglycosides); U.S. Pat. No. 4,656,160
(Aminoglycoside derivatives); U.S. Pat. No. 4,647,656
(Aminoglycoside compounds); U.S. Pat. No. 4,645,760 (Activated
aminoglycosides and aminoglycoside-aminocyclitols pharmaceutical
compositions and method of use); U.S. Pat. No. 4,617,293 (Flavonoid
phosphate salts of aminoglycoside antibiotics); U.S. Pat. No.
4,554,269 (Kasugamycin derivatives, pharmaceutical compositions and
method of use); U.S. Pat. No. 4,503,046 (1-Nitro-aminoglycoside
derivatives, pharmaceutical compositions containing them and such
derivatives for use as pharmaceuticals); U.S. Pat. No. 4,493,831
(Aminoglycoside derivatives); U.S. Pat. No. 4,486,418 (2'-Deamino
aminoglycosides and composition thereof); U.S. Pat. No. 4,468,513
(2'-N-Acylated and 2'-N-alkylated derivatives of
4-O-substituted-2-deoxys- treptamine aminoglycosides); U.S. Pat.
No. 4,468,512 (1-N-Acylated and 1-N-alkylated derivatives of
4-O-substituted-2-deoxystreptamine aminoglycosides); U.S. Pat. No.
4,438,107 (Aminoglycosides and use thereof); U.S. Pat. No.
4,424,345 (1-N-Acylated and 1-N-alkylated derivatives of
4-O-substituted-2-deoxystreptamine aminoglycosides and process);
U.S. Pat. No. 4,424,344 (2-N-Acylated and 2-N-alkylated derivatives
of 4-O-substituted-2-deoxystreptamine aminoglycosides and process);
U.S. Pat. No. 4,380,625 (Process for the preparation of purified
aminoglycoside antibiotics); U.S. Pat. No. 4,349,667
(Aminoglycoside antibiotic G-367-2); U.S. Pat. No. 4,347,354
(Preparation of
1'-N-[.omega.-amino-.alpha.-hydroxyalkanoyl]aminoglycoside
polysilylated antibiotics and products obtained therefrom); U.S.
Pat. No. 4,330,673 (Process for producing
3-O-demethylaminoglycoside and novel 3-O-demethylfortimicin
derivatives); U.S. Pat. No. 4,297,486 (Aminoglycoside antibiotic
G-367-1 and method for the production thereof); U.S. Pat. No.
4,297,485 (Production of a selectively protected N-acylated
derivative of an aminoglycosidic antibiotic); U.S. Pat. No.
4,279,997 (Process for production of aminoglycoside antibiotics);
U.S. Pat. No. 4,273,923 (Process for preparing aminoglycoside
derivatives); U.S. Pat. No. 4,255,421 (Fortimicin aminoglycosides,
process for production thereof, and use thereof); U.S. Pat. No.
4,252,972 (Fortimicin B-1,2:4,5-bis-carbamates); U.S. Pat. No.
4,250,170 (Antibacterial agents Bu-2349A and B and method of using
same); U.S. Pat. No. 4,248,865 (Novel aminoglycoside derivatives);
U.S. Pat. No. 4,242,331 (Aminoglycosides and method of use); U.S.
Pat. No. 4,230,847 (Aminoglycoside antibiotic compounds); U.S. Pat.
No. 4,226,978 (beta-Galactosyl-umbelliferone-labele- d
aminoglycoside antibiotics and intermediates in their preparation);
U.S. Pat. No. 4,223,022 Stabilized aminoglycoside antibiotic
formulations); U.S. Pat. No. 4,217,446 (omega.Amino-2-hydroxyalkyl
derivatives of aminoglycoside antibiotics); U.S. Pat. No. 4,214,074
Hydroxyalkyl derivatives of aminoglycoside antibiotics); U.S. Pat.
No. 4,212,859
(2'-Hydroxy-2'-desamino-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols,
methods for their manufacture, method for their use as
antibacterial agents, and compositions useful therefor: U.S. Pat.
No. 4,209,511 (Aminoglycoside antibiotics and process for
production thereof); U.S. Pat. No. 4,207,314 (Isofortimicin); U.S.
Pat. No. 4,201,774 (Novel aminoglycoside derivatives); U.S. Pat.
No. 4,200,628 (Novel aminoglycoside derivatives); U.S. Pat. No.
4,199,5701-N-Hetero containing aminoglycoside derivatives); U.S.
Pat. No. 4,189,569 (Seldomycin factor 5 derivatives); U.S. Pat. No.
4,187,372 (Seldomycin factor 5 derivative); U.S. Pat. No. 4,187,299
(Fortimicin E); U.S. Pat. No. 4,170,643
(Aminoglycoside-aminocyclitol derivatives and method of use); U.S.
Pat. No. 4,166,114 (Aminoglycoside antibiotic derivatives and
method of use); U.S. Pat. No. 4,146,617 (Desoxystreptamine
derivatives, salts, pharmaceutical compositions and method of use);
U.S. Pat. No. 4,136,254 (Process of selectively blocking amino
functions in aminoglycosides using transition metal salts and
intermediates used thereby); U.S. Pat. No. 4,125,707 (Protected
pseudotrisaccharide intermediate for paromomycin and neomycin
derivatives); U.S. Pat. No. 4,117,221 (Aminoacyl derivatives of
aminoglycoside antibiotics); U.S. Pat. No. 4,107,435 (Process for
.omega.-amino-2-hydroxyalkyl derivatives of aminoglycoside
antibiotics); U.S. Pat. No. 4,101,556 (Total synthesis of
2,5-dideoxystreptamines); U.S. Pat. No. 4,085,208 (Process for
preparing 4,6-di-O-(aminoglycosyl)-1- ,3-diaminocyclitols and novel
1-epimers and 1-N-alkyl derivatives produced thereby; methods for
the use of the 1-epimer derivatives as antibacterial agents and
compositions useful therefor); U.S. Pat. No.
4,066,7521-Desamino-1-hydroxy and
1-desamino-1-epi-hydroxy-4,6-di-o-(amin-
oglycosyl)-1,3-diaminocyclitols;
1-desamino-1-oxo-4,6-di-o-(aminoglycosyl)- -1,3-diaminocyclitols,
intermediates and use as antibacterial agents); U.S. Pat. No.
4,065,615 (Deoxyaminoglycoside antibiotic derivatives); U.S. Pat.
No. 4,064,339 (Antibiotic aminoglycosides, processes of preparation
and pharmaceutical compositions); U.S. Pat. No. 4,049,498 (Methods
for the preparation of semi-synthetic aminocyclitol aminoglycoside
antibiotics); U.S. Pat. No. 4,044,123
(6'-N-alkyl-4,6-di-O-(aminoglycosyl)-1,3-diaminocyclitols, methods
for their use as antibacterial agents and compositions useful
therefor); U.S. Pat. No. 4,038,478 (O-Glycoside ortho esters of
neamine containing compounds); U.S. Pat. No. 4,032,404
(Fermentation process for producing apramycin and nebramycin factor
V'); U.S. Pat. No. 4,031,210 (Antibiotic aminoglycosides, processes
of preparation and pharmaceutical compositions); U.S. Pat. No.
4,024,332 (Aminoglycoside antibiotics and intermediates therefor);
U.S. Pat. No. 4,020,269 (Epiminodeaminodeoxyamin- oglycoside
antibiotics and intermediates); U.S. Pat. No. 4,012,576 (Antibiotic
complex Bu 2183); U.S. Pat. No. 4,011,390 (Semi-synthetic
aminocyclitol aminoglycoside antibiotics and methods for the
preparation thereof); U.S. Pat. No. 4,009,328 (Aminoglycoside
66-40C, method for its manufacture, method for its use as an
intermediate in the preparation of known antibiotics and novel
antibacterials); U.S. Pat. No. 4,003,922 (Synthesis of
cis-1,4-cyclohexadiene dioxide); U.S. Pat. No. 4,002,608
(1-N-alkyl-aminoglycoside-XK-88 derivatives and methods for their
manufacture); U.S. Pat. No. 3,996,205 (Aminoglycoside antibiotics
and intermediates); U.S. Pat. No. 3,984,393 (Aminoglycoside
antibiotics); U.S. Pat. No. 3,981,861 (Antibiotic aminoglycosides,
processes of preparation and pharmaceutical compositions); U.S.
Pat. No. 3,978,214 (Novel
4,6-di-O-(aminoglycosyl)-2-deoxystreptamine, method for its
manufacture, method for its use as an antiprotozoal agent and
compositions useful thereof); U.S. Pat. No. 3,962,429 (Method for
reducing side effects of aminoglycoside antibiotics and composition
therefor); U.S. Pat. No. 3,959,255 (Antibiotic aminoglycosides, and
process of preparation); U.S. Pat. No. 3,953,422 (Deoxyglucose
derivatives); and U.S. Pat. No. 3,953,293 (Process for the
preparation of xylostasin).
[0189] Additional aminoglycosides are disclosed by Matsumoto et
al., "Synthesis of novel 13-methyl-13-dihydroanthracyclines", Chem
Pharm Bull (Tokyo), 34:4613-9, 1986; Israel et al., "Adriamycin
analogues. Preparation and biological evaluation of some
N-(trifluoroacetyl)-14-O-[(- N-acetylamino)acyl]adriamycin
derivatives", J Med. Chem., 2:1273-6, 1986; Takahashi et al.,
"Production of novel antibiotic, dopsisamine, by a new subspecies
of Nocardiopsis mutabilis with multiple antibiotic resistance", J
Antibiot (Tokyo), 39:175-83, 1986; Yasuda et al., "Total synthesis
of 3-O-demethylsporaricin A", J Antibiot (Tokyo), 38:1512-25, 1985;
Tsunakawa et al., "Inosamycin, a complex of new aminoglycoside
antibiotics. I. Production, isolation and properties", J Antibiot
(Tokyo), 38:1302-12, 1985; Matsuhashi et al., "In vitro and in vivo
antibacterial activities of dactimicin, a novel pseudodisaccharide
aminoglycoside, compared with those of other aminoglycoside
antibiotics", Antimicrob Agents Chemother, 27:589-94, 1985;
Matsunaga et al., "Bacterial uptake of habekacin, a novel
aminoglycoside antibiotic", J Antibiot (Tokyo), 37:596-601, 1984;
Tanaka et al., "Mechanism of action of habekacin, a novel amino
acid-containing aminoglycoside antibiotic", Antimicrob Agents
Chemother, 24;797-802, 1983; Dawson, "Activity of SC33428, a novel
bishydrazone-bridged derivative of 4-demethoxydaunorubicin, against
experimental tumors in mice", Cancer Res., 43:2880-3, 1983;
Digranes et al., "N-formimidoyl thienamycin: in vitro comparison
with cefoxitin and tobramycin against clinical, bacterial
isolates", Acta Pathol Microbiol Immunol Scand [B], 91:141-4, 1983;
FitzGerald et al., "3,4-Dihydroxybenzylamine: an improved dopamine
analog cytotoxic for melanoma cells in part through oxidation
products inhibitory to dna polymerase", J Invest Dermatol.,
80:119-23, 1983; Israel et al., "Adriamycin analogues. Novel
anomeric ribofuranoside analogues of daunorubicin", J Med. Chem.,
25:28-31, 1982; Tanaka, "Effects of habekacin, a novel
aminoglycoside antibiotic, on experimental corneal ulceration due
to Pseudomonas aeruginosa", J Antibiot (Tokyo), 34:892-7, 19881;
Fujiwara et al., "Production of a new aminoglycoside antibiotic by
a mutant of Bacillus circulans", J Antibiot (Tokyo), 33:836-41,
1980; Ohashi et al., "in vitro and in vivo antibacterial activity
of KW1070, a new aminoglycoside antibiotic", Antimicrob Agents
Chemother., 17:138-43, 1980; Inouye et al., "A novel aminoglycoside
antibiotic, substance SF-2052", J Antibiot (Tokyo), 32:1355-6,
1979; Perzynski et al., "Effects of apramycin, a novel
aminoglycoside antibiotic on bacterial protein synthesis", Eur J.
Biochem., 99:623-8, 1979; Suzuki et al., "Preparation and some
microbiological properties of novel kanamycin-glucoside
derivatives", J Antibiot (Tokyo), 32:753-5, 1979; Smith et al.,
"Synthesis of daunorubicin analogues with novel 9-acyl
substituents", J Med. Chem., 22:40-4, 1979; Davies et al.,
"Semisynthetic aminoglycoside antibacterials. 6. Synthesis of
sisomicin, Antibiotic G-52, and novel 6'-substituted analogues of
sisomicin from aminoglycoside 66-40C", J Med. Chem., 21:189-93,
1978; Egan et al., "Fortimicins A and B, new aminoglycoside
antibiotics. 111. Structural identification", J Antibiot (Tokyo),
30;552-63, 1977; Okachi et al., "Fortimicins A and B, new
aminoglycoside antibiotics. II. Isolation, physico-chemical and
chromatographic properties", J Antibiot (Tokyo), 30:541-51, 1977;
Kinumaki et al., "Macrolide antibiotics M-4365 produced by
Micromonospora. II. Chemical structures", J Antibiot (Tokyo),
30:450-4, 1977; Okutani et al., "Conversion of aminoglycosidic
antibiotics: Novel and efficient approaches to
3'-deoxyaminoglycosides via 3'-phosphoryl esters", J Am Chem Soc.,
99:1278-9, 1977; Hanessian et al., "Aminoglycoside antibiotics:
oxidative degradations leading to novel biochemical probes and
synthetic intermediates", J Antibiot (Tokyo), 28:835-7, 1975;
Reimann et al., "The structure of sisomicin, a novel unsaturated
aminocyclitol antibiotic from Micromonospora inyoensis", J Org
Chem., 39:1451-7, 1974; Kugelman et al., "Letter: The preparation
of garamine, a novel pseudodisaccharide from sisomycin", J Antibiot
(Tokyo), 26:394-5, 1973; Arcamone et al., "Adriamycin
(14-hydroxydaunomycin), a novel antitumor antibiotic", Tetrahedron
Lett., 13:1007-10, 1969; Matsumoto et al., "Synthesis of novel
13-methyl-13-dihydroanthracyclines"- , Chem Pharm Bull (Tokyo),
34:4613-9, 1986; Israel et al., "Adriamycin analogues. Preparation
and biological evaluation of some
N-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamycin
derivatives", J Med. Chem., 29:1273-6, 1986; Takahashi et al.,
"Production of novel antibiotic, dopsisamine, by a new subspecies
of Nocardiopsis mutabilis with multiple antibiotic resistance", J
Antibiot (Tokyo), 39:175-83, 1986; Yasuda et al., "Total synthesis
of 3-O-demethylsporaricin A", J Antibiot (Tokyo), 38:1512-25, 1985;
Tsunakawa et al., "Inosamycin, a complex of new aminoglycoside
antibiotics. I. Production, isolation and properties", J Antibiot
(Tokyo), 38:1302-12, 1985; Matsuhashi et al., "In vitro and in vivo
antibacterial activities of dactimicin, a novel pseudodisaccharide
aminoglycoside, compared with those of other aminoglycoside
antibiotics", Antimicrob Agents Chemother., 27:589-94, 1985;
Matsunaga et a I., "Bacterial uptake of habekacin, a novel
aminoglycoside antibiotic", J Antibiot (Tokyo), 37:596-601, 1984;
Tanaka et al., "Mechanism of action of habekacin, a novel amino
acid-containing aminoglycoside antibiotic", Antimicrob Agents
Chemother., 24:797-802, 1983; Dawson, "Activity of SC33428, a novel
bishydrazone-bridged derivative of 4-demethoxydaunorubicin, against
experimental tumors in mice", Cancer Res., 43:2880-3, 1983;
Digranes et al., "N-formimidoyl thienamycin: in vitro comparison
with cefoxitin and tobramycin against clinical, bacterial
isolates", Acta Pathol Microbiol Immunol Scand [B]., 91:141-4,
1983; FitzGerald et al., "3,4-Dihydroxybenzylamine: an improved
dopamine analog cytotoxic for melanoma cells in part through
oxidation products inhibitory to dna polymerase", J Invest
Dermatol., 80:119-23, 1983; Israel et al., "Adriamycin analogues.
Novel anomeric ribofuranoside analogues of daunorubicin", J Med.
Chem., 25:28-31, 1982; Tanaka, "Effects of habekacin, a novel
aminoglycoside antibiotic, on experimental corneal ulceration due
to Pseudomonas aeruginosa", J Antibiot (Tokyo), 34:892-7, 1981;
Fujiwara et al., "Production of a new aminoglycoside antibiotic by
a mutant of Bacillus circulans", J Antibiot (Tokyo), 33:836-41,
1980; Ohashi et al., "In vitro and in vivo antibacterial activity
of KW1070, a new aminoglycoside antibiotic", Antimicrob Agents
Chemother., 17:138-43, 1980; Inouye et al., "A novel aminoglycoside
antibiotic, substance SF-2052", J Antibiot (Tokyo), 32:1355-6,
1979; Perzynski et al., "Effects of apramycin, a novel
aminoglycoside antibiotic on bacterial protein synthesis", Eur J.
Biochem., 99:623-8, 1979; Suzuki et al., "Preparation and some
microbiological properties of novel kanamycin-glucoside
derivatives", J Antibiot (Tokyo), 32:753-5, 1979; Smith et al.,
"Synthesis of daunorubicin analogues with novel 9-acyl
substituents", J Med. Chem., 22:40-4, 1979; Davies et al.,
"Semisynthetic aminoglycoside antibacterials. 6. Synthesis of
sisomicin, Antibiotic G-52, and novel 6'-substituted analogues of
sisomicin from aminoglycoside 66-40C", J Med. Chem., 21:189-93,
1978; Egan et al., "Fortimicins A and B, new aminoglycoside
antibiotics. III. Structural identification", J Antibiot (Tokyo),
30:552-63, 1977; Okachi et al., "Fortimicins A and B, new
aminoglycoside antibiotics. II. Isolation, physico-chemical and
chromatographic properties", J Antibiot (Tokyo), 30:541-51, 1977;
Kinumaki et al., "Macrolide antibiotics M-4365 produced by
Micromonospora. II. Chemical structures", J Antibiot (Tokyo),
30:450-4, 1977; Okutani et al., "Conversion of aminoglycosidic
antibiotics: Novel and efficient approaches to
3'-deoxyaminoglycosides via 3'-phosphoryl esters", J Am Chem Soc.,
99:1278-9, 1977; Hanessian et al., "Aminoglycoside antibiotics:
oxidative degradations leading to novel biochemical probes and
synthetic intermediates", J Antibiot (Tokyo), 28:835-7, 1975;
Reimann et al., "The structure of sisomicin, a novel unsaturated
aminocyclitol antibiotic from Micromonospora inyoensis", J Org
Chem., 39:1451-7, 1974; Kugelman et al., "Letter: The preparation
of garamine, a novel pseudodisaccharide from sisomycin", J Antibiot
(Tokyo), 26:394-5, 1973; Arcamone et al., "Adriamycin
(14-hydroxydaunomycin), a novel antitumor antibiotic", Tetrahedron
Lett., 13:1007-10, 1969; Yew et al., "New antimycobacterial
agents", Monaldi Arch Chest Dis., 51:394-404, 1996; Urban et al.,
"Comparative in-vitro activity of SCH 27899, a novel everninomicin,
and vancomycin", J Antimicrob Chemother, 37:361-4, 1996; Lam et
al., "Production and isolation of two novel esperamicins in a
chemically defined medium", J Antibiot (Tokyo), 48:1497-501, 1995;
Pelyvas et al., "Novel aminocyclitol antibiotics derived from
natural carbohydrates", Carbohydr Res., 272:C5-9, 1995; Pelyvas et
al., "Synthesis of new pseudodisaccharide aminoglycoside
antibiotics from carbohydrates", J Antibiot (Tokyo), 48:683-95,
1995; Jones, "Isepamicin (SCH 21420, 1-N-HAPA gentamicin B):
microbiological characteristics including antimicrobial potency of
spectrum of activity", J Chemother., 2:7-16, 1995 Suppl; Abe et
al., "Novel antitumor antibiotics, saptomycins. II. Isolation,
physico-chemical properties and structure elucidation", J Antibiot
(Tokyo), 46:1536-49, 1993; Abe et al., "Novel antitumor
antibiotics, saptomycins. I. Taxonomy of the producing organism,
fermentation, HPLC analysis and biological activities", J Antibiot
(Tokyo), 46:1530-5, 1993; Phillipson et al., "Lanomycin and
glucolanomycin, antifungal agents produced by Pycnidiophora
dispersa. II. Structure elucidation", J Antibiot (Tokyo), 45:313-9,
1992; Mariani et al., "In vitro activity of novel sulphonic
derivatives of distamycin A", EXS., 61:455-8, 1992; Abe et al.,
"Novel antitumor antibiotics, saptomycins D and E", J Antibiot
(Tokyo), 44:908-11, 1991; Priebe et al., "3'-Hydroxyesorubicin.
Synthesis and antitumor activity", J Antibiot (Tokyo), 43:838-46,
1990; Brill et al., "Altromycins, novel pluramycin-like
antibiotics. II. Isolation and elucidation of structure", J
Antibiot (Tokyo), 43:229-37, 1990; Jackson et al., "Altromycins,
novel pluramycin-like antibiotics. I. Taxonomy of the producing
organism, fermentation and antibacterial activity", J Antibiot
(Tokyo), 43(3):223-8, 1990; Flynn et al., "The chiral synthesis and
biochemical properties of electron rich phenolic sulfoxide analogs
of sparsomycin", Biochem Biophys Res Commun., 166:673-80, 1990;
Kitamura et al., "Pirarubicin, a novel derivative of doxorubicin.
THP-COP therapy for non-Hodgkin's lymphoma in the elderly", Am J
Clin Oncol., 13 Suppl 1:S 15-9, 1990; Gu et al., "In vitro activity
of dactimicin, a novel pseudodisaccharide aminoglycoside, compared
with activities of other aminoglycosides", Antimicrob Agents
Chemother, 33:1998-2003, 1989; Lam, "Biosynthesis of elsamicin A, a
novel antitumor antibiotic", J Nat Prod., 52:1015-21, 1989; Rolston
et al., "in vitro activity of trospectomycin (U-63366F), a novel
spectinomycin analog, against gram-positive isolates from cancer
patients", Eur J Clin Microbiol Infect Dis., 8:254-60, 1989; Gupta
et al., "Synthesis, cytotoxicity, and antiviral activity of some
acyclic analogues of the pyrrolo[2,3-d]pyrimidine nucleoside
antibiotics tubercidin, toyocamycin, and sangivamycin", J Med.
Chem., 32:402-8, 1989; Hochlowski et al., "Phenelfamycins, a novel
complex of elfamycin-type antibiotics. II. Isolation and stricture
determination" J Antibiot (Tokyo), 41:1300-15, 1988; Jackson et
al., "Phenelfamycins, a novel complex of elfamycin-type
antibiotics. I. Discovery, taxonomy and fermentation", J Antibiot
(Tokyo), 41:1293-9, 1988; Saitoh et al., "Boholmycin, a new
aminoglycoside antibiotic. I. Production, isolation and
properties", J Antibiot (Tokyo), 41:855-61, 1988; Matsumoto et al.,
"Synthesis of novel 13-methyl-13-dihydroanthracyclines", Chem Pharm
Bull (Tokyo), 34:4613-9, 1986; Israel et al., "Adriamycin
analogues. Preparation and biological evaluation of some
N-(trifluoroacetyl)-14-O-[(- N-acetylamino)acyl]adriamycin
derivatives", J Med. Chem., 29:1273-6, 1986; Takahashi et al.,
"Production of novel antibiotic, dopsisamine, by a new subspecies
of Nocardiopsis mutabilis with multiple antibiotic resistance", J
Antibiot (Tokyo), 39:175-83, 1986; Yasuda et al., "Total synthesis
of 3-O-demethylsporaricin A", J Antibiot (Tokyo), 38:1512-25, 1985;
Tsunakawa et al., "Inosamycin, a complex of new aminoglycoside
antibiotics. I. Production, isolation and properties", J Antibiot
(Tokyo), 38:1302-12, 1985; Matsuhashi et al., "In vitro and in vivo
antibacterial activities of dactimicin, a novel pseudodisaccharide
aminoglycoside, compared with those of other aminoglycoside
antibiotics", Antimicrob Agents Chemother., 27:589-94, 1985;
Matsunaga et al., "Bacterial uptake of habekacin, a novel
aminoglycoside antibiotic", J Antibiot (Tokyo), 37:596-601, 1984;
Tanaka et al., "Mechanism of action of habekacin, a novel amino
acid-containing aminoglycoside antibiotic", Antimicrob Agents
Chemother., 24:797-802, 1983; Dawson, "Activity of SC33428, a novel
bishydrazone-bridged derivative of 4-demethoxydaunorubicin, against
experimental tumors in mice", Cancer Res., 43:2880-3, 1983;
Digranes et al., "N-formimidoyl thienamycin: in vitro comparison
with cefoxitin and tobramycin against clinical, bacterial
isolates", Acta Pathol Microbiol Immunol Scand [B], 91:141-4, 1983;
FitzGerald et al., "3,4-Dihydroxybenzylamine: an improved dopamine
analog cytotoxic for melanoma cells in part through oxidation
products inhibitory to dna polymerase", J Invest Dermatol.,
80:119-23, 1983; Israel et al., "Adriamycin analogues. Novel
anomeric ribofuranoside analogues of daunorubicin", J Med. Chem.,
25:28-31, 1982; Tanaka, "Effects of habekacin, a novel
aminoglycoside antibiotic, on experimental corneal ulceration due
to Pseudomonas aeruginosa", J Antibiot (Tokyo), 34:892-7, 1981;
Fujiwara et al., "Production of a new aminoglycoside antibiotic by
a mutant of Bacillus circulans", J Antibiot (Tokyo), 33:836-41,
1980; Ohashi et al., "In vitro and in vivo antibacterial activity
of KW1070, a new aminoglycoside antibiotic", Antimicrob Agents
Chemother., 17:138-43, 1980; Inouye et al., "A novel aminoglycoside
antibiotic, substance SF-2052", J Antibiot (Tokyo), 32:1355-6,
1979; Perzynski et al., "Effects of apramycin, a novel
aminoglycoside antibiotic on bacterial protein synthesis", Eur J.
Biochem., 99:623-8, 1979; Suzuki et al., "Preparation and some
microbiological properties of novel kanamycin-glucoside
derivatives", J. Antibiot (Tokyo), 32:753-5, 1979; Smith et al.,
"Synthesis of daunorubicin analogues with novel 9-acyl
substituents", J Med. Chem., 22:40-4, 1979; Davies et al.,
"Semisynthetic aminoglycoside antibacterials. 6. Synthesis of
sisomicin, Antibiotic G-52, and novel 6'-substituted analogues of
sisomicin from aminoglycoside 66-40C", 3 Med. Chem., 21:189-93,
1978; Egan et al., "Fortimicins A and B, new aminoglycoside
antibiotics. III. Structural identification", J Antibiot (Tokyo),
30:552-63, 1977; Okachi et al., "Fortimicins A and B, new
aminoglycoside antibiotics. II. Isolation, physico-chemical and
chromatographic properties", J Antibiot (Tokyo), 30:541-51, 1977;
Kinumaki et al., "Macrolide antibiotics M-4365 produced by
Micromonospora. II. Chemical structures", J Antibiot (Tokyo),
30:450-4, 1977; Okutani et al., "Conversion of aminoglycosidic
antibiotics: Novel and efficient approaches to
3'-deoxyaminoglycosides via 3'-phosphoryl esters", J Am Chem Soc.,
99:1278-9, 1977; Hanessian et al., "Aminoglycoside antibiotics:
oxidative degradations leading to novel biochemical probes and
synthetic intermediates", J Antibiot (Tokyo), 28:835-7, 1975;
Reimann et al., "The structure of sisomicin, a novel unsaturated
aminocyclitol antibiotic from Micromonospora inyoensis", J Org
Chem., 39:1451-7, 1974; Kugelman et al., "Letter: The preparation
of garamine, a novel pseudodisaccharide from sisomycin", J Antibiot
(Tokyo), 26:394-5, 1973; Arcamone et al., "Adriamycin
(14-hydroxydaunomycin), a novel antitumor antibiotic", Tetrahedron
Lett., 13:1007-10, 1969; Yew et al., "New antimycobacterial
agents", Monaldi Arch Chest Dis., 51:394-404, 1996; Urban et al.,
"Comparative in-vitro activity of SCH 27899, a novel eveminomicin,
and vancomycin", J Antimicrob Chemother., 37:361-4, 1996; Lam et
al., "Production and isolation of two novel esperamicins in a
chemically defined medium", J Antibiot (Tokyo), 48:1497-501, 1995;
Pelyvas et al., "Novel aminocyclitol antibiotics derived from
natural carbohydrates", Carbohydr Res., 272:C5-9, 1995; Pelyvas et
al., "Synthesis of new pseudodisaccharide aminoglycoside
antibiotics from carbohydrates", J Antibiot (Tokyo), 48:683-95,
1995; Jones, "Isepamicin (SCH 21420, 1-N-HAPA gentamicin B):
microbiological characteristics including antimicrobial potency of
spectrum of activity", J Chemother., 7 Suppl 2:7-16, 1995; Abe et
al., "Novel antitumor antibiotics, saptomycins. II. Isolation,
physico-chemical properties and structure elucidation", J Antibiot
(Tokyo), 46:1536-49, 1993; Abe et al., "Novel
antitumor antibiotics, saptomycins. I. Taxonomy of the producing
organism, fermentation, HPLC analysis and biological activities", J
Antibiot (Tokyo), 46:1530-5, 1993; Phillipson et al., "Lanomycin
and glucolanomycin, antifungal agents produced by Pycnidiophora
dispersa. II. Structure elucidation", J Antibiot (Tokyo), 45:313-9,
1992; Mariani et al., "in vitro activity of novel sulphonic
derivatives of distamycin A", EXS., 61:455-8, 1992; Abe et al.,
"Novel antitumor antibiotics, saptomycins D and E", J Antibiot
(Tokyo), 44:908-11, 1989; Priebe et al., "3'-Hydroxyesorubicin.
Synthesis and antitumor activity", J Antibiot (Tokyo), 43:838-46,
1990; Brill et al., "Altromycins, novel pluramycin-like
antibiotics. II. Isolation and elucidation of structure", J
Antibiot (Tokyo), 43:229-37, 1990; Jackson et al., "Altromycins,
novel pluramycin-like antibiotics. I. Taxonomy of the producing
organism, fermentation and antibacterial activity", J Antibiot
(Tokyo), 43:223-8, 1990; Flynn et al., "The chiral synthesis and
biochemical properties of electron rich phenolic sulfoxide analogs
of sparsomycin", Biochem Biophys Res Commun., 166:673-80, 1990;
Kitamura et al., "Pirarubicin, a novel derivative of doxorubicin.
THP-COP therapy for non-Hodgkin's lymphoma in the elderly", Am J
Clin Oncol., 13 Suppl 1:S15-9, 1990; Gu et al., "In vitro activity
of dactimicin, a novel pseudodisaccharide aminoglycoside, compared
with activities of other aminoglycosides", Antimicrob Agents
Chemother., 33:1998-2003, 1989; Lam et al., "Biosynthesis of
elsamicin A, a novel antitumor antibiotic", J Nat Prod. 52:1015-21,
1989; Rolston et al., "In vitro activity of trospectomycin
(U-63366F), a novel spectinomycin analog, against gram-positive
isolates from cancer patients", Eur J Clin Microbiol Infect Dis.,
8:254-60, 1989; Gupta et al., "Synthesis, cytotoxicity, and
antiviral activity of some acyclic analogues of the
pyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin,
toyocamycin, and sangivamycin", J Med. Chem., 32:402-8, 1989;
Hochlowski et al., "Phenelfamycins, a novel complex of
elfamycin-type antibiotics. II. Isolation and structure
determination", J Antibiot (Tokyo), 41:1300-15, 1988; Jackson et
al., "Phenelfamycins, a novel complex of elfamycin-type
antibiotics. I. Discovery, taxonomy and fermentation", J Antibiot
(Tokyo), 41(10):1293-9, 1988; Saitoh et al., "Boholmycin, a new
aminoglycoside antibiotic. I. Production, isolation and
properties", J Antibiot (Tokyo), 41:855-61, 1988; Yew et al., "New
antimycobacterial agents", Monaldi Arch Chest Dis., 51(5):394-404,
1996; Urban et al., "Comparative in-vitro activity of SCH 27899, a
novel eveminomicin, and vancomycin", J Antimicrob Chemother,
37:361-4, 1996; Lam et al., "Production and isolation of two novel
esperamicins in a chemically defined medium", J Antibiot (Tokyo),
148:1497-501, 1995; Pelyvas et al., "Novel aminocyclitol
antibiotics derived from natural carbohydrates", Carbohydr Res.,
272:C5-9, 1995; Plyvas et al., "Synthesis of new pseudodisaccharide
aminoglycoside antibiotics from carbohydrates", J Antibiot (Tokyo),
48:683-95, 1995; Jones, "Isepamicin (SCH 21420, 1-N-HAPA gentamicin
B): microbiological characteristics including antimicrobial potency
of spectrum of activity", J Chemother., 7 Suppl 2:7-16, 1995; Abe
et al., "Novel antitumor antibiotics, saptomycins. II. Isolation,
physico-chemical properties and structure elucidation", J Antibiot
(Tokyo), 46:15536-49, 1993; Abe et al., "Novel antitumor
antibiotics, saptomycins. I. Taxonomy of the producing organism,
fermentation, HPLC analysis and biological activities", J Antibiot
(Tokyo), 46:1530-5, 1993; Phillipson et at., "Lanomycin and
glucolanomycin, antifungal agents produced by Pycnidiophora
dispersa. II. Structure elucidation", J Antibiot (Tokyo), 45:313-9,
1992; Mariani et al., "In vitro activity of novel sulphonic
derivatives of distamycin A", EXS., 61:455-8, 1992; Abe et al.,
"Novel antitumor antibiotics, saptomycins D and E", J Antibiot
(Tokyo), 44:908-11, 1991; Priebe et al., "3'-Hydroxyesorubicin.
Synthesis and antitumor activity", J Antibiot (Tokyo), 43:838-46,
1990; Brill et al., "Altromycins, novel pluramycin-like
antibiotics. II. Isolation and elucidation of structure", J
Antibiot (Tokyo), 43:229-37, 1990; Jackson et al., "Altromycins,
novel pluramycin-like antibiotics. I. Taxonomy of the producing
organism, fermentation and antibacterial activity", J Antibiot
(Tokyo), 43:223-8, 1990; Flynn et al., "The chiral synthesis and
biochemical properties of electron rich phenolic sulfoxide analogs
of sparsomycin", Biochem Biophys Res Commun., 166:673-80, 1990;
Kitamura et al., "Pirarubicin, a novel derivative of doxorubicin.
THP-COP therapy for non-Hodgkin's lymphoma in the elderly", Am J
Clin Oncol., 13 Suppl 1:S15-9, 1990; Gu et al., "In vitro activity
of dactimicin, a novel pseudodisaccharide aminoglycoside, compared
with activities of other aminoglycosides", Antimicrob Agents
Chemother., 33:1998-2003, 1989; Lam et al., "Biosynthesis of
elsamicin A, a novel antitumor antibiotic", J Nat Prod.,
52:1015-21, 1989; Rolston et al., "In vitro activity of
trospectomycin (U-63366F), a novel spectinomycin analog, against
gram-positive isolates from cancer patients", Eur J Clin Microbiol
Infect Dis., 8:254-60, 1989; Gupta et al., "Synthesis,
cytotoxicity, and antiviral activity of some acyclic analogues of
the pyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin,
toyocamycin, and sangivamycin", J Med. Chem., 32:402-8, 1989;
Hochlowski et al., "Phenelfamycins, a novel complex of
elfamycin-type antibiotics. II. Isolation and structure
determination", J Antibiot (Tokyo), 41:1300-15, 1988; Jackson et
al., "Phenelfamycins, a novel complex of elfamycin-type
antibiotics. I. Discovery, taxonomy and fermentation", J Antibiot
(Tokyo), 41:1293-9, 1988; Saitoh et al., "Boholmycin, a new
aminoglycoside antibiotic. I. Production, isolation and
properties", J Antibiot (Tokyo), 41:855-61, 1988; Miller et al.,
"Clinical pharmacology and toxicity of
4'--O-tetrahydropyranyladriamycin"- , Cancer Res., 47:1461-5, 1987;
Stefani et al., "First microbiological approach to dactimicin, a
novel aminoglycoside antibiotic", Drugs Exp Clin Res., 13:727-9,
1987; Matsumoto et al., "Synthesis of novel
13-methyl-13-dihydroanthracyclines", Chem Pharm Bull (Tokyo),
34:4613-9, 1986; Israel et al., "Adriamycin analogues. Preparation
and biological evaluation of some
N-(trifluoroacetyl)-14-O-[(N-acetylamino)acyl]adriamyc- in
derivatives", J Med. Chem., 29:1273-6, 1986; Takahashi et al.,
"Production of novel antibiotic, dopsisamine, by a new subspecies
of Nocardiopsis mutabilis with multiple antibiotic resistance", J
Antibiot (Tokyo), 39:175-83, 1986; Yasuda et al., "Total synthesis
of 3-O-demethylsporaricin A", J Antibiot (Tokyo), 38:1512-25, 1985;
Tsunakawa et al., "Inosamycin, a complex of new aminoglycoside
antibiotics. I. Production, isolation and properties", J Antibiot
(Tokyo), 38:1302-12, 1985; Matsuhashi et al., "In vitro and in vivo
antibacterial activities of dactimicin, a novel pseudodisaccharide
aminoglycoside, compared with those of other aminoglycoside
antibiotics", Antimicrob Agents Chemother., 27:589-94, 1985;
Matsunaga et al., "Bacterial uptake of habekacin, a novel
aminoglycoside antibiotic", J Antibiot (Tokyo), 37:596-601, 1984;
Tanaka et al., "Mechanism of action of habekacin, a novel amino
acid-containing aminoglycoside antibiotic", Antimicrob Agents
Chemother., 24:797-802, 1983; Dawson, "Activity of SC33428, a novel
bishydrazone-bridged derivative of 4-demethoxydaunorubicin, against
experimental tumors in mice", Cancer Res., 43:2880-3, 1983;
Digranes et al., "N-formimidoyl thienamycin: in vitro comparison
with cefoxitin and tobramycin against clinical, bacterial
isolates", Acta Pathol Microbiol Immunol Scand [B], 91:141-4, 1983;
FitzGerald et al., "3,4-Dihydroxybenzylamine: an improved dopamine
analog cytotoxic for melanoma cells in part through oxidation
products inhibitory to dna polymerase", J Invest Dermatol.,
80:119-23, 1983; Israel et al., "Adriamycin analogues. Novel
anomeric ribofuranoside analogues of daunorubicin", J Med. Chem.
25:28-31, 1982; Tanaka. "Effects of habekacin, a novel
aminoglycoside antibiotic, on experimental corneal ulceration due
to Pseudomonas aeruginosa", J Antibiot (Tokyo), 34:892-7, 1981;
Fujiwara et al., "Production of a new aminoglycoside antibiotic by
a mutant of Bacillus circulans", J Antibiot (Tokyo), 33:836-41,
1980; Ohashi et al., "In vitro and in vivo antibacterial activity
of KW1070, a new aminoglycoside antibiotic", Antimicrob Agents
Chemother, 17:138-43, 1980; Inouye et al., "A novel aminoglycoside
antibiotic, substance SF-2052", J Antibiot (Tokyo), 32:1355-6,
1979; Perzynski et al., "Effects of apramycin, a novel
aminoglycoside antibiotic on bacterial protein synthesis", Eur J
Biochem, 99:623-8, 1979; Suzuki et al., "Preparation and some
microbiological properties of novel kanamycin-glucoside
derivatives", J Antibiot (Tokyo), 32:753-5, 1979; Smith et al.,
"Synthesis of daunorubicin analogues with novel 9-acyl
substituents", J Med. Chem., 22:40-4, 1979; Davies et al.,
"Semisynthetic aminoglycoside antibacterials. 6. Synthesis of
sisomicin, Antibiotic G-52, and novel 6'-substituted analogues of
sisomicin from aminoglycoside 66-40C", J Med. Chem., 21:189-93,
1978; Egan et al., "Fortimicins A and B, new aminoglycoside
antibiotics. III. Structural identification", J Antibiot (Tokyo),
30:552-63, 1977; Okachi et al., "Fortimicins A and B, new
aminoglycoside antibiotics. II. Isolation, physico-chemical and
chromatographic properties", J Antibiot (Tokyo), 30:541-51, 1977;
Kinumaki et al., "Macrolide antibiotics M-4365 produced by
Micromonospora. II. Chemical structures", J Antibiot (Tokyo),
30:450-4. 1977; Okutani et al., "Conversion of aminoglycosidic
antibiotics: Novel and efficient approaches to
3'-deoxyaminoglycosides via 3'-phosphoryl esters", J Am Chem Soc.,
99(4);1278-9, 1977; Hanessian et al., "Aminoglycoside antibiotics:
oxidative degradations leading to novel biochemical probes and
synthetic intermediates", J Antibiot (Tokyo), 28:835-7, 1975;
Reimann et al., "The structure of sisomicin, a novel unsaturated
aminocyclitol antibiotic from Micromonospora inyoensis", J Org
Chem., 39:1451-7, 1974; Kugelman et al., "Letter: The preparation
of garamine, a novel pseudodisaccharide from sisomycin", J Antibiot
(Tokyo), 26:394-5, 1973; Arcamone et al., "Adriamycin
(14-hydroxydaunoinycin), a novel antitumor antibiotic", Tetrahedron
Lett., 13:1007-10, 1969; Marchini et al.,
"4-Demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyl-
-daunorubicin (PNU-159548), a novel anticancer agent active against
tumor cell lines with different resistance mechanisms", Cancer
Res., 61:1991-5, 2001; Sunada et al., "Acetylation of
aminoglycoside antibiotics with 6'-methylamino group, istamycin B
and micronomicin, by a novel aminoglycoside 6'-acetyltransferase of
actinomycete origin", J Antibiot (Tokyo), 53:1416-9, 2000; Ganguly,
"Ziracin, a novel oligosaccharide antibiotic", J Antibiot (Tokyo),
53:1038-44, 2000; Nicolaou et al., "Total synthesis of
everninomicin 13,384-1--Part 2: synthesis of the FGHA2 fragment",
Chemistry, 6:3116-48, 2000; Nicolaou et al., "Total synthesis of
eveminomicin 13,384-1--Part 1: retrosynthetic analysis and
synthesis of the A1B(A)C fragment", Chemistry, 6:3095-115, 2000;
Wang et al., "In vivo activity and pharmacokinetics of ziracin
(SCH27899), a new long-acting everninomicin antibiotic, in a murine
model of penicillin-susceptible or penicillin-resistant
pneumococcal pneumonia", Antimicrob Agents Chemother., 44:1010-8,
2000; Ganguly et al., "Chemical modifications and structure
activity studies of ziracin and related eveminomicin antibiotics",
Bioorg Med Chem Lett., 9:1209-14, 1999; and Hotta et al., "The
novel enzymatic 3"-N-acetylation of arbekacin by an aminoglycoside
3-N-acetyltransferase of Streptomyces origin and the resulting
activity", J Antibiot (Tokyo), 51:73542, 1998.
[0190] Aminoglycosides Pharmacology
[0191] After injection, aminoglycosides are distributed mainly in
the ECF. Protein binding is low. Even with inflammation,
concentrations in tissues and secretions are much less than those
in plasma levels.
[0192] Aminoglycosides are excreted unchanged into the urine by
glomerular filtration. They generally have the same half-life in
plasma of 2 to 3 h; with renal insufficiency and in the elderly,
the half-life rises markedly. To avoid toxicity, the maintenance
dosages of aminoglycosides in patients with renal insufficiency
must be modified by either decreasing the dose or increasing the
interval between doses or both.
[0193] Because of the distribution properties of aminoglycosides,
dosing in obese patients should be based on a weight equal to lean
body weight plus 50% of the adipose mass. In patients with
excessive ECF, as in edema, the dose should be calculated based on
total body weight. Patients with burns and cystic fibrosis have
decreased plasma levels and may require higher doses. Anemia tends
to increase plasma levels.
[0194] One large aminoglycoside dose given once daily rather than
several divided doses given on multiple occasions throughout the
day is believed to result in less net transfer of aminoglycoside
from the blood into the tissue. This is believed to be accomplished
by saturating the rate by which aminoglycoside is moved into the
tissue.
[0195] Treatment of patients with certain aminoglycosides results
in the undesirable side effects of nephrotoxity and ototoxicity.
(For a review, see Mingeot-Leclerq et al., Antimicrobial Agents and
Chemotherapy 43:1003-1012, 1999). The compounds used in the methods
of the invention are preferably not toxic, in particular because
they would not typically be given for prolonged periods of time.
Compounds identified by the screening assays of the invention are
tested for their toxicity in animal models in order to identify
nontoxic compounds. Aminoglycosides having undesirable side-effects
can be administered to a patient with one or more agents that
ameriolate or prevent the undesirable side effects. Additionally or
alternatively, in the methods of the invention, the compounds are
administered using a dosage regimen that is designed to minimize or
avoid toxicity or any other undesirable side effects.
[0196] Modulation of the Sphingomyelin Signaling Pathway for
Therapeutic Benefit
[0197] The sphingomyelin signaling pathway (a.k.a. the SM pathway
or the ceramide signaling pathway) is a "cascadc" of biochemical
events in which proteins in the pathway are activated (by enzymatic
chemical modification or otherwise) with the end result that
sphingosine metabolism is affected. In most instances, activation
of the SM pathway leads to increased production of ceramide. For
reviews of the molecular biology of the sphingomyelin signaling
pathway, see Hannun et al., Adv. Lipid Res. 25:27-41, 1993; Liu et
al., Crit. Rev. Clin. Lab. Sci. 36:511-573, 1999; Igarashi, J.
Biochem. 122:1080-1087, 1997; and Oral et al., J. Biol. Chem.
272:4836-4842, 1997.
[0198] It has ben suggested that the sphingomyelin signal
transduction pathway is activated during cardiac ischemia/hypoxia
(Bielawska et al., Am. J. Pathol. 151:1257-1263, 1997; Meldrum, Am.
J. Physiol. 274:R577-R595, 1998; and Cain et al., J. Mol. Cell.
Cardiol. 31:931-947, 1999). If so, one or more factors or processes
may mediate the ischemia-induced SPH production. One likely
candidate for such a mediator is the pro-inflammatory cytokine,
tumor necrosis factor alpha (TNF.alpha.). In various animal models
of ischemia, the myocardium produces TNF.alpha. (Squadrito et al.,
Eur. J. Pharmacol. 237:223-230, 1993; Herrmann et al., European
Journal of Clinical Investigation 28:59-66, 1998; Meldrum et al.,
Ann. Thorac. Surg. 65:439-443, 1998). Recent evidence demonstrates
that the cardiomyocytes themselves produce TNF.alpha. and secrete
the cytokine into the extracellular fluid (Comstock et al., J. Mol.
Cell Cardiol. 30:2761-2775, 1998). Since TNF.alpha. receptors are
expressed by cardiomyocytes (Krown et al., FEBS Letters 376:24-30,
1995; Torre-Amione et al., Circulation 92:1487-1493, 1995), an
autocrine/paracrine role for TNF.alpha. has been suggested (Meldrum
et al., Ann. Thorac. Surg. 65:439-443, 1998). Significantly,
TNF.alpha. induces SPH production and apoptosis in cardiac myocytes
(Krown et al., J. Clin. Invest. 98:2854-2865, 1996), presumably by
acting by binding to the cardiomyocyte complement of TNF.alpha.
receptors Activation of the sphingomyelin signal transduction
cascade may be a key early event in the cytotoxic (apoptotic)
effects of the cytokine TNF.alpha. (Zhang et al., Endo.
136:4157-4160, 1995). TNF.alpha. can cause significant apoptosis in
cultured rat cardiomyocytes and it has been suggested that
TNF.alpha..alpha.-induced SPH production is responsible for the
cell death triggered by TNF.alpha. (Krown et al., J. Clin. Invest.
98:2854-2865, 1996).
[0199] The SM pathway, many steps of which occur intracellularly,
is induced by a variety of extracellular stimuli. In
sphingolipid-based cardiovascular therapy, such stimuli may be
inhibited or completely blocked. SM pathway-inducing agents, the
function of which may be modulated, include but are not limited to
cytokines. Cytokines of particular interest include but are not
limited to pro-inflammatory cytokines, interferons and
chemokines.
[0200] Methods of Screening for Novel Sphingolipid-Based
Therapeutic Agents
[0201] The sphingolipid targets of the invention are readily
adaptable for use in high-throughput screening assays for screening
candidate compounds to identify those which have a desired
activity, e.g., inhibiting an enzyme that catalyzes a reaction that
produces an undesirable sphingolipid, or blocking the binding of a
sphingolipid to a receptor therefor. The compounds thus identified
can serve as conventional "lead compounds" or can themselves be
used as therapeutic agents.
[0202] The methods of screening of the invention comprise using
screening assays to identify, from a library of diverse molecules,
one or more compounds having a desired activity. A "screening
assay" is a selective assay designed to identify, isolate, and/or
determine the structure of, compounds within a collection that have
a preselected activity. By "identifying" it is meant that a
compound having a desirable activity is isolated, its chemical
structure is determined (including without limitation determining
the nucleotide and amino acid sequences of nucleic acids and
polypeptides, respectively) the structure of and, additionally or
alternatively, purifying compounds having the screened activity).
Biochemical and biological assays are designed to test for activity
in a broad range of systems ranging from protein-protein
interactions, enzyme catalysis, small molecule-protein binding, to
cellular functions. Such assays include automated, semi-automated
assays and HTS (high throughput screening) assays.
[0203] In HTS methods, many discrete compounds are preferably
tested in parallel by robotic, automatic or semi-automatic methods
so that large numbers of test compounds are screened for a desired
activity simultaneously or nearly simultaneously. It is possible to
assay and screen up to about 6,000 to 20,000, and even up to about
100,000 to 1,000,000 different compounds a day using the integrated
systems of the invention.
[0204] Typically in HTS, target molecules are contained in each
well of a multi-well microplate; in the case of enzymes, reactants
are also present in the wells. Currently, the most widely
established techniques utilize 96-well microtiter plates. In this
format, 96 independent tests are performed simultaneously on a
single 8 cm.times.12 cm plastic plate that contains 96 reaction
wells. One or more blank wells contains all of the reactants except
the candidate compound. Each of the non-standard wells contain at
least one candidate compound.
[0205] These wells typically require assay volumes that range from
50 to 500 ul. In addition to the plates, many instruments,
materials, pipettors, robotics, plate washers and plate readers are
commercially available to fit the 96-well format to a wide range of
homogeneous and heterogeneous assays. Microtiter plates with more
wells, such as 384-well microtiter plates, are also used, as are
emerging methods such as the nanowell method described by Schullek
et al. (Anal Biochem., 30 246, 20-29, 1997).
[0206] In one modality, screening comprises contacting a
sphingolipid target with a diverse library of member compounds,
some of which are ligands of the target, under conditions where
complexes between the target and ligands can form, and identifying
which members of the libraries are present in such complexes. In
another non limiting modality, screening comprises contacting a
target enzyme with a diverse library of member compounds, some of
which are inhibitors (or activators) of the target, under
conditions where a product or a reactant of the reaction catalyzed
by the enzyme produce a detectable signal. In the latter modality,
inhibitors of target enzyme decrease the signal from a detectable
product or increase a signal from a detectable reactant (or
vice-versa for activators).
[0207] Chemical Libraries
[0208] Developments in combinatorial chemistry allow the rapid and
economical synthesis of hundreds to thousands of discrete
compounds. These compounds are typically arrayed in moderate-sized
libraries of small organic molecules designed for efficient
screening. Combinatorial methods, can be used to generate unbiased
libraries suitable for the identification of novel inhibitors. In
addition, smaller, less diverse libraries can be generated that are
descended from a single parent compound with a previously
determined biological activity. In either case, the lack of
efficient screening systems to specifically target therapeutically
relevant biological molecules produced by combinational chemistry
such as inhibitors of important enzymes hampers the optimal use of
these resources.
[0209] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks," such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide library, is formed by
combining a set of chemical building blocks (amino acids) in a
large number of combinations, and potentailly in every possible
way, for a given compound length (i.e., the number of amino acids
in a polypeptide compound). Millions of chemical compounds can be
synthesized through such combinatorial mixing of chemical building
blocks.
[0210] A "library" may comprise from 2 to 50,000,000 diverse member
compounds. Preferably, a library comprises at least 48 diverse
compounds, preferably 96 or more diverse compounds, more preferably
384 or more diverse compounds, more preferably, 10,000 or more
diverse compounds, preferably more than 100,000 diverse members and
most preferably more than 1,000,000 diverse member compounds. By
"diverse" it is meant that greater than 50% of the compounds in a
library have chemical structures that are not identical to any
other member of the library. Preferably, greater than 75% of the
compounds in a library have chemical structures that are not
identical to any other member of the collection, more preferably
greater than 90% and most preferably greater than about 99%.
[0211] The preparation of combinatorial chemical libraries is well
known to those of skill in the art. For reviews, sec Thompson et
al., Synthesis and application of small molecule libraries, Chem
Rev 96:555-600, 1996; Kenan et al., Exploring molecular diversity
with combinatorial shape libraries, Trends Biochem Sci 19:57-64,
1994; Janda, Tagged versus untagged libraries: methods for the
generation and screening of combinatorial chemical libraries, Proc
Natl Acad Sci USA. 91:10779-85, 1994; Lebl et al.,
One-bead-one-structure combinatorial libraries, Biopolymers
37:177-98, 1995; Eichler et al., Peptide, peptidomimetic, and
organic synthetic combinatorial libraries, Med Res Rev. 15:481-96,
1995; Chabala, Solid-phase combinatorial chemistry and novel
tagging methods for identifying leads, Curr Opin Biotechnol.
6:632-9, 1995; Dolle, Discovery of enzyme inhibitors through
combinatorial chemistry, Mol Divers. 2:223-36, 1997; Fauchere et
al., Peptide and nonpeptide lead discovery using robotically
synthesized soluble libraries, Can J Physiol Pharmacol. 75:683-9,
1997; Eichler et al., Generation and utilization of synthetic
combinatorial libraries, Mol Med Today 1:174-80, 1995; and Kay et
al., Identification of enzyme inhibitors from phage-displayed
combinatorial peptide libraries, Comb Chem High Throughput Screen
4:535-43, 2001.
[0212] Such combinatorial chemical libraries include, but are not
limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175,
Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton, et
al., Nature, 354:84-88 (1991)). Other chemistries for generating
chemical diversity libraries can also be used. Such chemistries
include, but are not limited to, peptoids (PCT Publication No. WO
91/19735); encoded peptides (PCT Publication WO 93/20242); random
bio-oligomers (PCT Publication No. WO 92/00091); benzodiazepines
(U.S. Pat. No. 5,288,514); diversomers, such as hydantoins,
benzodiazepines and dipeptides (Hobbs, et al., Proc. Nat. Acad.
Sci. USA, 90:6909-6913 (1993)); vinylogous polypeptides (Hagihara,
et al., J. Amer. Chem. Soc. 114:6568 (1992)); nonpeptidal
peptidomimetics with .beta.-D-glucose scaffolding (Hirschmann, et
al., J. Amer. Chem. Soc., 114:9217-9218 (1992)); analogous organic
syntheses of small compound libraries (Chen, et al., J. Amer. Chem.
Soc., 116:2661 (1994)); oligocarbamates (Cho, et al., Science,
261:1303 (1993)); and/or peptidyl phosphonates (Campbell, et al.,
J. Org. Chem. 59:658 (1994)); nucleic acid libraries (see, Ausubel,
Berger and Sambrook, all supra); peptide nucleic acid libraries
(see, e.g., U.S. Pat. No. 5,539,083); antibody libraries (see,
e.g., Vaughn, et al., Nature Biotechnology, 14(3):309-314 (1996)
and PCT/US96/10287); carbohydrate libraries (see, e.g., Liang, et
al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853);
small organic molecule libraries (see, e.g., benzodiazepines, Baum
C&E News, Jan. 18, page 33 (1993); isoprenoids (U.S. Pat. No.
5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No.
5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134);
morpholino compounds (U.S. Pat. No. 5,506,337); benzodiazepines
(U.S. Pat. No. 5,288,514); and the like.
[0213] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Bio
sciences, Columbia, Md., etc.).
[0214] Bioactive Lipid Libraries
[0215] Naturally occurring or synthetic lipids, particularly
sphingolipids, are known that modulate processes in sphingolipid
biosynthesis and intracellular signaling by ceramide, S-1-P and
other sphinglipids (for reviews of sphingolipid-mediated cell
signaling processes and consequences arising therefrom, see Linn et
al., "Regulation of de novo sphingolipid biosynthesis and the toxic
consequences of its disruption", Biochemical Society, 831-835,
2001; Luberto et al., "Sphingolipid Metabolism in the Regulation of
Bioactive Molecules", Lipids, 34:S5-S11, 1999; Kester,
"Sphingolipid Metabolites and the Cellular Phenotype", Trends in
Glycoscience and Glycotechnology, 9:447-460, 1997; Ariga et al.,
"Role of Sphingolipid-mediated cell death in neurodegenerative
diseases", Journal of Lipid Research, 39:1-16, 1998; Chan et al.,
"Ceramid Path in Human Lung Cell Death", Am. J. Respir. Cell Mol.
Biol., 22:460-468, 2000; and Hannun et al., "Ceramide in the
eukaryotic stress response", Cell Biology, 10:73-80, 2000; see also
Brownlee, Current Biology 11:R535-R538, 2001).
[0216] See, for example, Usta et al., "Structural Requirements of
Cermaide and Sphingosine Based Inhibitors of Mitochondrial
Ceramidase", Biochemistry, 40:9657-9668, 2000; Hannun et al.,
"Method of Inducing Cellular Differentiations and Altering Cell
Phenotype Using Ceramide Analogs", U.S. Pat. No. 5,369,030, issued
Nov. 29, 1994; Wei et al., "Pharmaceutically Active
Ceramide-Related Compounds", U.S. Pat. No. 5,631,394, issued May
20, 1997; Wei et al., "Methods of Treatment Using Pharmaceutically
Active Ceramide-Related Compositions", U.S. Pat. No. 5,677,337,
issued Oct. 14, 1997; Carson et al., "Compounds for Inhibition of
Ceramide-Mediated Signal Transduction", U.S. Pat. No. 6,323,201 B1,
issued Nov. 27, 2001; Bell et al., "Inhibition of Protein Kinase C
By Long-Chain Bases", U.S. Pat. No. 4,937,232, issued Jun. 26,
1990; Bell et al., "Inhibition of Protein Kinase C By Long-Chain
Bases", U.S. Pat. No. 4,816,450, issued Mar. 28, 1989; Hannun et
al., "Ceramidase Compositions and Methods Based Thereon",
PCT/US01/02866 published as WO01/55410 on Aug. 2, 2001; Kimura et
al., "Effect of N,N,N,-trimethylsphingosine on Protein Kinase-C
Activity; Melanoma Cell Growth In Vitro; Metastatic Potential In
Vivo and Human Platelet Aggregation"U.S. Pat. No. 5,331,014, dated
Jul. 19, 1994; Igarashi et al., "Effect of
N,N,N,-trimethylsphingos- ine on Protein Kinase C Activity Melanoma
Cell Growth In Vitro; Metastatic Potential In Vivo and Human
Platelet Aggregation", U.S. Pat. No. 5,137,919 dated Aug. 11, 1992;
Handa et al., "Effect of N,N,N,-trimethylsphingosine on Protein
Kinase-C Activity, Melanoma Cell Growth In Vitro, Metastatic
Potential In Vivo and Human Platelet Aggregation", U.S. Pat. No.
5,151,360 dated Sep. 29, 1992; Takesako et al., "Sphingosine
Analogues", PCT/JP98/01038, published as WO98/40349 on Sep. 16,
1998; Kobori et al., "Sphingosine Derivatives", PCT/JP00/08229,
published as WO01/38295 on May 31, 2001; Takesako et al.,
"Sphingosine Derivatives and Medicinal Composition",
PCT/JP98/04093, published as WO99/12890 on Mar. 18, 1999; Liotta et
al., "Sphingolipid Derivatives and Their Methods of Use",
PCT/US99/03093, published as WO99/41266 on Aug. 19, 1999; Macchia
et al., "Ceramide Analogs, Process for their Preparation and their
Use as Antitumor Agents", PCT/EP00/07023, published as WO01/07418
on Feb. 1, 2001; Shayman et al., "Amino Ceramide-Like Compounds and
Therapeutic Methods of Use", PCT/US00/18935, published as
WO01/04108 on Jan. 18, 2001; Bielawska et al.,
"(1S,2R)-D-erhthro-2-(N-My- ristoylamino)-1-phenyl-1-propanol as an
Inhibitor of Ceramidase", The Journal of Biological Chemistry, Vol.
271, May 24, 1996, pp. 12646-12654; Wanebo et al., "Ceramide and
Chemotherapeutic Agents for Inducing Cell Death", PCT/US00/09440,
published as WO00/59517 on Oct. 12, 2000; Ali et al., "Ceramide
Derivatives and Method of Use", PCT/US01/09894, published as
WO01/72701 on Oct. 4, 2001; Eibl et al., "Pseudoceramides",
PCT/EP99/07698, published as WO00/21919 on Apr. 20, 2000; Jonghe et
al., "Structure-Activity Relationship of Short-Chain Sphingoid
Bases As Inhibitors of Sphingosine Kinase", Bioorganic &
Medicinal Chemistry Letters 9:3175-3180, 1999; Arenz et al.,
"Synthese des ersten selektiven irreverilben Inhibitors der
neutralen Sphingomyelinase", Angew Chem., 112:1498-1500,2000; and
Abe et al., "Use of Sulfobutyl Ether-Cyclodextrin as a Vehicle for
D-threo-1-Phenyl-2-decanoylamino-3-morpholinopropanol-Re- lated
Glucosylceramide Synthase Inhibitors", Analytical Biochemistry,
287:344-347, 2000.
[0217] One aspect of the invention involves identifying
sphingolipids that are useful in sphingolipid-based therapy. This
can done by testing commercially available or otherwise obtainable
sphingolipids in assays that measure the activity of enzymes
involved in sphingolipid metabolism and/or intracellular
signalling.
[0218] Commercially available sphingolipids (Avanti Polar Lipids,
Inc., Alabaster, Ala.) include without limitation synthetic
D-erythro (C-18) derivatives of sphingosine, e.g., D-erythro
Sphingosine (synthetic), Sphingosine-1-Phosphate, D-erythro
Ceramide-1-Phosphate, N,N-Dimethylsphingosine,
N,N,N-Trimethylsphingosine, Sphingosylphosphorylcholine,
Sphingomyelin, and Ceramides; D-erythro (C-18) derivatives of
sphinganine (dihydrosphingosine), e.g., Sphinganine-1-Phosphate,
D-erythro Sphinganine, N-Acyl-Sphinganine C2, N-Acyl-Sphinganine
C8, N-acyl-Sphinganine C16, N-Acyl-Sphinganine C18,
N-Acyl-Sphinganine C24, and N-Acyl-Sphinganine C24:1; glycosylated
(C18) sphingosine and phospholipid derivatives, e.g., glycosylated
sphingosine, ceramide and phosphatidylethanolamine, beta
D-glucosyl-sphingosine, and beta D-galactosyl-sphingosine;
D-erythro (C17) derivatives, e.g., D-erythro Sphingosine and
D-erythro Sphingosine-1-phosphate; D-erythro (C20) derivatives,
such as D-erythro sphingosine; and L-threro (C18) derivatives such
as L-threo Sphingosine and L-threo Dihydrosphingosine (Safingol).
Phytosphingosine derivatives from yeast. e.g., Phytosphingosine,
D-ribo-Phytosphingosine-1-Phosphate, N-Acyl Phytosphingosine C2,
N-Acyl Phytosphingosine C8 and N-Acyl Phytosphingosine C18 may also
be used.
[0219] A variety of methods for synthesizing sphingolipids and
sphingolipid-related molecules are known. In addition to the
above-cited references, see Szule et al., "A facile regioselective
synthesis of sphingosine 1-phosphate and ceramide 1-phosphate,
Tetrahedron Letter 41:7821-7824, 2000; Igarashi et al.,
"Sphingosine-1-Phosphate Essentially Free of L-Threo Isomer, U.S.
Pat. No. 5,663,404, issued Sep. 2, 1997; Boumendjel et al., "Method
For Preparation of Sphingoid Bases", U.S. Pat. No. 5,430,160,
issued Jul. 4, 1995; Ito et al., "Process for the Preparation of
Sphingolipids and Sphingolipid Derivatives", PCT/JP97/02483,
published as WO98/03529 on Jan. 29, 1998; and Igarashi et al.,
"Method of Preparing N,N,N,-trimethylsphingosine", U.S. Pat. No.
5,248,824 dated Sep. 28, 1993.
[0220] In a preferred embodiment, sphingolipids having a desired
activity are identified by high throughput screening (HTS) of
combinatorial libraries of sphingolipid-related compounds.
Combinatorial sphingolipid libraries are prepared according to
methods known in the art, or may be purchased commercially. One
type of combinatorial sphingolipid library that may be used is the
BIOMOL Bioactive Lipid Library (Affiniti Research Products Ltd.,
Mamhead, U.K.).
[0221] High Throughput Screening (HTS) Assays
[0222] HTS typically uses automated assays to search through large
numbers of compounds for a desired activity. Typically HTS assays
are used to find new drugs by screening for chemicals that act on a
particular enzyme or molecule. For example, if a chemical
inactivates an enzyme it might prove to be effective in preventing
a process in a cell which causes a disease. High throughput methods
enable researchers to try out thousands of different chemicals
against each target very quickly using robotic handling systems and
automated analysis of results.
[0223] As used herein, "high throughput screening" or "HTS" refers
to the rapid in vitro screening of large numbers of compounds
(libraries); generally tens to hundreds of thousands of compounds,
using robotic screening assays. Ultra high-throughput Screening
(uHTS) generally refers to the high-throughput screening
accelerated to greater than 100,000 tests per day.
[0224] To achieve high-throughput screening, it is best to house
samples on a multicontainer carrier or platform. A multicontainer
carrier facilitates measuring reactions of a plurality of candidate
compounds simultaneously. Multi-well microplates may be used as the
carrier. Such multi-well microplates, and methods for their use in
numerous assays, are both known in the art and commercially
available.
[0225] Screening assays may include controls for purposes of
calibration and confirmation of proper manipulation of the
components of the assay. Blank wells that contain all of the
reactants but no member of the chemical library are usually
included. As another example, a known inhibitor (or activator) of
an enzyme for which modulators are sought, can be incubated with
one sample of the assay, and the resulting decrease (or increase)
in the enzyme activity determined according to the methods herein.
It will be appreciated that modulators can also be combined with
the enzyme activators or inhibitors to find modulators which
inhibit the enzyme activation or repression that is otherwise
caused by the presence of the known the enzyme modulator.
Similarly, when ligands to a sphingolipid target are sought, known
ligands of the target can bc present in control/calibration assay
wells.
[0226] Measuring Enzymatic and Binding Reactions During Screening
Assays
[0227] Techniques for measuring the progression of enzymatic and
binding reactions in multicontainer carriers are known in the art
and include, but are not limited to, the following.
[0228] Spectrophotometric and spectrofluorometric assays are well
known in the art. Examples of such assays include the use of
colorimetric assays for the detection of peroxides, as disclosed in
Example 1(b) and Gordon, A. J. and Ford, R. A., The Chemist's
Companion: A Handbook Of Practical Data, Techniques, And
References, John Wiley and Sons, N.Y., 1972, Page 437.
[0229] Fluorescence spectrometry may be used to monitor the
generation of reaction products. Fluorescence methodology is
generally more sensitive than the absorption methodology. The use
of fluorescent probes is well known to those skilled in the art.
For reviews, see Bashford et al., Spectrophotometry and
Spectrofluorometry: A Practical Approach, pp. 91-114, IRL Press
Ltd. (1987); and Bell, Spectroscopy In Biochemistry, Vol. I, pp.
155-194, CRC Press (1981).
[0230] In spectrofluorometric methods, enzymes are exposed to
substrates that change their intrinsic fluorescence when processed
by the target enzyme. Typically, the substrate is nonfluorescent
and converted to a fluorophore through one or more reactions. As a
non-limiting example, SMase activity can be detected using the
Amplex.RTM. Red reagent (Molecular Probes, Eugene, Oreg.). In order
to measure sphingomyelinase activity using Amplex Red, the
following reactions occur. First, SMase hydrolyzes sphingomyelin to
yield ceramide and phosphorylcholine. Second, alkaline phosphatase
hydrolyzes phosphorylcholine to yield choline. Third, choline is
oxidized by choline oxidase to betaine. Finally, H2O2, in the
presence of horseradish peroxidase, reacts with Amplex Red to
produce the fluorescent product, Resorufin, and the signal
therefrom is detected using spectrofluorometry.
[0231] Fluorescence polarization (FP) is based on a decrease in the
speed of molecular rotation of a fluorophore that occurs upon
binding to a larger molecule, such as a receptor protein, allowing
for polarized fluorescent emission by the bound ligand. FP is
empirically determined by measuring the vertical and horizontal
components of fluorophore emission following excitation with plane
polarized light. Polarized emission is increased when the molecular
rotation of a fluorophore is reduced. A fluorophore produces a
larger polarized signal when it is bound to a larger molecule (i.e.
a receptor), slowing molecular rotation of the fluorophore. The
magnitude of the polarized signal relates quantitatively to the
extent of fluorescent ligand binding. Accordingly, polarization of
the "bound" signal depends on maintenance of high affinity
binding.
[0232] FP is a homogeneous technology and reactions are very rapid,
taking seconds to minutes to reach equilibrium. The reagents are
stable, and large batches may be prepared, resulting in high
reproducibility. Because of these properties, FP has proven to be
highly automatable, often performed with a single incubation with a
single, premixed, tracer-receptor reagent. For a eview, see Owicki
et al., Application of Fluorescence Polarization Assays in
High-Throughput Screening, Genetic Engineering News, 17:27,
1997.
[0233] FP is particularly desirable since its readout is
independent of the emission intensity (Checovich, W. J., et al.,
Nature 375:254-256, 1995; Dandliker, W. B., et al., Methods in
Enzymology 74:3-28, 1981) and is thus insensitive to the presence
of colored compounds that quench fluorescence emission. FP and FRET
(see below) are well-suited for identifying compounds that block
interactions between sphingolipid receptors and their ligands. See,
for example, Parker et al., Development of high throughput
screening assays using fluorescence polarization: nuclear
receptor-ligand-binding and kinase/phosphatase assays, J Biomol
Screen 5:77-88, 2000.
[0234] Fluorophores derived from sphingolipids that may be used in
FP assays are commercially available. For example, Molecular Probes
(Eugune, Oreg.) currently sells sphingomyelin and one ceramide
flurophores. These are, respectively,
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-inda-
cene-3-pentanoyl)sphingosyl phosphocholine (BODIPY.RTM. FL
C5-sphingomyelin);
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-inda-
cene-3-dodecanoyl)sphingosyl phosphocholine (BODIPY.RTM. FL
C12-sphingomyelin); and
N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-
-indacene-3-pentanoyl)sphingosine (BODIPY.RTM. FL C5-ceramide).
U.S. Pat. No. 4,150,949, (Immunoassay for gentamicin), discloses
fluorescein-labelled gentamicins, including fluoresceinthiocarbanyl
gentamicin. Additional fluorophores may be prepared using methods
well known to the skilled artisan.
[0235] Exemplary normal-and-polarized fluorescence readers include
the POLARION fluorescence polarization system (Tecan A G,
Hombrechtikon, Switzerland). General multiwell plate readers for
other assays are available, such as the VERSAMAX reader and the
SPECTRAMAX multiwell plate spectrophotometer (both from Molecular
Devices).
[0236] Fluorescence resonance energy transfer (FRET) is another
useful assay for detecting interaction and has been described
previously. See, e.g., Heim et al., Curr. Biol. 6:178-182, 1-996;
Mitra et al., Gene 173:13-17 1996; and Selvin et al., Meth.
Enzymol. 246:300-345, 1995. FRET detects the transfer of energy
between two fluorescent substances in close proximity, having known
excitation and emission wavelengths. As an example, a protein can
be expressed as a fusion protein with green fluorescent protein
(GFP). When two fluorescent proteins are in proximity, such as when
a protein specifically interacts with a target molecule, the
resonance energy can be transferred from one excited molecule to
the other. As a result, the emission spectrum of the sample shifts,
which can be measured by a fluorometer, such as a fMAX multiwell
fluorometer (Molecular Devices, Sunnyvale Calif.).
[0237] Scintillation proximity assay (SPA) is a particularly useful
assay for detecting an interaction with the target molecule. SPA is
widely used in the pharmaceutical industry and has been described
(Hanselman et al., J. Lipid Res. 38:2365-2373 (1997); Kahl et al.,
Anal. Biochem. 243:282-283 (1996); Undenfriend et al., Anal.
Biochem. 161:494-500 (1987)). See also U.S. Pat. Nos. 4,626,513 and
4,568,649, and European Patent No. 0,154,734. One commercially
available system uses FLASHPLATE scintillant-coated plates (NEN
Life Science Products, Boston, Mass.).
[0238] The target molecule can be bound to the scintillator plates
by a variety of well known means. Scintillant plates are available
that are derivatized to bind to fusion proteins such as GST, His6
or Flag fusion proteins. Where the target molecule is a protein
complex or a multimer, one protein or subunit can be attached to
the plate first, then the other components of the complex added
later under binding conditions, resulting in a bound complex.
[0239] In a typical SPA assay, the gene products in the expression
pool will have been radiolabeled and added to the wells, and
allowed to interact with the solid phase, which is the immobilized
target molecule and scintillant coating in the wells.
[0240] The assay can be measured immediately or allowed to reach
equilibrium. Either way, when a radiolabel becomes sufficiently
close to the scintillant coating, it produces a signal detectable
by a device such as a TOPCOUNT NXT microplate scintillation counter
(Packard BioScience Co., Meriden Conn.). If a radiolabeled
expression product binds to the target molecule, the radiolabel
remains in proximity to the scintillant long enough to produce a
detectable signal.
[0241] In contrast, the labeled proteins that do not bind to the
target molecule, or bind only briefly, will not remain near the
scintillant long enough to produce a signal above background. Any
time spent near the scintillant caused by random Brownian motion
will also not result in a significant amount of signal. Likewise,
residual unincorporated radiolabel used during the expression step
may be present, but will not generate significant signal because it
will be in solution rather than interacting with the target
molecule. These non-binding interactions will therefore cause a
certain level of background signal that can be mathematically
removed. If too many signals are obtained, salt or other modifiers
can be added directly to the assay plates until the desired
specificity is obtained (Nichols et al., Anal. Biochem.
257:112-119, 1998).
[0242] Assays for Enzymes Involved in Sphingolipid Metabolism
[0243] SMase: A variety of methods are available to measure SMase
activity. It is possible to assay the SMase activity in vivo
through labeling the cells with a radioactive substrate for SMase
and then determining the level of radiolabel in the enzymatic
products. Liu, B., and Y. A. Hannun. "Sphingomyclinase Assay Using
Radiolabeled Substrate." Sphingolipid Metabolism and Cell
Signaling, Pt a., 164-67. Methods in Enzymology, vol. 311, 2000.
HTS assays of SMase activity are described by Lin et al.,
"Sphingomyclinase Assay Using Radiolabeled Substrate", p. 164, Liu
et al., Barbone et al., "Robotic Assay of Sphingomyelinase for
High-Throughput Screening", p. 168, and Hessler et al., "A High
Throughput Sphingomyelinase Assay", p. 176 In: Sphingolipid
Metabolism and Cell Signaling, Hannun, Yusaf A. (editor); Merrill,
Alfred H. (editor), Academic Press (1999). The activity of SMase
can be also determined in vitro either using radiolabelled SM, or a
chromogenic analog of SM or colored and fluorescent derivatives of
natural SM. Torley et al. (A turbidometric assay for phospholipase
C and sphingomyelinase, Anal Biochem 222:461-464, 1994) describe a
turbidometric assay for SMase suitable for high-volume screening
using unmodified substrates.
[0244] SPH Kinase: Assays for SPH kinase are described by Olivera
et al., "Assaying Sphingosine Kinase Activity", Methods in
Enzymology, 311:215-223, 1999; and Caligan et al., "A
High-Performance Liquid Chromatographic Method to Measure
Sphingosine 1-Phosphate and Related Compounds from Sphingosine
Kinase Assays and Other Biological Samples", Analytical
Biochemistry, 281:36-44, 2000.
[0245] CER Kinase: Assays for CER kinase are disclosed by Bajjalieh
et al., "Ceramide Kinase", Methods in Enzymology, 311:207-215,
1999.
[0246] Ceramidase: Assays for Ceramidase are disclosed by Zhang et
al., "Human Acid Ceramidase Gene: Novel Mutations in Farber
Disease", Molecular Geneetics and Metabolism, 70:301-309, 2000.
[0247] CER Synthase: Assays for CER synthase are disclosed by Bose
et al., "Measurement of Ceramide Synthase Activity", Methods in
Enzymology, 322:378-382,2000.
[0248] Glucosylceramide Synthase: Assays for glucosylceramide
synthase are disclosed by Shayman et al., "Glucosylceramide
Synthase: Assay and Properties", Methods in Enzymology, 311:42-49,
1999.
[0249] Assays for Sphingolipids:
[0250] S-1-P assays are disclosed by Ruwisch et al., "An improved
high-performance liquid chromatographic method for the
determination of sphingosine-1-phosphate in complex biological
materials", Naunyn-Schmiedeberg's Arch Pharmacol, 363:358-363,
2001; Edsall et al., "Enzymatic Measurement of Sphingosine
1-Phosphate", Analytical Biochemistry, 272:80-86, 1999; and Edsall
et al., "Enzymatic Method for Measurement of Sphingosine
1-Phosphate", Methods in Enzymology, 312:9-16, 2000.
[0251] SPH assays are disclosed by Chmura et al. (Down-Regulation
of Ceramide Production Abrogates Ionizing Radiation-Induced
Cytochrome c Release and Apoptosis, Molecular Pharmacology,
57:792-796, 2001); U.S. Pat. No. 5,677,189, and Shephard et al.
(Liquid chormatographic determination of the
sphinganine/sphingosine ratio in serum, Journal of Chromatography
B, 710:291-222, 1998).
[0252] Pharmaceutical Compositions
[0253] Another aspect of the invention is drawn to compositions,
including but not limited to pharmaceutical and/or biological
compositions. According to the invention, a "composition" refers to
a mixture comprising at least one carrier, preferably a
physiologically acceptable carrier, and one or more therapeutic
agents according to the invention. The term "carrier" defines a
chemical compound that does not inhibit or prevent the
incorporation of therapeutic agents into cells or tissues. A
carrier typically is an inert substance that allows an active
ingredient to be formulated or compounded into a suitable dosage
form (e.g., a pill, a capsule, a gel, a film, a tablet, a
microparticle (e.g., a microsphere), a solution etc.). A
"physiologically acceptable carrier" is a carrier suitable for use
under physiological conditions that does not abrogate (reduce,
inhibit, or prevent) the biological activity and properties of the
compound. For example, dimethyl sulfoxide (DMSO) is a carrier as it
facilitates the uptake of many organic compounds into the cells or
tissues of an organism. Preferably, the carrier is a
physiologically acceptable carrier, preferably a pharmaceutically
or veterinarily acceptable carrier, in which the therapeutic agent
is disposed. A "pharmaceutical composition" refers to a composition
wherein the carrier is a pharmaceutically acceptable carrier, while
a "veterinary composition" is one wherein the carrier is a
veterinarily acceptable carrier. The term "pharmaceutically
acceptable carrier" or "veterinarily acceptable carrier" includes
any medium or material that is not biologically or otherwise
undesirable, i.e., the carrier may be administered to an organism
along with a therapeutic agent, composition or compound without
causing any undesirable biological effects or interacting in a
deleterious manner with the complex or any of its components or the
organism. Examples of pharmaceutically acceptable reagents are
provided in The United States Pharmacopeia, The National Formulary,
United States Pharmacopeial Convention, Inc., Rockville, Md. 1990,
hereby incorporated by reference herein into the present
application. The terms "therapeutically effective amount" or
"pharmaceutically effective amount" mean an amount sufficient to
induce or effectuate a measurable response in the target cell,
tissue, or organism. What constitutes a therapeutically effective
amount will depend on a variety of factors which the knowledgeable
practitioner will take into account in arriving at the desired
dosage regimen.
[0254] The compositions of the invention can further comprise other
chemical components, such as diluents and excipients. A "diluent"
is a chemical compound diluted in a solvent, preferably an aqueous
solvent, that facilitates dissolution of the therapeutic agent in
the solvent, and it may also serve to stabilize the biologically
active form of the therapeutic agent or one or more of its
components. Salts dissolved in buffered solutions are utilized as
diluents in the art. For example, preferred diluents are buffered
solutions containing one or more different salts. A preferred
buffered solution is phosphate buffered saline (particularly in
conjunction with compositions intended for pharmaceutical
administration), as it mimics the salt conditions of human blood.
Since buffer salts can control the pH of a solution at low
concentrations, a buffered diluent rarely modifies the biological
activity of a therapeutic agent.
[0255] An "excipient" is any more or less inert substance that can
be added to a composition in order to confer a suitable property,
for example, a suitable consistency or to form a drug. Suitable
excipients and carriers include, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, agar, pectin, xanthan gum, guar gum, locust bean gum,
hyaluronic acid, casein potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethyl-cellulose, polyacrylate,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
If desired, disintegrating agents can also be included, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Other suitable excipients and
carriers include hydrogels, gellable hydrocolloids, and chitosan.
Chitosan microspheres and microcapsules can be used as carriers.
See WO 98/52547 (which describes microsphere formulations for
targeting compounds to the stomach, the formulations comprising an
inner core (optionally including a gelled hydrocolloid) containing
one or more active ingredients, a membrane comprised of a water
insoluble polymer (e.g., ethylcellulose) to control the release
rate of the active ingredient(s), and an outer layer comprised of a
bioadhesive cationic polymer, for example, a cationic
polysaccharide, a cationic protein, and/or a synthetic cationic
polymer; U.S. Pat. No. 4,895,724. Typically, chitosan is
cross-linked using a suitable agent, for example, glutaraldehyde,
glyoxal, epichlorohydrin, and succinaldehyde. Compositions
employing chitosan as a carrier can be formulated into a variety of
dosage forms, including pills, tablets, microparticles, and
microspheres, including those providing for controlled release of
the active ingredient(s). Other suitable bioadhesive cationic
polymers include acidic gelatin, polygalactosamine, polyamino acids
such as polylysine, polyhistidine, polyornithine, polyquaternary
compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE),
DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran,
DEAE-cellulose, poly-p-aminostyrene, polyoxethane,
copolymethacrylates, polyamidoamines, cationic starches,
polyvinylpyridine, and polythiodiethylaminomethylethylene.
[0256] The compositions of the invention can be formulated in any
suitable manner. Therapeutic agents may be uniformly
(homogeneously) or non-uniformly (heterogeneously) dispersed in the
carrier. Suitable formulations include dry and liquid formulations.
Dry formulations include freeze dried and lyophilized powders,
which are particularly well suited for aerosol delivery to the
sinuses or lung, or for long term storage followed by
reconstitution in a suitable diluent prior to administration. Other
preferred dry formulations include those wherein a composition
according to the invention is compressed into tablet or pill form
suitable for oral administration or compounded into a sustained
release formulation. When the composition is intended for oral
administration but the therapeutic agent is to be delivered to
epithelium in the intestines, it is preferred that the formulation
be encapsulated with an enteric coating to protect the formulation
and prevent premature release of the therapeutic agents included
therein. As those in the art will appreciate, the compositions of
the invention can be placed into any suitable dosage form. Pills
and tablets represent some of such dosage forms. The compositions
can also be encapsulated into any suitable capsule or other coating
material, for example, by compression, dipping, pan coating, spray
drying, etc. Suitable capsules include those made from gelatin and
starch. In turn, such capsules can be coated with one or more
additional materials, for example, and enteric coating, if desired.
Liquid formulations include aqueous formulations, gels, and
emulsions.
[0257] In one embodiment, the pharmaceutical composition is
formulated for rapid cardiac delivery. One type of pharmaceutical
composition that is formulated for rapid cardiac delivery is an
injectable pharmaceutical composition. Liquid pharmaceutical
compositions which are sterile solutions or suspensions can be
utilized by for example, intramuscular, intrathecal, epidural,
intraperitoneal or subcutaneous injection. Sterile solutions can
also be administered intravenously. The active ingredient may be
prepared as a sterile solid composition which may be dissolved or
suspended at the time of administration using sterile water,
saline, or other appropriate sterile injectable medium. Carriers
are intended to include necessary and inert binders, suspending
agents, lubricants, flavorants, sweeteners, preservatives, dyes,
and coatings.
[0258] Some preferred embodiments concern compositions that
comprise a bioadhesive, preferably a mucoadhesive, coating. A
"bioadhesive coating" is a coating that allows a substance (e.g., a
composition or therapeutic agent according to the invention) to
adhere to a biological surface or substance better than occurs
absent the coating. A "mucoadhesive coating" is a preferred
bioadhesive coating that allows a substance, for example, a
composition according to the invention, to adhere better to mucosa
occurs absent the coating. For example, micronized particles (e.g.,
particles having a mean diameter of about 0.01, 0.1, 0.2, 0.3, 0.4,
0.5, 1, 5, 10, 25, 50, or 100 um) can be coated with a
mucoadhesive. In instances wherein the therapeutic agent is a
soluble molecule, including but not limited to soluble receptor
fragments, antibodies and antibody derivatives or other soluble
polypeptides, preferred diameters include but are not limited to
about 0.1, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 um. In instances
where the therapeutic agent is a soluble molecule, including but
nor limited to soluble receptor fragments and derivatives,
antibodies and antibody derivatives and other soluble polypeptides,
preferred diameters are about 0.1, 0.3, 0.5, 0.6, 0.7, 0.8, 0.9 and
1.0 um. The coated particles can then be assembled into a dosage
form suitable for delivery to an organism. Preferably, and
depending upon the location where the cell surface transport moiety
to be targeted is expressed, the dosage form is then coated with
another coating to protect the formulation until it reaches the
desired location, where the mucoadhesive enables the formulation to
be retained while the therapeutic agents interact with the target
cell surface transport moiety.
[0259] Those skilled in the art will appreciate that when the
compositions of the present invention are administered as agents to
achieve a particular desired biological result, which may include a
therapeutic or protective effect(s) (including vaccination), it may
be necessary to combine the therapeutic agents of the invention
with a suitable pharmaceutical carrier. The choice of
pharmaceutical carrier and the preparation of the therapeutic agent
as a therapeutic or protective agent will depend on the intended
use and mode of administration. Suitable formulations and methods
of administration of therapeutic agents include those for oral,
pulmonary, nasal, buccal, occular, dermal, rectal, or vaginal
delivery.
[0260] Depending on the mode of delivery employed, the
context-dependent functional entity can be delivered in a variety
of pharmaceutically acceptable forms. For example, the
context-dependent functional entity can be delivered in the form of
a solid, solution, emulsion, dispersion, micelle, liposome, and the
like, incorporated into a pill, capsule, tablet, suppository,
aerosol, droplet, or spray. Pills, tablets, suppositories,
aerosols, powders, droplets, and sprays may have complex,
multilayer structures and have a large range of sizes. Aerosols,
powders, droplets, and sprays may range from small (1 micron) to
large (200 micron) in size.
[0261] Pharmaceutical compositions of the present invention can be
used in the form of a solid, a solution, an emulsion, a dispersion,
a micelle, a liposome, and the like, wherein the resulting
composition contains one or more of the compounds of the present
invention, as an active ingredient, in admixture with an organic or
inorganic carrier or excipient suitable for enteral or parenteral
applications. The active ingredient may be compounded, for example,
with the usual non-toxic, pharmaceutically acceptable carriers for
tablets, pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, mannose, gum acacia,
gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn
starch, keratin, colloidal silica, potato starch, urea, medium
chain length triglycerides, dextrans, and other carriers suitable
for use in manufacturing preparations, in solid, semisolid, or
liquid form. In addition auxiliary, stabilizing, thickening and
coloring agents and perfumes may be used. Examples of a stabilizing
dry agent includes triulose, preferably at concentrations of 0.1%
or greater (See, e.g., U.S. Pat. No. 5,314,695).
[0262] Pharmaceutical compositions that preferentially target
cardiac tissues are generally preferred. As a non-limiting example,
U.S. Pat. No. 5,876,747 to Stracher et al. Claims liposomes that
preferentially travel to cardiac and skeletal muscle.
[0263] Medical Devices and Kits
[0264] Medical devices that incorporate the therapeutic agents of
the invention may be prepared and used. Such devices and kits may
be designed for use by trained medical personnel in, e.g.,
hospitals, ambulances, and the like. Additionally or alternatively,
such devices and kits may be designed to be used by untrained
individuals, including a patient in need of treatment, in
situations where trained medical personnel are not available.
Non-limiting examples of such devices and kits are disclosed in
U.S. Pat. No. 4,658,830 to Sarnoff. Such devices and kits may
further include other (supplementary) devices and formulations
useful for treating cardiac disorders. A non-limiting example of
one such supplementary device is a portable defibrillator and
similar devices, such as is disclosed in U.S. Pat. No. RE 30,750 to
Diack et al. A non-limiting example of a supplementary formulation
is one that includes compounds useful for treating conditions
associated with cardiac disorders, such as those that are disclosed
in U.S. Pat. No. 6,130,235 to Mavunkel et al.
[0265] Medical devices that incorporate sphingolipid-binding
ligands, such as antibodies, according to the invention include
those that are commonly referred to as "dialysis machines" (see,
e.g., U.S. Pat. No. 6,080,321). In this type of sphingolipid-based
cardiac medical device, sphingolipid ligands are immobilized onto a
surface in the device. A patient's blood is pumped into the device
in such a way as to bring blood into contact with the sphingolipid
ligands. Undesirable, toxic and/or cardiotoxic sphingolipids and/or
their metabolic precursors in the blood bind to the immobilized
sphingolipid ligands, thus removing undesirable, toxic and/or
cardiotoxic sphingolipids and/or their less toxic metabolic
precursors from the blood. The blood, which is returned to the
patient after passing through the device, leaves the device while
the targeted sphingolipid remains bound to, and thus retained by,
the immobilized sphingolipid ligands. The patient's blood re-enters
the patient's body relatively or completely depleted of the
targeted sphingolipid. The passage of a patient's blood through the
device is repeated as many times as is needed in order to achieve
the desired effect of lowering the concentration of undesirable,
toxic and/or cardiotoxic sphingolipids and their metabolic
precursors.
[0266] An in-dwelling catheter would be inserted to the area at
risk in the heart. The catheter would have a smaller inner catheter
which would subsequently inserted. The smaller catheter would be
coated with an antibody or other sphingolipid binding ligands which
would act as a sink to remove the sphingolipids in the focused
area. This leads to a localized depletion or complete removal of
undesirable sphingolipids.
[0267] The invention provides for diagnostic and therapeutic kits
related to sphingolipid-based therapy. In one embdiment, the
invention relates to kits for determining the diagnosis or
prognosis of a patient. These kits preferably comprise devices and
reagents for measuring one or more marker levels in a test sample
from a patient, and instructions for performing the assay.
Optionally, the kits may contain one or more means for converting
marker level(s) to a prognosis. Such kits preferably contain
sufficient reagents to perform one or more such determinations.
[0268] More specifically, a diagnostic kit of the invention
comprises any of the following reagents and/or components in any
combination.
[0269] 1. A detectable or detectably labeled first reagent that
binds a spingolipid or spingolipid metabolite of interest. The
sphingolipid-binding reagent can, but need not, be an antibody or
an antibody derivative comprising a detectable moiety. The
sphingolipid-binding reagent is stored in an openable container in
the kit, or is bound to a surface of a substrate such that it is
accessible to other reagents. Examples of the latter include test
strips.
[0270] 2. If the first reagent in neither detectable nor detectably
labeled, the kit may comprise a detectable or detectably labeled
second reagent that binds to the first reagent (e.g., a secondary
antibody) or which produces a detectable signal when in close
proximity to the first reagent (e.g., as results from fluorescent
resonance energy transfer FRET). In either case, the signal
produced from the second reagent correlates with the amount of
sphingolipid in the sample.
[0271] 3. One or more positive control reagents. Typically, these
reagents comprise a compound that is known to produce a signal in
the assay. In one embodiment, the positive control reagents are
standards, i.e., comprise a known amount of a detectable or
detcetably labeled compound, the signal from which may be compared
to the signal from a test sample. In addition to serving as
positive control reagents, they may be used to develop calibration
curves that relate the amount of signal to the known concentration
of a detectable or detectably labeled compound. The signal from a
test sample is compared to the calibration curve in order to
determine what concentration of the detectable or detectably
labeled compound corresponds to the signal from the test sample. In
this embodiment, the kit provides quantitative measurements of the
amount of a sphingolipid in a test sample.
[0272] 4. One or more negative control reagents. Typically, these
control reagents may comprise buffer or another solution that does
not contain any of the detectable or detectably labeled first or
second reagents and should thus not produce any detectable signal.
Any signal that is detected reflects the background level of
"noise" in the assay. Another type of negative control reagent
contains most of the components necessary for the signal of the
assay to be produced, but lacks at least one such component and
therefor should not produce a signal. Yet another type of negative
control reagent contains all of the components necessary for the
signal of the assay to be produced, but also contains an inhibitor
of the process that produced the signal.
[0273] 5. One or more auxiliary reagents for use in the diagnostic
assays of the kit, e.g., buffers, alcohols, acid solutions, etc.
These reagents are generally available in medical facilities and
thus are optional components of the kit. However, these reagents
preferably are included in the kit to ensure that reagents of
sufficient purity and sterility are used, since the resulting
protein conjugates are to be administered to mammals, including
humans, for medical purposes, and to provide kits that can be used
in situations where medical facilities are not readily available,
e.g., when hiking in places located far from medical facilities, or
in situations where the presence of these auxiliary reagents allows
for the immediate treatment of a patient outside of a medical
facility as opposed to treatment that arrives at some later time
(e.g.,
[0274] 6. Instructions to a person using a kit for its use. The
instructions can be present on one or more of the kit components,
the kit packaging and/or a kit package insert.
[0275] A therapeutic kit of the invention comprises any of the
following reagents and/or components in any combination.
[0276] 1. One or more therapeutic agents.
[0277] 2. If the therapeutic agent(s) are not formulated for
delivery via the alimentary canal, which includes but is not
limited to sublingual delivery, a device capable of delivering the
therapeutic agent through some other routes. One type of device for
parenteral delivery is a syringe that is used to inject the
therapeutic agent into the body of an animal in need of the
therapeutic agent. Inhalation devices may also be used. A device
for delivering gentamicin to a patient via inhalation is disclosed
in U.S. patent.
[0278] 3. Separate containers, each of which comprises one or more
reagents of the kit. In a preferred embodiment, the containers are
vials contain sterile, lyophilized formulations of a therapeutic
composition that are suitable for reconstitution.
[0279] 4. Instructions to a person using a kit for its use. The
instructions can be present on one or more of the kit components,
the kit packaging and/or a kit package insert.
[0280] For a better understanding of the present invention,
reference is made to the accompanying drawings and detailed
description and its scope will be pointed out in the appended
claims. All references cited herein are hereby incorporated by
reference.
EXAMPLES
[0281] The following examples are non-limiting and are merely
representative of various aspects and features of the present
invention.
Example 1
Sphingosine Production in Rabbits Increases in Cardiac Ischemia
[0282] Tissue levels of sphingosine (SPH) in adult rabbit hearts
under various conditions were determined as follows. Rabbits were
subjected to retrograde coronary perfusion with hypoxic (low
oxygen) conditions (i.e., 95% CO.sub.2; 5% O.sub.2) or normal
Kreb's buffers (equilibrated with 95% O.sub.2; 5% CO.sub.2). The
rabbits were sacrificed, and hearts were removed and quickly
homogenized. Sphingolipids were extracted from homogenates using
protocols essentially as described by Sabbadini et al., Biochem.
Biophys. Res. Comm. 193:752-758, 1993. HPLC analysis of the
extracted lipids revealed significant increases in tissue SPH
levels for hearts perfused with CO.sub.2 when compared to hearts
exposed to control conditions (20-fold, p<0.001). These
increases occurred after only 5 minutes of hypoxia.
Example 2
Sphingosine Production 1N Humans
[0283] Serum levels of SPH levels in human patients experiencing
cardiac ischemia were examined as follows. Serum samples were taken
from patients presenting themselves to the emergency department of
the Naval Medical Center of San Diego using strict human subjects
protocols. Sphingolipids in the blood samples were extracted and
analyzed as described above. Patients with confirmed myocardial
ischemia had significantly higher SPH levels than any of the
control groups.
[0284] Serum SPH levels in six "well-conditioned athletes" were
combined as one control group. These subjects were Navy special
forces (Navy Seals) and Olympic athletes from the Olympic Training
Center who were exercised to exhaustion on treadmills at 49.degree.
C. In the case of the Olympic athletes, individuals at rest were
also evaluated. The well-conditioned athletic control group had
serum SPH levels of 4.18.+-.1.8 pmol/mL, ranging from the lower
limit of detection (5 pmol/mL) to 16.4 pmol/mL.
[0285] An age-matched (47-79 yrs) control group consisted of
fifteen subjects enrolled in the San Diego State University (SDSU)
Adult Fitness Program who tested negative for exercise stress
(treadmill) and no symptoms of acute coronary syndrome (e.g., chest
pain). The age-matched control group had a mean serum level of SPH
of 99.3.+-.32.4 pmol/mL, ranging from the lower limit of detection
to 369 pmol/mL.
[0286] A group of ischemic patients (n=19) were individuals who had
myocardial ischemia, tested positive for exercise stress for
exercise treadmill testing and/or were referred to the
catherization lab for percutaneous coronary revascularization
(angioplasty). The ischemic patients had a mean serum SPH level of
697.+-.80.7 pmol/mL.
[0287] The ischemic patient subgroup with angina symptoms who
underwent angioplasty had average pre-procedure serum SPH levels of
885.+-.123 pmol/mL (ranging from 447 to 1122 pmol/mL). The patient
serum SPH levels were significantly (p<0.001) higher than the
SPH levels of the age-matched control group. When the control group
of ischemic patients was examined, an average serum SPH level of
697.+-.80.7 pmol/mL was obtained. This value is .about.7-fold
higher than the age-matched control group (p<0.001) and
.about.160-fold higher than well-conditioned athletes.
Example 3
Hypoxic Effects on the Sphingomyelin Signal Transduction
Cascade
[0288] TNF.alpha., acting via TNF.alpha. receptors, has been shown
to utilize the sphingomyelin signal transduction cascade in
cardiomyocytes (Oral et al., J. Biol. Chem. 272:4836-4842, 1997;
Krown et al., J. Clin. Invest. 98:2854-2865, 1996). The following
experiments were carried out in order to determine if this
signaling system could also be activated by hypoxia, and if
sphingolipids were produced before cell death as is the case for
TNF.alpha., as described in the Detailed Description of the
Invention.
[0289] Adult rat cardiomyocytes were subjected to hypoxic
conditions as described above and assayed for their ability to
produce sphingolipid bases involved in the sphingomyelin signal
transduction cascade.
[0290] Cardiomyocytes were cultured on plastic dishes that were
placed in a humidified modular incubator chamber (ICN Biomedicals,
Aurora, Ohio) pressurized to 0.04-0.05 psi by the particular gas
mixture used in the treatment. Warm (37.degree. C.) Tyrode's
solution containing 0.2 mM BSA, ampicillin (50 mg/mL), kanamycin
(100 mg/mL) and fungizone (20 .mu.g/mL) was gassed in a 50 mL
sterile conical for 15 minutes with 95% N.sub.2/5% CO.sub.2 prior
to adding the solution to the cultured cells. The pO.sub.2 was
monitored by a Micro pO.sub.2 System oxygen electrode (Lasar
Research Labs, Los Angeles, Calif.) and found to be 4.0 PPM for the
duration of the hypoxia condition. The chamber was maintained at
37.degree. C. for the designated times. Control (normoxia) cells
were treated the same except for the use of 95% O.sub.2/5% CO.sub.2
and incubated in a standard incubator. The pO.sub.2 of the normoxia
treatment was 7.3 PPM. The pH of control and experimental cell
cultures was monitored with a micro pH electrode (Bectrode pH
Electrodes, Sarasota, Fla.) and remained constant at pH=7.26+/-0.02
for normoxic cells and 7.15+/-0.03 for hypoxic cells throughout the
experiment. Because the cells were incubated in Tyrode's lacking
glucose, these experiments represent a model of hypoxia with
metabolic inhibition. In selected experiments with neonatal rat
cardiomyocytes, a third treatment following hypoxia was employed to
simulate reperfusion. This treatment consisted of 5 hours of
reoxygenation with 95% O.sub.2/5% CO.sub.2 in the incubation
chamber. At the end of each incubation period, the cell-conditioned
media was aspirated and saved for analysis. In selected
experiments, the cells were scraped in the presence of 800 .mu.l
1-butanol for the determination of both cellular and extracellular
(cell-conditioned media) content of sphingolipids.
[0291] For the extraction of sphingolipids, 100-300 .mu.l samples
of tissue, serum or cell extracts were deproteinized by adding
warmed butanol (70.degree. C., 800 .mu.l), vortexing and incubating
at 70.degree. C. while rocking. The mixture was then placed in a
sonicating water bath for 10 minutes. Denatured protein and aqueous
phase were separated from the butanol layer by centrifugation at
15,300.times.g. The upper butanol layer was transferred into a new
extraction tube and saponified by the addition 0.5 M KOH (200
.mu.l). After vortexing, samples were incubated at 70.degree. C.
while rocking for 1 hour with intermittent vortexing and
sonicating. Nanopure water (400 .mu.l) was added to each sample and
returned to the incubator for 10 minutes. After sonicating for 1
minute, the layers were separated by centrifuging at 15,300.times.g
for 3 minutes. The butanol layers was transferred to a new tube and
dried down using a Savant (Holbrook, N.Y.) SpeedVac Plus. Dried
samples were completely resuspended in methanol (375 .mu.l) and
agitated in a bath sonicator for 2 minutes. The extracts were then
derivatized with O-phthalaldehyde (OPA) (Molecular Probes, Eugene,
Oreg.). In brief, 50 mg of OPA were dissolved in ethanol (1.0 mL).
Into 0.5 M boric acid (24.75 mL), 0.25 mL of the OPA in ethanol was
added and mixed. Finally, 2-mercaptoethanol (13 .mu.l) was added
and mixed to make up the OPA derivatization solution. To each
sample 10 mM disodium EDTA (50 .mu.l) was added followed by 0.5 M
boric acid (50 .mu.l) and OPA solution (25 .mu.l). Samples were
incubated at room temperature protected from light for 20
minutes.
[0292] The HPLC analysis was performed using a Beckman (Fullerton,
Calif.) System Gold 118 Solvent Module and 507e Autosampler. A
Jasco (Easton, Md.) FP-920 Intelligent Fluorescence Detector was
used with an excitation wavelength of 330 nm and emission
wavelength of 455 nm. The derivatized samples (50 .mu.l injection)
were separated on a Beckman (Fullerton, Calif.) Ultrasphere ODS 4.6
mm.times.25 cm column with a 1.5 cm Perkin Elmer (Norwalk, Conn.)
NewGuard RP-18 guard column. The solvent system was methanol,
glacial acetic acid, 1 M tetrabutlyammonium dihydrogen phosphate,
Nanopure water (82.9:1.5:0.9:14.7, v/v) run at 1.5 mL/minute.
Chromatograms were analyzed using the Beckman System Gold Nouveau
software. Results are shown in Table 1.
[0293] The only sphingolipid base that accumulated in
cardiomyocytes in response to hypoxia was sphingosine (SPH). Levels
of S-1-P and SPC were not increased by hypoxia. Cumulative data
from several experiments demonstrated that hypoxia produces a
6.4-fold increase in SPH production. The increase in total cell SPH
is not reflected in an increased intracellular content. Instead,
the majority of the SPH produced in response to hypoxia is released
from the cells into the cell-conditioned media. These data
demonstrate a 18-fold increase in the extracellular SPH content of
hypoxic cardiomyocytes.
[0294] The short time (5 hrs) of hypoxia employed did not result in
appreciable necrotic or apoptotic cell death but was associated
with significant TNF.alpha. release. Pretreatment with the
TNFRII:Fc receptor fragment (Mohler et al., J. Immunol.
151:1548-1561, 1993), resulted in the significant (p<0.001)
reduction (.about.3-fold) of the SPH release. TNF.alpha.: receptor
fragment pretreatment did not mitigate SPH-triggered apoptosis (SPH
only, no TNF), indicating that SPH production is a step in the
signal cascade that is "downstream" from TNF.alpha. binding to its
receptors.
Example 4
Blood SPH Is Converted to S-1-P
[0295] Studies with human blood obtained from normal subjects
suggest that blood platelets are capable of converting SPH to S-1-P
because of their rich source of sphingosine kinase (Yatomi et al.,
J. Biochem. 121:969-973, 1997; Yatomi et al., J. Biol. Chem.
272:5291-5297, 1997; Yatomi et al., Blood 86:193-202, 1995). In
such experiments, commercially supplied SPH was added to blood
serum which, in the absence of cellular components, was found to be
unable to convert SPH to S-1-P. Without wishing to be bound by any
particular theory, applicants believe that the ischemic heart is
the major source of serum SPH and that cardiac-derived SPH could be
converted to S-1-P by blood platelets (U.S. Pat. No. 6,210,976 B1,
and published PCT patent application WO 98/57179).
[0296] Experiments were carried out to determine the fate of any
SPH that might be released from cells or platelets into the
extracellular compartment. Whole blood samples were incubated in
vitro for up to 15 hours with .sup.3H-SPH, followed by thin layer
chromatography (TLC) to examine which, if any, of the known
metabolic products of the sphingomyelin pathway were radiolabeled
as a result of metabolism of radiolabeled SPH.
[0297] The results (FIG. 3) indicate that, in whole blood, the
radiolabeled SPH was not converted to ceramide or sphingomyelin,
nor was it metabolized to dimethylsphingosine or dihydrosphingosine
(see FIG. 2). The major metabolite of the pathway that was
appreciably labeled was sphingosine-1-phosphate (S-1-P), presumably
due to the action of the very active sphingosine kinase present in
blood platelets (Yatomi et al., J. Biochem. 121:969-973, 1997;
Yatomi et al., J. Biol. Chem. 272:5291-5297, 1997; Yatomi et al.,
Blood 86:193-202, 1995).
[0298] A substantial amount of the time-dependent conversion of
.sup.3H-1-SPH to .sup.3H-S-1-P occurred within 20 minutes and
represented a conversion of 86%.
3TABLE 1 Sphingolipid Production in Cultured Cardiomyocytes During
Normoxia, Hypoxia and Reoxygenation Hypoxia 12 hours for neonates
Reoxygenation Normoxia 7 hours for adults 5 hrs Neonatal
cardiomyocytes % Permeabilized cells 1.7 .+-. 1.3 3.15 .+-. 1.3
7.45 .+-. 2.5 % Apoptotic cells 1.17 .+-. 0.37 4.58 .+-. 0.74 *23.5
.+-. 2.7 TNF.alpha. (pg/106 cells) 561 .+-. 13.4 *979 .+-. 26.7 --
SPH (pmol/106 cells) 5.8 .+-. 0.43 *77.6 .+-. 6.24 -- Adult
cardiomyocytes % Permeabilized cells 4.71 .+-. 1.18 9.5 .+-. 2.9 --
% Apoptotic cells 8.12 .+-. 1.76 *26.7 .+-. 2.9 -- TNF.alpha.
(pg/106 cells) 810 .+-. 155 *8660 .+-. 3150 -- SPH (pmol/106 cells)
238 .+-. 39 *3860 .+-. 547 -- *Significant at the p < 0.05 level
or greater.
[0299] The data also demonstrate that S-1-P was very stable in
whole blood for the 15-hour time course of the experiment. Only
after several hours was it evident that a small but measurable
amount of t-2-hexadecanal (a.k.a. palmitaldehyde) was produced as a
consequence of the low level of S-1-P lyase present in platelets
that can convert S-1-P to hexadecanal and ethanolamine phosphate
(Yatomi et al., J. Biochem. 121:969-973, 1997; Yatomi et al., J.
Biol. Chem. 272:5291-5297, 1997; Yatomi et al., Blood 86:193-202,
1995).
[0300] It is possible that blood platelets were the major reservoir
of SPH-derived S-1-P (Yatomi et al., J. Biochem. 121:969-973,
1997). Another possible reservoir of S-1-P could be heart cells.
Some studies have indicated that SPH is produced intracellularly by
cultured cardiomyocytes, although these studies did not present
evidence of S-1-P release/secretion (U.S. Pat. No. 6,210,976 B1 and
Published PCT Application WO 98/57179).
Example 5
Blood S-1-P is Cardiotoxic and Dependent on S-1-P Receptors
[0301] During ischemia-induced myocardial infarction, several
important events occur as a consequence of the pathophysiology. In
74% of the cases of AMI, the sudden cardiac death is associated
with significant thrombus (blood clot) in the coronary artery
supplying blood to the infarcted region of the myocardium (Davies
et al., N. Engl. J. Med. 310:1137-1140, 1984). "Infarcted" areas
are those in which cells are dead or have sustained so much damage
that they are fated to die. There is a profound negative inotropic
effect (i.e., loss of contractility) on the myocardial cells
induced by the ischemia itself. Intracellular calcium control is
deregulated with diastolic (resting) calcium steadily rising in an
uncontrolled fashion accompanied by a decrease in systolic
(contractile) calcium leading to an eventual `calcium
overload`.
[0302] Without wishing to bound by any particular theory,
applicants believe that these effects are a consequence of the
initial SPH production by pre-AMI and post-AMI ischemic cardiac
cells and the subsequent production of S-1-P by nearby platelets.
It is also believed that S-1-P activates nearby platelets through
calcium deregulation of the platelets and that platelet-derived
S-1-P is released into the serum.
[0303] Exogenously applied S-1-P is capable of activating blood
platelets (Yatomi et al., J. Biol. Chem. 272:5291-5297, 1997;
Yatomi et al., Blood 86:193-202, 1995). It has been suggested that
the increased serum S-1-P has two major actions, both of which are
cardiotoxic. The first is to act on the platelets to promote the
clotting response and further exacerbate the ischemia. Secondly,
the platelet-derived S-1-P acts in a paracrine fashion on
neighboring endothelial and myocardial cells to promote calcium
deregulation and apoptosis. On the cardiomyocytes, the consequence
is to promote calcium overload. The endothelial cell response is to
promote vasoconstriction and further limit the blood supply through
the coronary vasculature. It has demonstrated that S-1-P applied to
cultured cardiomyocytes resulted in apoptosis and dramatic
increases in diastolic calcium followed in minutes by decreases in
systolic calcium and eventual calcium overload reminiscent of what
happens to the ischemic myocardium. It has also demonstrated that
both rat and human cardiac tissue express genes for the recently
identified S-1-P receptors of the EDG (endothelial differentiation
gene) family, and that these receptors mediate the calcium
deregulation (Nakajima et al., Biophysical J. 78:319 A, 2000).
[0304] Extracellular S-1-P is the ligand for a novel class of G
protein-coupled receptors (GPCRs). Such receptors were first
described as an orphan GPCRs cloned from human umbilical vein
endothelial cells (Hla et al., J. Biol. Chem. 265:9308-9313, 1990;
Lynch et al., Trends Pharmacol. Sci. 20:473-475, 1999). Tissue
expression of the EDG-1/3/5 genes in the murine system indicates
that heart and lung have the highest overall expression of these
three genes (Zhang et al., Gene 227:89-99, 1999). However, cardiac
tissues are composed of diverse cell types, including endothelial
cells, which have high levels of EDG-1 expression (Hla et al., J.
Biol. Chem. 265:9308-9313, 1990). Although EDG-1/3/5 are expressed
in C2C12 skeletal muscle myoblasts (Meacci et al., FEBS Letters
457:184-188, 1999), cardiomyocyte-specific expression of the EDG
genes has not been elucidated. Recently a EDG-1 gene and its
protein expression were determined in primary cultures of neonatal
rat cardiomyocytes and in adult rat ventricular tissue (Nakajima et
al., Biophysical J. 78:319 A, 2000). Cultured cardiomyocytes were
used to evaluate the functional role of EDG receptors, including
the ability of S-1-P to modulate intracellular calcium levels.
[0305] Importantly, the calcium deregulatory responses evoked by
S-1-P in cultured cardiomyocytes are quite similar to the calcium
deregulation seen in models of acute myocardial infarction (Lee et
al., Circ. 78:1047-1059, 1988; Smith et al., Amer. Heart J
103:716-723, 1982; Kihara et al., Circ. Res. 65:1029-1044, 1989).
These characteristic responses include: increases in diastolic
calcium (and calcium overload), decreases in systolic calcium (the
negative inotropic state) and the production of abnormal
oscillatory beating behavior (arrhythmias) and cessation of
activity, all of which were seen in the cardiomyocytes when treated
with S-1-P. Additionally, the average calcium level combining both
diastolic and systolic responses is significantly increased by
S-1-P treatment. Taken together, these data suggest that S-1-P,
possibly acting via EDGRs, increases the influx of extracellular
calcium which then causes the calcium overload.
[0306] It is well known that calcium deregulation is a prerequisite
to apoptosis (Magnelli et al., Bioch. Biophys. Res. Comm.
204:84-90, 1994). Previously published data indicate that rat
cardiomyocytes in culture undergo apoptotic cell death when exposed
to sphingosine (Krown el al., J. Clin. Invest. 98:2854-2865, 1996).
It is also suggested that the S-1-P present in putative high levels
in cardiac circulation could act on cardiac cell S-1-P receptors to
produce profound negative inotropic effects and cell death by
apoptosis.
[0307] Thus, it is likely that the pre-AMI ischemic myocardium
produces the initial instruments of its own destruction, namely,
various cytokines and, importantly, SPH. The secreted SPH, acting
indirectly through platelet-derived S-1-P, promotes the activation
of S-1-P receptors on platelets, endothelial cells and
cardiomyocytes. Blood clotting, vasoconstriction and myocardial
calcium overload are the consequences with myocardial infarction as
the ultimate result. As the growing ischemia and the area of
infarction challenges more cardiac tissue, additional SPH is
released and a positive feedback loop results until substantial
cell death occurs.
[0308] Although not wishing to be bound to any particular theory,
it is possible that during low levels of ischemia and cardiac
stress (e.g., hyptertension), the cardiac cells produce cytokines
and spbingolipids as extracellular signaling molecules that serve
to precondition the heart to these stresses. Protection can come
from preconditioning the heart itself via protection against
calcium overload, cell death and arrhymthias or by producing a
hypernatating myocardial state to lower energy demands during
ischemia.
Example 6
Use of Antibodies 1N Sphingolipid-Based Therapy
[0309] This Example describes how sphingolipid-based cardiovascular
therapy can be realized by the use of antibodies and derivatives
thereof (single-chain Fv's, CDR's, etc.) that specifically bind
certain molecules as therapeutic agents. Such antibodies are
directed to, by way of non-limiting example, antibodies to
sphingolipids and receptors thereof.
[0310] Antibodies to Sphingolipid Receptors
[0311] One type of therapeutic antibody specifically sphingolipid
receptors that carry out the cellular internalization of
undesirable sphingolipids. In some cases, the delivery into the
cell of an undesirable sphingolipid results in a sequence of events
having an undesirable effect. Antibodies to such receptors prevent
the entry of the undesirable sphingolipid into cells, thus avoiding
the undesirable consequences of such entry. For example, the
undesirable, toxic and/or cardiotoxic sphingolipid S-1-P has many
actions that are dependent upon binding to sphingolipid receptors,
including without limitation Edg receptors (Example 15). Antibodies
to receptors that block the binding of a undesirable, toxic and/or
cardiotoxic sphingolipid are developed and tested for their ability
to inhibit the binding of S-1-P to its receptors, as well as for
their ability to block post-binding events that lead to cardiotoxic
effects. Antibodies to Edg receptors are known, and are in some
instances commercially available. For example, antibodes to Edg-1,
-7 and -8 are available from Oncogene Research Products; antibodies
to Edg-2 are available from Calbiochem; antibodies to Edg-4 are
available from Antibody Solutions (Palo Alto, Calif.); and
antibodies to Edg-5 are available from Exalpha Biologicals, Inc.
(Boston, Mass.).
[0312] Antibodies to Sphingolipids
[0313] One type of therapeutic antibody specifically binds
undesirable sphingolipids. Such antibodies bind sphingolipids in
order to achieve beneficial effects such as, e.g., (1) lowering the
effective concentration of available (i.e., unbound) undesirable,
toxic and/or cardiotoxic sphingolipids (and/or the concentration of
their metabolic precursors) that would otherwise be free to exert
their harmful effects on cells (including, by way of non-limiting
example, removing undesirable, toxic and/or cardiotoxic
sphingolipids and their metabolic precursors from blood via ex vivo
treatments); (2) to inhibit the binding of an undesirable, toxic
and/or cardiotoxic sphingolipid to a cellular receptor therefor,
and/or to lower the concentration of a sphingolipid that is
available for binding to such a receptor; and/or (3) preventing the
metabolic conversion of a first sphingolipid into a second and more
undesirable, toxic and/or cardiotoxic sphingolipid, and/or to lower
the concentration of such a precursor that is available for
enzymatic conversion into a undesirable, toxic and/or cardiotoxic
sphingolipid.
[0314] Examples of such therapeutic effects include but are not
limited to the use of (i) anti-S-1-P antibodies to lower the
concentration of available S-1-P, thereby blocking or at least
limiting S-1-P's cardiotoxic and thrombogenic effects, and/or (ii)
anti-SPH antibodies to prevent the metabolic conversion of SPH to
the more undesirable, toxic and/or cardiotoxic sphingolipid
S-1-P.
[0315] To produce mAb to phospholipids, acid-treated Salmonella
minnesota are administered directly into a mouse spleen using
protocols essentially according to the methods of Umeda et al. that
have been used to make mAbs to phosphatidylserine (J. Immunol.
143:2273-2279, 1989; see also Reza et al., FEBS Lett. 339:229-233,
1994).
[0316] For production of anti-SPH antibodies, the acid-treated S.
minnesota are coated with SPH and injected into the mouse spleen
prior to cell fusion to produce a hybridoma that secretes anti-SPH
mAb. Similar methods are used to produce anti-S-1-P mAb and
anti-SPC mAb.
[0317] Additionally or alternatively, fatty acid free
BSA-sphingolipid conjugates are used as the immunogen in order to
present unique epitopes to the animal. Appropriate steps are taken
to ensure that the mAbs produced in this fashion are directed to
sphingolipids of choice and not to oxidized lipid or protein-lipid
adducts (Horkko et al., J. Clin. Invest. 98:815-825, 1996; Palinski
et al., J. Clin. Invest. 98:800-814, 1996).
[0318] Antibodies to S-1-P
[0319] In order to develop antibodies to S-1-P, guinea pigs were
immunized IP once a week for 4 weeks with 1 mg of KLH-derivatized
sphingolipid. The protocols that were used are essentially those of
Horkko et al. (J. Clin. Invest. 98:815-825, 1996) and Palinski et
al. (J. Clin. Invest. 98:800-814, 1996).
[0320] In brief, the animals were given weekly injections over a
period of several weeks. In the first week, 150 ug of immunogen in
Complete Freund's adjuvant was injected. During the second, third,
fourth, fifth and sixth weeks, 100 ug of immunogen in Incomplete
Freund's adjuvant was injected into the guinea pigs. Serum samples
collected from the immunized guinea pigs were shown to contain
antibodies to S-1-P by use of an ELISA assay. The ELISA was carried
out essentially according to the procedures described by Horkko et
al. (J. Clin. Invest. 98:815-825, 1996) and Palinski et al. (J.
Clin. Invest. 98:800-814, 1996). The serum samples had a titer of
140,000 Relative Lumiscent Units/100 ms.
Example 7
Modulation of Sphingosine-1-Phosphate (S-1-P) Metabolism
[0321] The concentration of the undesirable, toxic and/or
cardiotoxic sphingolipid S-1-P is lowered (i) by stimulating
reactions that utilize S-1-P as a reactant (i.e., reactions that
degrade S-1-P, e.g., Rxns. #1 and #2 in FIG. 2) and, additionally
or alternatively, (ii) by inhibiting chemical reactions that yield
S-1-P as a product (i.e., reactions that produce S-1-P, e.g. Rxn.
#3 in FIG. 2). Such stimulation and/or inhibition is achieved by,
for example, (1) increasing the amount of, and/or enhancing the
activity of, enzymes that catalyze the catabolism (degradation) of
S-1-P and, additionally or alternatively, (2) reducing the amount
of, and/or or inhibiting or completely blocking the activity of,
enzymes that catalyze the anabolism (production) of S-1-P.
[0322] In instances where the goal is to increase the concentration
enzymes that degrade S-1-P, pharmaceutical formulations of such
enzymes are administered to a patient. S-1-P-degrading enzymes are
purified from a variety of mammals and other animals, or produced
in vitro from cells using recombinant DNA techniques.
[0323] Inhibition of Production of S-1-P
[0324] The inhibition of enzymes that catalyze reactions that yield
S-1-P (i.e., reactions that have S-1-P as a product) is expected to
result in the reduction or complete inhibition of the production of
S-1-P. Such enzymes include but are not limited to the
following:
[0325] Sphingosine Kinase (SPH kinase) catalyzes the conversion of
SPH to S-1-P (Rxn. #3 in FIG. 2; see also FIG. 1). A genetic
sequence encoding human SPH-kinase has been described (Melendez et
al., Gene 251:19-26, 2000). Three human homologs of SPH kinase
(SKA, SKB and SKC) have been described (published PCT patent
application WO 00/52173). Murine SPH kinase has also been described
(Kohama et al., J. Biol. Chem. 273:23722-23728, 1998; and published
(PCT patent application WO 99/61581). Published PCT patent
application WO 99/61581 to Spiegel is stated to describe nucleic
acids encoding a sphingosine kinase. Published PCT patent
application WO 00/52173 to Munroe et al. is stated to describe
nucleic acids encoding homologues of sphingosine kinase. Other SPH
Kinases are described by Pitson et al., "Human sphingosine kinase:
purification, molecular cloning and characterization of the native
and recombinant enzymes", Biochem J. 350:429-441, 2000; and
published PCT application WO 00/70028 to Pitson et al.; and Liu et
al., "Molecular Cloning and Functional Characterization of a Novel
Mammalian Sphingosine Kinase Type 2 Isoform", The Journal of
Biological Chemistry, 275:19513-19520, 2000; Vadas et al.,
"Sphingosine Kinase and Uses Thereof", PCT/AU01/00539, published as
WO 01/85953 on Nov. 15, 2001; Rastelli, "Novel Sphingosine
Kinases", PCT/US01/04789, published as WO 01/60990 on Aug. 23,
2001; Allen et al., "Human Sphingosine Kinase Gene",
PCT/EP00/09498, published as WO 01/31029 on May 3, 2001.
[0326] Inhibitors of SPH kinase include but are not limited to
N,N-dimethylsphingosine (DMS) (Edsall et al., Biochem.
37:12892-12898, 1998); D-threo-dihydrosphingosine (Olivera et al.,
Nature 365:557-560, 1993); and Sphingoid bases (Jonghe et al.,
"Structure-Activity Relationship of Short-Chain Sphingoid Bases As
Inhibitors of Sphingosine Kinase", Bioorganic & Medicinal
Chemistry Letters 9:3175-3180, 1999).
[0327] Assays of SPH kinase useful for evaluating these and other
known or potential SPH kinase inhibitors include those disclosed by
Olivera et al., "Assaying Sphingosine Kinase Activity", Methods in
Enzymology, 311:215-223, 1999; Caligan et al., "A High-Performance
Liquid Chromatographic Method to Measure Sphingosine 1-Phosphate
and Related Compounds from Sphingosine Kinase Assays and Other
Biological Samples", Analytical Biochemistry, 281:36-44, 2000.
[0328] Pharmaceutical compositions of these and other inhibitors of
SPH kinase, especially those that are formulated for rapid cardiac
delivery, are used for this form of sphingolipid-based
cardiovascular therapy.
[0329] Inhibition of SPH kinase may lead to an accumulation of its
substrate, SPH, which is also an undesirable sphingolipid, albeit
generally less harmful than S-1-P. In order to avoid or mitigate
this effect should it occur, additional agents are concurrently
administered to (i) stimulate an enzyme that has SPH as a
substrate, with the proviso that the enzyme should not be one that
has S-1-P as a product (such as, e.g., ceramide synthase; see
below); and, additionally or alternatively, (ii) inhibit an enzyme
that has SPH as a product.
[0330] Stimulation of Destruction of S-1-P
[0331] The stimulation of enzymes that catalyze reactions that
degrade S-1-P (i.e., reactions that have S-1-P as a reactant) is
expected to result in the stimulation of degradation of S-1-P
molecules. Such enzymes include but are not limited to the
following:
[0332] S-1-P Lyase catalyzes the conversion of S-1-P to
ethanolamine-P and (a.k.a. t-2-hexadecanal) palmitaldehyde
(Veldhoven et al., Adv. Lipid Res. 26:67-97, 1993; Van Veldhoven,
"Sphingosine-1-phosphate Lyase" Methods in Enzymology, 311:244-254,
1999; Rxn. #1 in FIG. 2). Yeast (Lanterman et al., Biochem. J.
332:525-531, 1998), murine (Zhou et al., Biochem. Biophys. Res.
Comm. 242:502-507, 1998) and human (published PCT patent
applications WO 99/38983 and WO 99/16888) S-1-P lyase genes have
been described. Published PCT patent application WO 99/16888 to
Saba et al. is stated to describe S-1-P lyase DNA and protein
sequences. U.S. Pat. No. 6,187,562, Published PCT patent
application WO 99/38983 to Duckworth et al. is stated to describe a
S-1-P lyase. See also Van Veldhoven et al., "Human
sphingosine-1-phosphate lyase: cDNA cloning, functional expression
studies and mapping to chromosome 10q22.sup.1", Biochimica et
Biophysica Acta 1487:128-134, 20000); and Mandala et al.,
"Molecular cloning and characterization of a lipid phosphohydrolase
that degrades sphingosine-1-phosphate and induces cell death",
PNAS, 97:7859-7864, 2000.
[0333] Pharmaceutical compositions of agents that are stimulators
of S-1-P lyase, especially those that are formulated for rapid
cardiac delivery, are used for this form of sphingolipid-based
cardiovascular therapy.
[0334] S-1-P Phosphatase (a.k.a. SPP phosphohydrolase) is a
mammalian enzyme that catalyzes the conversion of S-1-P to
sphingosine (Rxn. #2 in FIG. 2) (Mandala et al., Proc. Nat. Acad.
Sci. 95:150-155, 1998; Mandala et al., Proc. Nat. Acad. Sci.
97:7859-7864, 2000; Mandala, "Sphingosine-1-Phosphate
Phosphatases", Prostaglandins & other Lipid Mediators,
64:143-156, 2001; Brindley et al., "Analysis of Ceramide
1-phosphate and Sphingosine-1-phosphate Phosphatase Activities",
Methods in Enzymology, 311:233-244, 1999). Two S-1-P phosphatases,
LBP1 and LB P2, have been isolated from yeast (Mandala et al., J.
Biol. Chem. 272:32709-32714, 1997). Mandala et al., "Mammalian
Sphingosine-1-Phosphate Phosphatase", PCT/UW01/03879, published as
WO01/57057 on Aug. 9, 2001.
[0335] Pharmaceutical compositions of agents that are stimulators
of S-1-P phosphatase, especially those that are formulated for
rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
Example 8
Modulation of Sphingosine (SPH) Metabolism
[0336] The concentration of the undesirable, toxic and/or
cardiotoxic sphingolipid SPH is lowered (i) by stimulating
reactions that utilize SPH as a reactant (i.e., reactions that
degrade SPH without producing S-1-P) and, additionally or
alternatively, (ii) by inhibiting chemical reactions that yield SPH
as a product (i.e., reactions that produce SPH, e.g., Rxn. #5 in
FIG. 2). Such stimulation and/or inhibition is achieved by, for
example, (1) increasing the amount of, and/or enhancing the
activity of, enzymes that catalyze the catabolism (degradation) of
SPH and, additionally or alternatively, (2) reducing the amount of,
and/or or inhibiting or completely blocking the activity of,
enzymes that catalyze the anabolism (production) of SPH. Because
SPH is converted into S-1-P by enzymes such as SPH kinase (Rxn. #3
in FIG. 2), lowering the concentration of the undesirable, toxic
and/or cardiotoxic sphingolipid SPH is not only therapeutic in its
own right but, if done without converting SPH to S-1-P, has the
additional therapeutic benefit of lowering the production of the
more undesirable, toxic and/or cardiotoxic sphingolipid S-1-P.
[0337] In instances where the goal is to increase the concentration
of enzymes that degrade SPH, pharmaceutical formulations of such
enzymes are administered to a patient. SPH-degrading enzymes, such
as SPH Kinase and ceramide synthase (Rxns. #3 and 4, respectively,
in FIG. 2) are purified from a variety of mammals, including
humans, and other animals; or are produced in vitro using, e.g.,
recombinant DNA techniques.
[0338] Inhibition of Production of SPH
[0339] The inhibition of enzymes that catalyze reactions that yield
SPH (i.e., reactions that have SPH as a product) is expected to
result in the reduction or complete inhibition of the production of
SPH. Such enzymes include but are not limited to the following:
[0340] Ceramidase (CDase) catalyzes the conversion of ceramide to
SPH (Rxn. #5 in FIG. 2; see also FIG. 1). For a review, see
Nikolova-Karakashian et al., "Ceramidases", Methods in Enzymology,
311:194-201, 1999. At least two types of ceramidases are known in
the art, ceramidase I and ccramidase II, which differ in terms of
pH optima (Sugita et al., Biochim. Biophys. Acta. 398:125-131,
1975; Yada et al., J. Biol. Chem. 270:12677-12684, 1995).
Ceramidases are disclosed by Tani et al., "Purification and
Characterization of a Neutral Ceramidase from Mouse Liver: A Single
Protein Catalyzes the Reversible Reaction in Which Ceramide is Both
Hydrolyzed and Synthesized", The Journal of Biological Chemistry
275:3462-3468, 2000; Mao et al., "Cloning and Characterization of a
Saccharomyces cerevisiae Alkaline Ceramidase with Specificity for
Dihydroceramide", The Journal of Biological Chemistry
275:31369-31378, 2000; Zhang et al., "Human Acid Ceramidase Gene:
Novel Mutations in Farber Disease", Molecular Geneetics and
Metabolism 70:301-309, 2000; Mao et al., "Cloning of an Alkaline
Ceramidase from Saccharomyces cerevisiae: An Enzyme with Reverse
(CoA-Independent) Ceramide Synthase Activity", The Journal of
Biological Chemistry 275:6876-6884, 2000; Okino et al., "Molecular
Cloning, Sequencing, and Expression of the Gene Encoding Alkaline
Ceramidase from Pseudomonas aeruginosa: Cloning of A Ceramidase
Homologue from Mycobacterium Tuberculosis", 274:36616-36622, 1999;
Kita et al., "Reverse hydrolysis reaction of a recombinant alkaline
ceramidade of Pseudomonas aeruginosa" Biochimica et Biophysica Acta
1485:111-120, 2000; Li et al., "The Human Acid Ceramidase Genes
(ASAH): Structure, Chromosomal Location, Mutation Analysis, and
Expression", Genomics, 62:223-231, 1999; Mao et al., "Cloning and
Characterization of a Novel Human Alkaline Ceramidase: A Mammalian
Enzyme That Hydrolyzes Phytoceramide", The Journal of Biological
Chemistry, 276:26577-26588, 2001; Mitsutake et al., "Purification,
Characterization, Molecular Cloning, and Subcellular Distribution
of Neutral Ceramidase of Rat Kidney", The Journal of Biological
Chemistry, 276:26249-26259, 2001; Ito et al., "Ceramidase Gene",
PCT/JP00/01802, published as WO00/58448 on Oct. 5, 2000; Okino et
al., "Molecular Cloning, Sequencing, and Expression of the Gene
Encoding Alkaline Ceramidase from Pseudomonas aeruginosa", The
Journal of Biological Chemistry, Vol. 274, Dec. 17, 1999, pp.
36616-36622; and Bawab et al., "Molecular Cloning and
Characterization of a Human Mitochondrial Ceramidase", The Journal
of Biological Chemistry, 275:21508-21513, 2000.
[0341] Inhibitors of ceramidase include but are not limited to
D-erythro-MAPP and L-erythro-MAPP (Bielawska et al., J. Biol. Chem.
271:12646-12654, 1996; Hannun et al., "Inhibitor of Ceramidase",
PCT/US96/17769, published as WO97/44019 on Nov. 27, 1997); and
N-oleoyl-ethanolamine (NOE; Sugita et al., 1975; Yada et al., J.
Biol. Chem. 270:12677-12684, 1995; Meroni et al., "Effect of
N-Acetylsphingosine (C2) and the Ceramidase Inhibitor
(1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol on the
Regulation of Sertoli Cell Function", Journal of Andrology,
20:619-625, 1999). As CDase is inhibited by endogenous
sphingolipids such as SPH and SM (Hise et ul., J. Clin. Invest.
77:768-773, 1986); nontoxic synthetic sphingolipids that inhibit
CDase are also used. See also Hannun et al., "Inhibitors of
Ceramidase", U.S. Pat. No. 5,851,782, issued Dec. 22, 1998.
[0342] Methods for assaying ceramidase activity are disclosed by He
et al.; "A Fluorescence-Based High-Performance Liquid
Chromatography Assay to Determine Acid Ceramidase Activity",
Analytical Biochemistry, 274:264-269, 1999. Pharmaceutical
compositions of these and other inhibitors of CDase, especially
those that are formulated for rapid cardiac delivery, are used for
this form sphingolipid-based cardiovascular therapy.
[0343] S-1-P Phosphatase (a.k.a. SPP phosphohydrolase) catalyzes
the conversion of S-1-P to sphingosine (Rxn. #2 in FIG. 2) and is
described in more detailed in the preceding Example 8. Inhibition
of S-1-P phosphatase has the beneficial result of lowering SPH
production; however, inhibition of S-1-P phosphatase potentially
includes the undesirable effect of inhibiting the degradation of
S-1-P. It is thus useful to include one or more stimulators of an
enzyme that degrades S-1-P (such as, e.g., S-1-P lyase; see above),
in pharmaceutical compositions used for this form
sphingolipid-based cardiovascular therapy.
[0344] Stimulation of Destruction of SPH
[0345] The stimulation of enzymes that catalyze reactions that
degrade SPH (i.e., reactions that have SPH as a reactant, e.g.,
Rxns. #3 and #4 in FIG. 2) is expected to result in the stimulation
of degradation of SPH molecules. In general, it is preferable that
such reactions do not yield a undesirable, toxic and/or cardiotoxic
sphingolipid, especially S-1-P, as a product (e.g., Rxn. #4 in FIG.
2, which is catalyzed by ceramide synthase). SPH kinase may be
stimulated to enhance the degradation of SPH but the reaction it
catalyzes produces the undesirable sphingolipid S-1-P; accordingly,
stimulators of SPH kinase are preferably combined with stimulators
of enzymes that degrade S-1-P, e.g., S-1-P lyase (Rxn. # 1 in FIG.
2).
Example 9
Modulation of Ceramide (CER) Metabolism
[0346] The concentration of CER is lowered (i) by stimulating
reactions that utilize CER as a reactant (i.e., reactions that
degrade CER), preferably those reactions that do not yield SPH as a
product (e.g., Rxn. # 5 in FIG. 2); and, additionally or
alternatively, (ii) by inhibiting chemical reactions that yield CER
as a product (i.e., reactions that produce CER), preferably those
reactions that do not use SPH as a reactant (e.g., Rxn. #4 in FIG.
2). Such stimulation and/or inhibition is achieved by, for example,
(1) increasing the amount of, and/or enhancing the activity of,
enzymes that catalyze the catabolism (degradation) of CER and,
additionally or alternatively, (2) reducing the amount of, and/or
or inhibiting or completely blocking the activity of, enzymes that
catalyze the anabolism (production) of CER. However, such
CER-producing enzymes do not use SPH as a substrate, as inhibition
of such enzymes (e.g., ceramide synthase) is expected to result in
an increase in the level of SPH. Because CER is directly converted
into the more undesirable, toxic and/or cardiotoxic sphingolipid
SPH by enzymes such as ceramidase (Rxn. # 5 in FIG. 2), lowering
the concentration of CER has the therapeutic benefit of lowering
the production of the more undesirable, toxic and/or cardiotoxic
sphingolipid SPH; this effect which results in a lowered production
of S-1-P by SPH kinase.
[0347] In instances where the goal is to increase the concentration
of enzymes that degrade SPH, pharmaceutical formulations of such
enzymes are administered to a patient. SPH-degrading enzymes are
purified from a variety of mammals, including humans, and other
animals; or produced in vitro from cells using recombinant DNA
techniques.
[0348] Inhibition of Production of CER
[0349] The inhibition of enzymes that catalyze reactions that yield
CER (i.e., reactions that have CER as a product) is expected to
result in the reduction or complete inhibition of the production of
CER. Such enzymes include but are not limited to the following:
[0350] Ceramide Synthase (CER synthase), also known as sphingosine
N-acyltransferase, catalyzes the acetylation of dihydrosphingosine
(Rxn. #10 in FIG. 2) which leads to the production of ceramide.
[0351] Inhibitors of CER synthase include the fungal toxin
Fumonisin B1 (Merrill et al., J. Lipid Res. 26:215-234A, 1993; Wang
et al., Adv. Lipid Res. 26:215-234, 1993; Lee et al., Biochem. J.
334:457-461, 1998; Xu et al., J. Biol. Chem. 273:16521-16526, 1998;
Lochhead et al., Kidney Int. 54:373-381, 1998; Tsunoda et al., J.
Biochem. Mol. Toxicol. 12:281-289, 1998); derivatives of fumonisin
(Humpf et al., J. Biol. Chem. 273:19060-19064, 1998); alternaria
toxins (Id. and Mandala et al., J. Antibiot. 48:349-356, 1995);
viridiofungins (Merrill et al., J Lipid Res. 26:215-234A, 1993;
astralifungins (Mandala et al., J. Antiobiot. 48:349-356, 1995;
Furneisen et al., Biochim. Biophys. Acta. 1484:71-82, 2000); and
D-erythro-N-myristoyl 2-amino-1-phenylpropanol (Hunnan, Science
274:1855-1859, 1996). Pharmaceutical compositions of these and
other inhibitors of CER synthase, especially those that are
formulated for rapid cardiac delivery, are used for this form
sphingolipid-based cardiovascular therapy.
[0352] Ceramide-1-P Phosphatase (CER-1-P phosphatase) catalyzes the
production of ceramide from ceramide 1-P (Rxn. #17 in FIG. 2). See
Shinghal et al., "Ceramide 1-Phosphate Phosphatase Activity in
Brain", Journal of Neurochemistry, 61:2279-2285, 1993; Boudker et
al., "Detection and Characterization of Ceramide-1-phosphate
Phosphatase Activity in Rat Liver Plasma Membrane", The Journal of
Biological Chemistry, 268:22150-22155, 1993.
[0353] Inhibitors of CER-1-P phosphatase include but are not
limited to manganese; cobalt; NaF; propranolol; phenylglyoxal; and
n-ethylmaleimide (Fureisen et al., Biochim. Biophys. Acta.
1484:71-82, 2000). Pharmaceutical compositions of these and other
inhibitors of CER-1-P phosphatase, especially those that are
formulated for rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0354] Sphingomyelinase (SMase) catalyzes the conversion of
sphingomyelin to ceramide (Rxn. #7 in FIG. 2; see also FIG. 1).
Various isoforms of SMase have been described. These include
nSMases (n for neutral pH isoform), aSMases (a for acidic pH
isoform), and alkaline SMases including an SMase isoform found in
the gut. Both the acidic and neutral forms of SMase are endogenous
to cardiac tissue (Andrieu-Abadie et al., FASEB J 13:1501-1510,
1999), and a novel form of a high turnover sphingomyelinase
localized in the junctional T-tubule membranes (Ghosh et al., Mol.
Cellular Biochem. 189:161-168, 1998). The neutral form of SMase is
exposed to the extracellular surface of the membrane (Mohan et al.,
Biochem Biophys Acta 777:339-342, 1984) and would thus be
accessible to lipid-insoluble agents. SMases are described in
greater detail in Example 13.
[0355] It has been demonstrated that the sphingomyelinase
inhibitor, L-carnitine, blocks doxorubicin-induced apoptosis
coincident with the inhibition of ceramide production
(Andrieu-Abadie et al., FASEB J 13:1501-1510, 1999; Katircioglu, et
al., J. Cardiovasc. Surg. 41:45-50, 1999; Gunther, Eur. J. Pharma.
406:123-126, 2000.
[0356] Inhibitors of SMase include but are not limited to
gentamicin (Ghosh et al., J. Biol. Chem. 262:12550-12556, 1987) and
gentamicin derivatives, and other aminoglycosides, as is described
in more detail elsewhere herein.
[0357] Several inhibitors of SMase have been prepared that are
based on the structures of the naturally occuring compounds
Scyphostatin and Manumycin. In addition to naturally occuring
compounds such as Scyphostatin and Manumycins A-D, several
inhibtors have been synthesized in vitro. These include without
limitation those described by Arenz et al., "Synthesis and
Biochemical Investigation of Scyphostatin Analogues as Inhibitors
of Neutral Sphingomyelinase", Bioorganic & Medicinal Chemistry,
9:2901-2904, 2001; Arenz et al., "Synthesis of the First Selective
Irreversible Inhibitor of Neutral Sphingomyelinase", Eur. J. Org.
Chem., 137-140, 2001; Arenz et al., "Synthese des ersten selektiven
irreverilben Inhibitors der neutralen Sphingomyelinase", Angew
Chem., 112:1498-1500, 2000; Tanaka et al., "Structural Elucidation
of Scyphostatin, an Inhibitor of Membrane-Bound Neutral
Sphingomyelinase", J. Am. Chem. Soc. 199:7871-7872, 1997; Saito et
al., "Absolute Configuration of Scyphostatin", Organic Letters,
2:505-506, 2000; Hoye et al., "Synthesis (and Alternative Proof of
Configuration) of the Scyphostatin C(1')-C(20') Trienoyl Fragment",
Organic Letters, 2:1481-1483, 2000; Izuhara et al., "Studies toward
the Total Synthesis of Scyphostatin: First Entry to the Highly
Functionalized Cyclohexenone Segment", Organic Letters,
3:1653-1656, 2001; Runcie et al., "A Short and Efficient Route to
Novel Scyphostatin Analogues", Organic Letters, 3:3237-3239, 2001;
Chau et al., "Synthesis of Simple Aryl Neutral Sphingomyclinase
Inhibitors", Asbtr. Pap.--Am. Chem. Soc., 2001; Arenz et al.,
"Manumycin A and Its Analogues Are Irreversible Inhibitors of
Neutral Sphingomyelinase", ChemiBiochem., 2:141-143, 2001; Zeeck et
al., "Manumycin derivatives and the use thereof", U.S. Pat. No.
5,079,263, issued Jan. 7, 1992; and Patel et al., "Manumycin
Compounds", U.S. Pat. No. 5,444,087, issued Aug. 22, 1995.
[0358] Other inhibitors of SMase that may serve as therapeutic
agents or lead compounds, or may provide a chemical framework for
preparing focused chemical libraries that can be screened for
inhibitors of SMase, include but are not limited to L-carnitine
(Andrien-Abadie et al., FASEB J. 13:1501-1510, 1999), and related
compounds (U.S. Pat. No. 6,284,798); ubiquinol and ubiquinone
homologs (Martin et al., J. Bioenerg Biomember 33:143-153, 2001);
antioxidants such as ascorbate and alpha-tocoperol (Hernandez et
al., Circ. Res. 86: 198-204, 2000); glutathione (oxidized form)
(Liu et al., J. Biol. Chem. 272:16281-16287, 1997; Liu et al., J.
Biol. Chem. 273:11313-11320, 1998; Yoshimura et al., J. Neurochem.
73:675-683, 1999; and Yoshimura et al., "Inhibition of Neutral
Sphingomyelinase Activation and Ceramide Formation by Glutathione
in Hypoxic PC 12 Cell Death", Journal of Neurochemistry,
73:675-683, 1999); Alutenusin, a protein produced by Penicillium
spp. (Uchida et al., J. Antibiotics 52:572-574, 1999); SR 3357
(2-isopropyl-1-4-[3-N-methyl-N-3,4-
-dimethoxy-phenethylamino]propyloxy benzenesulfonylindolizine)
(Higuchi et al., J. Immunol. 157:297-304, 1996; Lee et al., Biochem
J. 334:457-461, 1998); desipramine (Lee et al., Biochem J.
334:457-461, 1998; Xu et al., J. Biol. Chem. 273:16521-16526,
1998); DTT (Yamanaka et al., J. Neurochem. 38:1753-1764, 1982);
sphingomyelin methylene analogs (Hakogi et al., Stereocontrolled
synthesis of a sphingomyelin methylene analogue as a
sphingomyelinase inhibitor, Org Lett 2:2627-2629, 2000); and
substituted amino acids (U.S. Pat. No. 6,306,911 B1 to Wachter et
al. Published PCT applications WO 99/41265 and WO 00/58491 disclose
compounds named KF-1040A, KF-1040B, KF-1040T4A, KF-1040T4B,
KF-1040T5A and KF-10407TB, which are stated to be inhibitors of
SMase. Other SMase inhibitors are disclosed in published PCT
application WO 00/72833 A2. Sphingomyelin derivatives that are
inhibitors of SMase are disclosed by Lister et al. (Biochimicha et
Biophysica Acta 1256:25-30, 1995); Tazabekova et al. (Bioorg Khim
1987 May;13:648-653, 1987); and Hakogi et al. (Stereocontrolled
synthesis of a sphingomyelin methylene analogue as a
sphingomyelinase inhibitor, Org Lett 2:2627-2629, 2000).
[0359] Pharmaceutical compositions of these and other inhibitors of
SMase, especially those that are formulated for rapid cardiac
delivery, are used for this form sphingolipid-based cardiovascular
therapy.
[0360] Desaturase catalyzes the conversion of dihydroceramide to
ceramide (Rxn. #9 in FIG. 2). Dihydroceramidase desaturase are
disclosed in Geeraert et al., "Conversion of dihydroceramide into
ceramide: involvement of a desaturase", Biochem J., 327:125-132,
1997; Triola et al., "Synthesis of a Cyclopropene Analogue of
Ceramide, a Potent Inhibitor of Dihydroceramide Desaturase", Angew.
Chem. Int. Ed., 40:1960-1962, 2001; Heinz t al.,
"Sphingolipid-Desaturase", PCT/DE99/01859, published as WO00/00593
on Jan. 6, 2000; Michel et al., "Characterization of Ceramide
Synthesis: A Dihydroceramide Desaturase Introduces The
4,5-TRANS-Double Bond of Sphingosine at the Level of
Dihydroceramide", 272:22432-22437, 1997. Inhibitors of desaturase
are disclosed in Triola et al., "Synthesis of a Cyclopropene
Analogue of Ceramide, a Potent inhibitor of Dihydroceramide
Desaturasc", Angew. Chem. Int. Ed., 40:1960-1962, 2001.
[0361] Pharmaceutical compositions of inhibitors of desaturase,
especially those that are formulated for rapid cardiac delivery,
are used for this form sphingolipid-based cardiovascular
therapy.
[0362] Cerebrosidases catalyze the production of ceramide from
glucosylceramide (Rxn. #14 in FIG. 2). Pharmaceutical compositions
of inhibitors of one or more cerebrosidases, especially those that
are formulated for rapid cardiac delivery, are used for this form
of sphingolipid-based cardiovascular therapy.
[0363] Stimulation of Destruction of CER
[0364] The stimulation of enzymes that catalyze reactions that
degrade CER (i.e., reactions that have CER as a reactant) is
expected to result in the stimulation of degradation of CER
molecules. In general, it is preferable that such reactions do not
yield a undesirable, toxic and/or cardiotoxic sphingolipid, such as
SPH, as a product (an example of an enzyme of this type is
ceramidase). Other enzymes that may be stimulated to enhance the
degradation of CER include but are not limited to the
following:
[0365] Sphingomyelin Synthase catalyzes the conversion of ceramide
to sphingomyelin (Rxn. #6 in FIG. 2); see, e.g., Luberto, et al.,
"Sphingomyelin synthase, a potential regulator of intracellular
levels of ceramide and diacylglycerol during SV40 transformation.
Does sphingomyelin synthase account for the putative
phosphatidylcholine-speci- fic phopholipase C? PubMed, J. Biol
Chem, 273:14550-14559, 1998. Inhibitors of SM synthase include
sphingomyelin metabolites; Vivekananda et al., "Sphingomyelin
metabolites inhibit sphingomyelin synthase and CTP:phophocholine
cytidylyltransferase", Am J. Physiol Lung Cell Mol Physiol,
2281:L91-L107, 2001. CER kinases are disclosed in Bajjalieh et al.,
"Ceramide Kinase", Methods in Enzymology, 311:207-215, 1999;
Kolesnick et al., "Characterization of a Ceramide Kinase Activity
from Human Leukemia (HL-60) Cells: Separation From Diacylglycerol
Kinase Activity", The Journal of Biological Chemistry,
265:18803-18808, 1990. Pharmaceutical compositions of stimulators
of sphinomyelin synthase, especially those that are formulated for
rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0366] Ceramide (CER kinase) catalyzes the conversion of ceramide
to ceramide-1-P (Rxn. # 16 in FIG. 2). Pharmaceutical compositions
of stimulators of CER kinase, especially those that are formulated
for rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0367] Glucosylceramide Synthase catalyzes the conversion of
ceramide to glucosylceramide (Rxn. #13 in FIG. 2). For reviews, see
Shayman et al., "Glucosylceramide Synthase: Assay and Properties",
Methods in Enzymology, 311:42-49, 1999 and Marks et al., "Methods
for Studying Glucosylceramide Synthase", Methods in Enzymology,
311:50-59, 1999. Pharmaceutical compositions of stimulators of
glucosylceramide synthase, especially those that are formulated for
rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0368] Inhibitors of glucosylceramide synthase are known. See U.S.
Pat. No. 6,051,598 to Shayman et al.; Rani et al., "Cell Cylc
Arrest Induced by an Inhibitor of Glucosylceramide Synthase", The
Journal of Biological Chemistry, 270:2859-2867, 1995; Abe et al.,
"Use of Sulfobutyl Ether .beta.-Cyclodextrin as a Vehicle for
D-threo-1-Phenyl-2-decanoylamino-3-m- orpholinopropanol-Related
Glucosylceramide Synthase Inhibitors", Analytical Biochemistry,
287:344-347, 2000; Oshefski et al., "Glucosylceramide Synthase
Inhibition Enhances Vincristine-Induced Cytotoxicity", Int. J.
Cancer, 93:131-138, 2001; Abe et al., "Glycosphingolipid depletion
in Fabry disease lymphoblasts with potent inhibitors of
glucosylceramide synthase", Kidney International, 57:446-454, 2000;
Lee et al., "Improved Inhibitors of Glucosylceramide Synthase", The
Journal of Biological Chemistry, 274:14662-14669, 1999; Abe et al.,
"Structural and stereochemical studies of potent inhibitors and
glucosylceramide synthase and tumor cell growth", Journal of Lipid
Research, 36:611-621, 1995; Shayman et al., "Inhibitors of
Glucosylceramide Synthase", Methods in Enzymology, 311:373-387,
1999; Jimbo et al., "Development of a New Inhibitor of
Glucosylceramide Synthase", J. Biochem 127:485-491, 2000; and Marks
et al., "Methods for Studying Glucosylceramide Synthase", Methods
in Enzymology, 311:50-59, 1999.
[0369] Enzymes that catalyze the production of galactosylceramide
from CER (Rxn. # 15 in FIG. 2) are stimulated to enhance the
degradation of CER. Pharmaceutical compositions of stimulators of
such enzymes, especially those that are formulated for rapid
cardiac delivery, are used for this form of sphingolipid-based
cardiovascular therapy.
Example 10
Modulation of Metabolic Precursors of CER
[0370] The concentration of harmful sphingolipids is lowered by
inhibiting reactions that yield metabolic precursors of ceramide
(CER), which is a metabolic precursor of SPH and S-1-P. Enzymes
that catalyze such reactions include but are not limited to the
following.
[0371] Inhibition of Production of Metabolic Precursors of CER
[0372] The concentration of harmful sphingolipids is lowered by
inhibiting reactions that yield metabolic precursors of ceramide
(CER), which is a metabolic precursor of SPH and S-1-P. Enzymes
that catalyze such reactions include but are not limited to the
following.
[0373] Serine Palmitoyl Transferase catalyzes the production of
3-ketosphinganine (Rxn. #12 in FIG. 2), a precursor in ceramide
synthesis. For a review, see Dickson et al., "Serine
Palmitoyltransferase", Methods in Enzymology, 311:1-9, 1999.
[0374] Inhibitors of serine palmitoyl transferase include but are
not limited to viridiofungins (Mandala et al., J. Antibiot. (Tokyo)
50:339-343, 1997; and Mandala et al., "Isolation and
Characterization of Novel Inhibitors of Sphingolipid Synthesis:
Australifungin, Viridiofungins, Rustmicin, and Khafrefungin,
Methods in Enzymology, 311:335-348, 1999), lipoxamycin (Mandala et
al., J. Antibiot. (Tokyo) 47:376-379, 1994), and sphingofungins E
and F (Horn et al., J. Antibiot. (Tokyo) 45:1692-1696, 1992). Other
SPT inhibitors are disclosed by Hanada et al., "Specificity of
Inhibitors of Serine Palmitoyltransferase (SPT), a Key Enzume in
Sphingolipid Biosynthesis, in Intact Cells", Biochemical
Pharmacology, 59:1211-1216, 2000; Zweerink et al.,
"Characterization of a Novel, Potent, and Specific Inhibitor of
Serine Palmitoyltransferase", The Journal of Biological Chemistry,
267:25032-25038, 1992; and Riley et al., "Fermentation, Partial
Purification, and Use of Serine Palmitoyltransferase Inhibitors
from Isaria (=Cordyceps) sinclairii, Methods in Enzymology,
311:348-361, 1999.
[0375] Pharmaceutical compositions of inhibitors of serine
palmitoyl transferase, especially those that are formulated for
rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0376] 3-Ketosphiganine Reductase catalyzes the production of
sphinganine (dihydrosphingosine) (Rxn. #11 in FIG. 2), a precursor
in ceramide synthesis. See Beeler et al., "The Saccharomyces
cerevisiae TSC10/YBR265.omega. Gene Encoding 3-Ketosphinganine
Reductase Is Identified in a Screen for Temperature-sensitive
Suppressors of the CA.sup.2+-sensitive csg2.DELTA. Mutant", The
Journal of Biological Chemistry, 273:30688-30694, 1998.
Pharmaceutical compositions of inhibitors of 3-ketosphiganine
reductase, especially those that are formulated for rapid cardiac
delivery, are used for this form of sphingolipid-based
cardiovascular therapy.
[0377] Dihydroceramide Synthase catalyzes the acetylation of
dihydrosphingosine (Rxn. #10 in FIG. 2) which leads to the
production of dihydroceramide, a direct precursor of ceramide.
Without wishing to be bound by any particular theory,
dihydroceramide synthase may be the same enzyme as ceramide
synthase (Rxn. #4 in FIG. 2).
[0378] Inhibitors of ceramide synthase include Fumonisin B1 (a
fungal toxin) (Merrill et al., J. Lipid Res. 26:215-234A, 1993;
Wang et al., Adv. Lipid Res. 26:215-234, 1993; Tsunoda et al., J.
Biochem. Mol. Toxicol. 12:281-289, 1998); derivatives of fumonisin
(Humpf et al., J. Biol. Chem. 273:19060-19064, 1998); alternaria
toxins (Id. and Mandala et al., J. Antibiot. 48:349-356, 1995);
viridiofungins (Merrill et al., J. Lipid Res. 26:215-234A, 1993);
astralifungins (Mandala et al., J. Antibiot. 48:349-356, 1995;
Furneisen et al., Biochim. Biophys. Acta. 1484:71-82, 2000); and
D-erythro-N-myristoyl 2-amino-1-phenylpropanol (Hunnan, Science
274:1855-1859, 1996). Pharmaceutical compositions of inhibitors of
dihydroceramide synthase, especially those that are formulated for
rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
[0379] Stimulation of Destruction of Metabolic Precursors of
CER
[0380] The concentration of harmful sphingolipids is lowered by
stimulating reactions that degrade metabolic precursors of ceramide
(CER), which is a metabolic precursor of SPH and S-1-P. Enzymes
that catalyze such reactions include but are not limited to the
following.
[0381] Sphingomyelin Deacylase (SM deacylase) catalyzes the
production of sphingoylphosphorylcholine (SPC) from sphingomyelin
(Rxn. #8 in FIG. 2; see also FIG. 1). Pharmaceutical compositions
of stimulators of SM deacylase, especially those that are
formulated for rapid cardiac delivery, are used for this form of
sphingolipid-based cardiovascular therapy.
Example 11
An Inhibitor of Sphingomyelinase Blocks Hypoxia-Induced Production
of Sphingosine in a Cellular Model
[0382] L-carnitine is a known inhibitor of SMase (Andrien-Abadie et
al., FASEB J. 13:1501-1510, 1999; see also U.S. Pat. No.
6,284,798). This Example demonstrates that L-carnitine blocks the
hypoxia-induced production of sphingosine in a cellular model.
[0383] Cardiomyocytes were cultured on plastic dishes that were
placed in a humidified modular incubator chamber (ICN Biomedicals,
Aurora, Ohio) pressurized to 0.04-0.05 psi by the particular gas
mixture used in the treatment. Warm (37.degree. C.) Tyrode's
solution containing 0.2 mM BSA, ampicillin (50 mg/mL), kanamycin
(100 mg/mL) and fungizone (20 .mu.g/mL) was gassed for 15 minutes
with 95% N2/5% CO2 prior to cell treatment. The pO2 was monitored
by a Micro pO2 System oxygen electrode (Lasar Research Labs, Los
Angeles, Calif.) and found to be 4.0 mmHg for the duration of the
hypoxia condition. The chamber was maintained at 37.degree. C. for
the designated times. Control (normoxia) cells were treated the
same except for the use of 95% O2/5% CO2 and incubated in a
standard incubator. The pO2 of the normoxia treatment was 7.3 mmHg.
The pH of control and experimental cell cultures was monitored with
a micro pH electrode (Beetrode pH Electrodes, Sarasota, Fla.) and
remained constant at pH=7.26+/-0.02 for normoxic cells and
7.15+/-0.03 for hypoxic cells throughout the experiment. Adult
cardiomyocytes were cultured under either normoxia or hypoxia
conditions for 5 hours.
[0384] The cell-conditioned Tyrode's solution was collected and the
cells were scraped from the culture dish. Both cellular and
extracellular sphingolipids were extracted and then quantified by
reverse-phase HPLC. The retention times of the key sphingolipids
(SPH, SPC, S1P, DHSPH) followed in these experiments are shown in
FIG. 4. Hypoxia resulted in a substantial increase in a peak
corresponding to D-crythrosphingosine (SPH).
[0385] The fold-increases in SPH and TNF-alpha (inset) in response
to hypoxia are shown. Cells were pretreated for 30 min with either
TNFRII:Fc (0.5 ng/mL) or L-carnitine (20 ng/mL) prior to hypoxia.
Data are means+/-SEM from 19 separate experiments. FIG. 4 shows the
amount of SPH and TNF-alpha (inset) released into the
cell-conditioned media and is expressed in terms of fold increases
associated with hypoxia. These data demonstrate that the SPH
response (.about.20-fold increase over normoxia) was more profound
than the TNF-alpha response (.about.10-fold increase over normoxia,
inset). Further, FIG. 4 shows that both TNFRII:Fc and the
sphingomyelinase inhibitor L-carnitine were capable of
significantly (p<0.01) reducing the amount of total SPH.
Example 12
An Inhibitor of Sphingomyelinase Blocks The Effects of Global
Ischemia in an Animal Model
[0386] A rat model of ischemia and reperfusion was used to evaluate
the therapeutic potential of a gentamicin, an amino glycoside that
inhibits sphingomyelinase.
[0387] Reagents
[0388] Krebs-Henseleit (KH) Buffer was prepared by combining the
following components:
4 Chemical Conc. (mM) Amount per liter (g) NaHCO3 25.0 2.10 NaCl
118 6.90 KCl 4.7 0.35 MgSO4 (anhy) 1.2 0.145 NaH2PO4 1.2 0.145
CaCl2 1.2 0.175 Glucose 11 1.98
[0389] The following steps are taken to prepare KH buffer. First,
95% O2/5% CO2 is bubbled through sterile water (Gibco Cat. No.
15230-147), which is concurrently warmed to 37.degree. C. Second,
the above ingredients are added and the pH is adjusted to 7.4.
Finally, the buffer is sterilized by filtration.
[0390] Gentamicin vehicle solution was prepared as follows. A stock
solution of 0.1 M Gentamicin was prepared by adding 70 mg of
Gentamicin Sulfate (Sigma, G1264) to 1 ml of KH Buffer. KH buffer
(150 ml) was combined with 150 .mu.l of the stock solution to yield
a 0.1 mM solution.
[0391] Protocol
[0392] The protocol used was essentially that described by Sakai et
al. (A device for recording left ventricular contraction and
electrocardiogram in nonworking isolated perfused rat heart, Jpn J
Pharmacol 28:223-9, 1978), and Zelinski et al.,
(Phosphatidylcholine biosynthesis in isolated hamster heart, J
Biol. Chem. 255:11423-8, 1980) with the exception, in the latter
instance, that rat studies were used in the experiments described
herein instead of hamster hearts.
[0393] In brief, rats were anesthetized and their hearts were
excised. The isolated hearts were cannulated and attached to
Langendorff perfusion apparatus, and perfused with KH buffer. The
hearts were allowed to stabilize for 5-10 min. After stabilization,
the hearts should be paceable and drip rate should be between 5 and
10 mls per minute.
[0394] A small hole was cut in the left atrium of each animal to
expose the mitral valve. A deflated transducer balloon was
introduced into the left ventricle through the hole in left atrium.
The transducer tube was taped and adjusted so that there was no
pressure on the heart. Left ventricular pressure was measured and
recorded using the MacLab program. The transducer balloon was
filled by adjusting amount of water using a syringe attached to the
transducer via a stop-cock so that diastolic pressure was between 0
and 5 mm Hg. The hearts were allowed to stabilize for 10 min. Once
stabilized, the hearts should be paceable with a minimum of 100
beats per minute (bpm). The systolic pressure should be at least
about 80 mm Hg, resulting in a developed left ventricular pressure
of at least about 80 mm Hg.
[0395] The flow of KH buffer was switched to Gentamicin vehicle
solution using the stop-cock at the top of the canula. The
Gentamicin vehicle solution was allowed to recirculate for 30 min.,
after which time two 1.5 mL aliquots were collected and flash
frozen in liquid nitrogen.
[0396] Next, all perfusate was turned off for 40 min. in order to
begin global ischemia. The set up of the Langendorf apparatus was
adjusted so that the peristaltic pump was securely attached and all
the air was moved out of the line to begin reperfusion. The flow of
KH buffer was turned off after 40 min., and two 1.5 mL aliquots of
perfusate dripping from the heart were collected and flash frozen.
The heart was then reperfused for 45 min. Perfusate was collected
at 2 min. intervals and flash frozen.
[0397] After about 45 min., the perfusate was turned off and the
ventricular pressure was no longer recorded. The atria were cut
off, the pressure transducer was removed, and the ventricles were
frozen in a 50 mL conical tube.
[0398] The results, shown in FIG. 5, show that the neutral
sphingomyelinase inhibitor gentamicin mitigated ischemia and
reperfusion injury in the Langendorff rat heart model of
ischemia.
Example 13
HTS Screening for Thereaputic Agents That Inhibit
Sphingomyelinase
[0399] This Example describes an exemplary screening assay of the
invention that is used to screen for and identify compounds useful
in the therapeutic methods of the invention. The screening assay of
this Example, which is designed to isolate inhibitors of
sphingomyelinase (SMase) from a library of compounds based on the
structure of aminoglycosides, may be in used high throughput
screening (HTS) formats.
[0400] This Example describes a high-throughput screening (HTS)
scintillation proximity assay (SPA) designed to identify
aminoglyosidic inhibitors of sphingomyelinase (SMase). The HTS-SPA
of the Example involves high-volume, homogeneous radiometric
assays, based upon the principle of scintillation proximity.
Although assays of a single plate are described in this Example, it
is understood that many such plates would be prepared and tested in
high-throughput screening.
[0401] Sphingomyelinases
[0402] Sphingomyelinase (sphingomyelin choline phosphohydrolase)
(SMase) catalyzes the hydrolytic cleavage of sphimgomyelin (SM) via
reaction which lead to ceramide and phosphocholine generation
(Brady, R. O., Kanfer, J. N., Moek, M. B., and Fredrickson, D. S.
1966. The metabolism of sphingomyelin. II. Evidence of an enzymatic
deficiency in Niemann-1-Pick disease. Proc. Natl. Acad. Sci. USA
55:366). Any sphingomyelinase can be used in the HTS assays,
although mammalian SMases, particularly neutral SMases, are of
particular interest.
[0403] Several different types of mammalian SMases have been
identified, i.e.,
[0404] (1) Neutral, membrane-associated, Mg2+-stimulated SMases
found predominantly in brain and kidney (Spence, M.W. 1993.
Sphingomyelinases. Adv. Lipid Res. 26:3-23), which are known to
arise from a separate gene from lysosomal SMase (Gatt, S., Dinur,
T., and Kopolovic, J. 1978. Niemann Pick disease: presence of the
magnesium-dependent sphingomyelinase in brain of the infantile form
of the disease. J. Neurochem. 31:547-550). For review of neutral
SMases, see Chatterjee, "Neutral Sphingomyelinase", Advances in
Lipid Research, 26:25-49, 1993; and Chatterjee, "Neutral
sphingomyelinase: past, present and future", Chemistry and Physics
of Lipds, 102: 79-96, 1999. At least two structurally different
types of mammalian neutral SMase (nSMase) have been cloned:
[0405] (a) The nSMase described by Stoffel et al., i.e., a cloned
human neutral SMase described in Tomiuk et al., Cloned mammalian
neutral sphingomyelinase: Functions in sphingolipid signaling?
Proc. Natl. Acad. Sci. (U.S.A.) 95: 3638-3643; Hofman et al.,
Cloning and characterization of the mammalian brain-specific,
Mg2+-dependent neutral sphingomyelinase Proc. Natl. Acad. Sci.
(U.S.A.) 97:5895-5900,2000; and published PCT application WO
99/07855 to Stofel et al., entitled "Neutral Sphingomyelinase," was
filed Aug. 11, 1998 and was published on Feb. 18, 1999.
[0406] (b) The nSMase described in U.S. Pat. No. 5,919,687 to
Chatterjee, entitled "Recombinant N-SMases and Nucleic Acids
Encoding Same", Published PCT application WO 98/28445 to
Chatterjee, entitled "Recombinant N-SMases and Nucleic Acids
Encoding Same", and Chatterjee, Molecular Cloning,
Characterization, and Expression of a Novel Human Neutral
Sphingomyelinase, J. Biol. Chem. 274:37407-37412, 1999). In the
latter reference, the nSMase of Chatterjee is stated to be
unrelated to the nSMase of Stoffel et al. as the two nSMases have
different amino acid sequences (p. 37412, left column, 11.
33-34).
[0407] (2) Lysosomal SMases that act optimally at low pH and show
no dependence on divalent cations (Kanfer, J. N., Young, O.,
Shapiro, D., and Brady, R. O. 1966. The metabolism of
sphingomyelin. I. Purification and properties of a
sphingomyelin-cleaving enzyme from rat liver tissue. J. Biol. Chem.
241:1081; Levade, T., Salvayre, R., and Blazy-Douste, L. 1986.
Sphingomyelinases and Niemann-Pick disease. J. Clin. Chem. Biochem.
24:205-220);
[0408] (3) Acidic, Zn2+-stimulated SMases present in fetal bovine
serum and to a lesser degree in newborn human serum (Spence, M. W.,
Byers, D. M., Palmer, F. B. St. C., and Cook, H. W. 1989. A new
Zn2+-stimulated sphingomyelinase in fetal bovine serum. J. Biol.
Chem. 264:5358-5363), and also secreted by several human cell types
during several pathophysiological processes (Schissel, S. L.,
Shuchman, E. H., Williams, K. J., and Tabas, l. 1996.
Zn2+-stimulated sphingomyelinase is secreted by many cell types and
is a product of the acid sphingomyelinase gene. J. Biol. Chem.
271:18431-18436);
[0409] (4) Cytosolic SMases that, like Mg2+-dependent neutral
SMases, have a neutral pH optimum but no dependence on divalent
cations (Okazaki, T., Bielawska, A., Domae, N., Bell, R. M., and
Hannun, Y. A. 1994. Characteristics and partial purification of a
novel cytosolic magnesium-independent, neutral sphingomyelinase
activated in the early signal transduction of
1a,25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J.
Biol. Chem. 269:4070-4077).
[0410] (5) Placental SMases (Garcia-Ruiz, Human placenta
sphingomyelinase, an exogenous acidic pH-optimum sphingomyelinase,
induces oxidative stress, glutathione depletion, and apoptosis in
rat hepatocytes, Hepatology 32:56-65, 2000).
[0411] (6) Brain-specific SMases have been described (Yamanaka et
al., J. Neurochem. 38:1753-1764, 1982); Hofmann et al., "Cloning
and characterization of the mammalian brain-specific,
Mg.sup.2+-dependent neutral sphingomyelinase", PNAS, 97:5895-5900,
2000; Bernardo et al., "Purification and Characterization of a
Magnesium-dependent Neutral Sphingomyelinase from Bovine Brain",
The Journal of Biological Chemistry, 275:7641-7647, 2000; Liu et
al., "Purification and Characterization of a Membrane Bound Neutral
pH Optimum Magnesium-dependent and Phosphatidylserine-stimulated
Sphingomyelinase from Rat Brain", The Journal of Biological
Chemistry, 273:34472-34479, 1998. A rat brain SMase is commercially
available (MDS Panlabs).
[0412] SMases from Non-Mammalian Species Include:
[0413] (1) Bacterial SMases, such as the well-characterized
Phospholipase C from Bacillus cereus (Ikezawa et al., Studies on
sphingomyelinase of Bacillus cereus. I. Purification and
properties, Biochim Biophys Acta 1978 Feb. 27;528(2):247-56;
Hetland et al., Phospholipase C from Bacillus cereus has
sphingomyelinase activity, Scand J Clin Lab Invest 1982
February;42(1):57-61; Gilmore et al., A Bacillus cereus cytolytic
determinant, cereolysin AB, which comprises the phospholipase C and
sphingomyelinase genes: nucleotide sequence and genetic linkage, J
Bacteriol 1989 February;171(2):744-53; Yamada et al., Nucleotide
sequence and expression in Escherichia coli of the gene coding for
sphingomyelinase of Bacillus cereus, Eur J Biochem 1988 Aug.
1;175(2):213-20; Johansen et al., Bacillus cereus strain SE-1:
nucleotide sequence of the sphingomyelinase C gene, Nucleic Acids
Res. 16:103770, 1998; Fujii et al., Mg2+ binding and catalytic
function of sphingomyelinase from Bacillus cereus, J Biochem
(Tokyo) 124:1178-1187, 1998; Gavrilenko et al., Nucleotide sequence
of phospholipase C and sphingomyelinase genes from Bacillus cereus
BKM-B164, Bioorg Khim 19:133-138, 1993; Tomita et al., Secondary
structure of sphingomyelinase from Bacillus cereus, J Biochem
(Tokyo) 108:811-815, 1990; and Tamura et al., Mass production of
sphingomyelinase of Bacillus cereus by a protein-hyperproducing
strain, Bacillus brevis 47, and its purification, J Biochem (Tokyo)
112:488-491, 1992).
[0414] Other bacterial SMases are known and include, by way of
non-limiting examples, those from Helicobacter pylori (Chan et al.,
Purification and characterization of neutral sphingomyelinase from
Helicobacter pylori, Biochemistry 39:4838-4845, 2000; Lin et al.,
Identification of neutral and acidic sphingomyelinases in
Helicobacter pylori, FEBS Lett 423:249-253, 1998); Listeria
ivanovii (Gonzalez-Zorn et al., The smcL gene of Listeria ivanovii
encodes a sphingomyelinase C that mediates bacterial escape from
the phagocytic vacuole, Mol Microbiol 33:510-523, 1999);
Staphylococcus aureus (Walev et al., Selective killing of human
monocytes and cytokine release provoked by sphingomyelinase
(beta-toxin) of Staphylococcus aureus, Infect Immun 64:2974-2979,
1996); and Clostridium perfringens (Saint-Joanis et al., Gene
cloning shows the alpha-toxin of Clostridium perfringens to contain
both sphingomyelinase and lecithinase activities, Mol Gen Genet
1989 November;219(3):453-60).
[0415] (2) Arachnoid SMases, such as the cytotoxic sphingomyelinase
D from the brown recluse spider (Loxosceles reclusa) (Gates et al.,
Serum amyloid p component: its role in platelet activation
stimulated by sphingomyelinase d purified from the venom of the
brown recluse spider (Loxosceles reclusa). Toxicon 28: 1303-1315,
1990).
[0416] (3) SMase homologs, such as ISC1 (YER019w) of Saccharomyces
cerevisiae (Sawai et al., Identification of ISC1 (YER019w) as
Inositol Phosphosphingolipid Phospholipase C in Saccharomyces
cerevisiae, J Biol Chem 275:39793-39798, 2000), which may have
SMase activity in vitro even though sphingomyelin is not present in
cells of the organism from which the SMase homolog is prepared.
That is, an SMase homolog may act on substrates other than
sphingomylein in vivo but nevertheless have bona fide SMase
activity in vitro.
[0417] Other SMases that may be used to practice the invention
include without limitation Romiti et al., "Characterization of
sphingomyelinase activity released by thrombin-stimulated
platelets", Molecular and Cellular Biochemistry, 205:75-81, 200;
Sawai et al., "Function of the Cloned Putative Neutral
Sphingomyelinase as Lyso-platelet Activating Factor-Phospholipase
C", The Journal of Biological Chemistry, Vol. 274, No. 53, Dec. 31,
1999, pp. 38131-38139; Fensome et al., "A Neutral
Magnesium-dependent Sphingomyelinase Isoform Associated with
Intracellular Membranes and Reversibly Inhibited by Reactive Oxygen
Species", The Journal of Biological Chemistry, 275:1128-1136, 2000;
Holopainen et al., "Sphingomyelinase Activity Associated with Human
Plasma Low Density Lipoprotein", The Journal of Biological
Chemistry, 275:16484-16489, 2000; and Hinkovska-Glacheva et al.,
"Activation of a Plasma Membrane-Associated Neutral
Sphingomyelinase and Concomitant Ceramide Accumulation During
IgC-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes",
Blood, 91:4761-4769, 1998.
[0418] Chemical Libraries Based on Aminoglycoside Structures
[0419] Combinatorial chemical libraries that are used in this
screening assay are preferably "biased" in the sense that they are
prepared using the basic aminoglycoside structure as a framework to
produce aminoglyosidic molecules having a multitude of positions
where alternate "R" groups may be incorporated. The structure shown
in FIG. 6, which has 13 "R" groups, is used. In the following
naturally occuring aminoglycoside antibiotics, the structure shown
in FIG. 6 has the following R groups that may be substituted; in
all of these compounds, R11 is NH2, and R12 is OH, and R13 is
OH.
5 Aminoglycoside R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 Kanamycin A OH OH
OH H NH2 H CH2OH OH H H Kanamycin B NH2 OH OH H NH2 H CH2OH OH H
CH3 Kanamycin C NH2 OH OH H OH H CH2OH OH H H Gentamicin C1 NH2 H H
NHCH3 NHCH3 H H CH3 OH CH3 Gentamicin C1a NH2 H H NH2 NH2 H H CH3
OH CH3 Gentamicin C2 NH2 H H NH2 NH2 H H CH3 OH CH3 Gentamicin C26
NH2 H H NHCH3 NHCH3 H H CH3 OH CH3 Gentamicin B NH2 H H NH2 NH2 H H
CH3 OH CH3 Tobramycin NH2 H OH H NH2 H H OH H H Dibekacin NH2 H H H
NH2 H H OH H H Aberkacin NH2 H H H NH2 COR' COR' OH H H Isepamicin
OH OH OH H NH2 COR COR CH3 OH CH3
[0420] A combinatorial library of aminoglycosides and
aminoglycoside derivatives is prepared using methods discussed in
Sucheck et al. (Combinatorial synthesis of aminoglycoside
libraries, Curr Opin Drug Discov Devel 4:462-70, 2001); Hofstadler
et al., (Multiplexed screening of neutral mass-tagged RNA targets
against ligand libraries with electrospray ionization FTICR MS: a
paradigm for high-throughput affinity screening, Anal Chem
71:3436-40, 1999), and references cited therein.
[0421] Pre-Assay Studies
[0422] A FlashPlate.RTM. microtiter plate (NEN Life Science
Products, Boston, Mass.) is used in the assay. The interior of each
well of the microtiter plate is permanently coated with a thin
layer of polystyrene-based scintillant that produces a signal when
the surface of the well is in close proximity to any of a variety
of isotopes (e.g., 3H, 125I, 14C and 33P). Because the scintillant
is permanently bound to the wells of the plate, a liquid
scintillation cocktail does not need to be added to the wells
during the assay.
[0423] A radiolabeled substrate for SMase is coated or bound onto
the surface of wells in a FlashPlate. The radioactive decay
associated with the radiolabeled causes a microplate surface
scintillation effect detectable on a microplate scintillation
counter. Radiolabel that is released from the radiolabeled
substrate by the enzymatic activity of SMase does not activate the
scintillant. Thus, after SMase is added, the signal from the
radiolabel decreases over time. If an inhibitor of SMase is present
in a well, the radiolabel is released at a lower rate, and the
scintillant-mediated signal thus decreases at a slower rate.
[0424] In the present Example, the radiolabelled SMase substrate
that is coated onto the surface of wells in a FlashPlate is
[3H]sphingomyelin (NEN Life Sciences). Before HTS is begun, studies
are done to determine the optimal means and conditions for coating
the wells with the substrate. Aliquots of [3H]sphingomyelin, having
concentrations ranging from 0.1 to 10 u Ci/ml, are prepared in a
constant volume of Tris buffer. The aliquots are added at 0.2
ml/well at room temperature and the signal from the coated
microtiter plates is read at regular intervals using a TopCount NXT
Microplate Scintillation and Luminescence Counter (Packard
BioScience). Two parameters are optimized in these studies. First,
the time at which the maximum counts/well is achieved before the
signal hits a plateau; typically, this occurs after 10 hours.
Second, the range of [3H]sphingomyelin that can be added to the
wells and still exhibit a linear increase in signal; typically, 0.2
.mu.Ci/well is used.
[0425] Next, studies are done to determine the amount of time
needed to hydrolyze the maximum amount of substrate at a chosen
concentration. A plate is coated using the optimized conditions
described above, i.e., 0.2 .mu.Ci/well with 10 hours of pre-assay
incubation. The SMase being used in the assay is added (0.2 u/ml,
diluted in PBS with 1 mg/ml CaCl2 and 1 mg/ml MgCl12). The plate is
incubated at room temperature and counts are read at regular time
points over a period of 24 hours. The hydrolysis of the substrate
occurs rapidly at first before reaching a plateau, typically after
about the first 3 hours of incubation.
[0426] In these initial studies, and in plates used in HTS, several
control wells are used. In one type of control, commerically
available (Sigma) SMases are added to control wells to confirm the
activity of the assay, including bacterial SMases (from
Staphylococcus aureus or Bacillus cereus) or a human placental
SMase. If the target SMase is dependent on a divalent cation, a
chelator such as EDTA is added to one or more control wells.
[0427] HTS Assays
[0428] A target SMase of interest is prepared by recombinant DNA
technology. The target SMases in this Example are mammalian neutral
SMases. The nSMase described by Chatterjee (U.S. Pat. No.
5,919,687) and the nSMase described by Stoffel et al. (WO 98/28445)
are both tested because they have unrelated amino acid sequences,
and either or both may be involved in sphingolipid metabolism
and/or signaling in cardiovascular tissues.
[0429] In the HTS-SPA, many microtiter plates are prepared as
described above. In non-control wells, a member of the
aminoglycoside chemical library is added to each well, followed by
the addition of the target SMase, and the signals from the wells of
the plates are read. When large numbers of plates are to be
screened, an external plate stacker can be added to the TopCount
NXT Microplate Scintillation and Luminescence Counter, which allows
for more than 15,000 samples to be loaded and tested unattended.
Wells in which the signal from the unhydrolyzed substrate does not
decrease, or decreases less rapidly than in a control well
comprising the target SMase only, identify members of the library
that are candidate inhibitors of the target SMase. The activity of
these candidate compounds is confirmed by repeated testing.
[0430] The compounds are further characterized in terms of other
desirable attributes. For example, the safety and efficacy of the
compositions and methods for sphingolipid-based cardiovascular
therapy are initially evaluated in cell culture (e.g., cultured
cardiomyocytes) and animal models. Non-limiting examples of such
animal models include rat and pig models of infarction (Olivetti et
al., Cardioscience 6:101-106, 1995; Jacobs et al., J. Mol. Cell
Cardiol. 31:1949-1959, 1999; Gunther et al., Eur. J. Pharma.
406:123-126, 2000; and Holmes et al., Circulation 90:411-420,
1994).
Example 14
Modulation of the Sphingomyelin Signaling Pathway
[0431] The sphingomyelin signaling pathway (a.k.a. the SM pathway)
is a "cascade" of biochemical events in which proteins in the
pathway are activated (by enzymatic chemical modification or
otherwise) with the end result that sphingosine metabolism is
affected. In most instances, activation of the SM pathway leads to
increased production of ceramide. For reviews of the molecular
biology of the sphingomyelin signaling pathway, see Hannun et al.,
Adv. Lipid Res. 25:27-41, 1993; Liu et al., Crit. Rev. Clin. Lab.
Sci. 36:511-573, 1999; Igarashi, Y., J. Biochem. 122:1080-1087,
1997; and Oral et al., J. Biol. Chem. 272:4836-4842, 1997.
[0432] It has suggested that the sphingomyelin signal transduction
pathway is activated during cardiac ischemia/hypoxia (Bielawska et
al., Am. J. Pathol. 151:1257-1263, 1997; Meldrum, Am. J. Pathol.
274:577-595, 1998; and Cain et al., J. Mol. Cell. Cardiol.
31:931-947, 1999). If so, there must be a factor or process that
mediates the ischemia-induced SPH production. The most likely
candidate for the mediator is the pro-inflammatory cytokine, tumor
necrosis factor alpha (TNF.alpha.). In various animal models of
ischemia, the myocardium produces TNF.alpha. (Squadrito et al.,
Eur. J. Pharmacol. 237:223-230, 1993; Herrmann et al., European
Journal of Clinical Investigation 28:59-66, 1998; Meldrum, Ann.
Thorac. Surg. 65:439-443, 1998). Recent evidence demonstrates that
the cardiomyocytes themselves produce TNF.alpha. and secrete the
cytokine into the extracellular fluid (Comstock et al., J. Mol.
Cell Cardiol. 30:2761-2775, 1998). Since TNF.alpha. receptors are
expressed by cardiomyocytes (Krown et al., FEBS Letters 376:24-30,
1995; Torre-Amione et al., Circulation 92:1487-1493, 1995), an
autocrine/paracrine role for TNF.alpha. has been suggested
(Meldrum, Ann. Thorac. Surg. 65:439-443, 1998). Significantly,
TNF.alpha. induces SPH production and apoptosis in cardiac myocytes
(Krown et al., J. Clin. Invest. 98:2854-2865, 1996), presumably by
acting by binding to the cardiomyocyte complement of TNF.alpha.
receptors.
[0433] Activation of the sphingomyelin signal transduction cascade
is a key early event in the cytotoxic (apoptotic) effects of the
cytokine TNF.alpha. (Zhang et al., Endo. 136:4157-4160, 1995).
TNF.alpha. can cause significant apoptosis in cultured rat
cardiomyocytes and it has been suggested that TNF.alpha.-induced
SPH production is responsible for the cell death triggered by
TNF.alpha. (Krown et al., J. Clin. Invest. 98:2854-2865, 1996).
[0434] Inhibitors of Cytokines that Activate the Sphingomyelin
Signaling Pathway
[0435] The SM pathway, many steps of which occur intracellularly,
is induced by a variety of extracellular stimuli. In
sphingolipid-based cardiovascular therapy, such stimuli are at
least partially blocked. SM pathway-inducing agents that are
desirably interfered with include but are not limited to cytokines.
Cytokines of particular interest include but are not limited to
pro-inflammatory cytokines, interferons and chemokines.
Pro-inflammatory cytokines of particular interest include but are
not limited to TNF-alpha; interleukins such as IL-1beta, IL-2,
IL-10, interferons of particular interest include but are not
limited to gamma-IFN. Chemokines of particular interest are those
involved in the ischemic process including but not limited to
interleukin-8 (IL-8), and the monocyte chemotaxic proteins MCP-1
and MCP-2. One non-limiting example of an agent that may be used to
modulate the SM pathway (in particular, stroke-induced apoptosis)
is the immunosuppresant FK506 (Herr et al., Brain Res. 826:210-219,
1999).
[0436] Agents that Block Cytokine-Induced Activation of the SM
Pathway
[0437] Sphingolipid-based cardiac therapeutic agents that are used
to inhibit the actions of cytokines include but are not limited to
an antibody directed to a cytokine or to a cytokine receptor; a
cytokine receptor fragment that binds a cytokine but is otherwise
biologically inactive; and a cytokine analog that binds cytokine
receptor but is otherwise biologically inactive.
[0438] As one non-limiting example of this aspect of the invention,
an antibody directed to a cytokine or a cytokine receptor is used
as a therapeutic agent in sphingolipid-based cardiovascular
therapy. Such antibodies are generated in an immunoreactive
response to a cytokine, a cytokine receptor, or a synthetic
polypeptide derived therefrom. A preferred type of antibody is a
monoclonal antibody, which is initially isolated from a hybridoma;
more preferred is a monoclonal antibody that has been "humanized"
via molecular genetic manipulation. Also preferred are fragments,
preferably soluble, which are derived from antibodies to a cytokine
and retain the ability to bind a cytokine, such as, e.g.,
single-chain Fv analogs (scFv). The isolation, production,
humanization and derivatization of antibodies is described in
Ramnarayan et al., Am. Biotechnol. Lab.:26-28, 1995; Gavilondo et
al., BioTechniques 29:128-145, 2000; Kling, J., Modern Drug
Discovery 2:33-45, 1999; Morrow, K. J. Jr., American Laboratory
32:15-19, 2000; Huston et al., Methods in Enzymology 203:46-88,
1991; Johnson et al., Methods in Enzymology 203:88-98, 1991; Gussow
et al., Methods in Enzymology 203:99-121, 1991; and references
cited therein.
[0439] A preferred antibody that is used as a therapeutic agent in
sphingolipid-based cardiovascular therapy is one that blocks the
binding of a cytokine to its receptor. Assays for determining the
degree of inhibition of binding of a cytokine to its receptor (see,
e.g., Murata et al., Anal. Biochem. 282:11-120, 2000) are used in
initial assessments of the effectiveness of such antibodies.
[0440] As another non-limiting example of this aspect of the
invention, a receptor fragment that binds a cytokine but is
otherwise biologically inactive is used as a therapeutic agent in
sphingolipid-based cardiovascular therapy. For example, therapeutic
inhibition of the SM pathway is achieved by blocking the binding of
extracellular TNF-alpha to a cellular receptor (TNFR); this in turn
prevents the activation of the SM pathway. The binding of TNF-alpha
to a TNFR is directly or competitively inhibited. One example of an
agent for competitive inhibition of the binding is a soluble
TNF-alpha receptor fragment such as TNRFII:Fc. TNFRII:Fc receptor
fragment, which is sold as Embrel.sup.R (from Immunex Corporation,
Seattle, Wash.). This soluble fusion protein was made from the
extracellular binding domain of the TNF type II receptor and an
iimmunoglobulin Fc portion of IgG1. This soluble fusion protein has
a very high affinity for TNF (Im et al., J. Biol. Chem
275:14281-14286, 2000).
[0441] As another non-limiting example of this aspect of the
invention, small molecules that serve as inhibitors and/or
antagonists of sphingolipid receptors are used as therapeutic
agents in sphingolipid-based cardiovascular therapy. Such molecules
include sumarin, which blocks EDG-3 action (Mandala et al., Proc.
Natl. Acad. Sci. U.S.A. 97:7859-7864, 2000), and pertusis toxin,
which blocks EDG receptors that use Gi (Gonda et al., Biochem. J.
337:67-75, 1999).
[0442] In addition to TNF and Fas, other examples of extracellular
agents that activate the SM pathway by interacting with their
receptors include but are not limited to Fas and the Fas receptor
(Brenner et al., J. Biol. Chem. 272:22173-22181, 1997; and nerve
growth factor (NGF) and the p75 neutrotrophin receptor (p75NTR)
(Dobrowsky et al., Science 265:1596-1599, 1994).
[0443] Other Agents Directed to the Sphingomyelin Signaling
Pathway
[0444] Sphingolipid-based cardiovascular therapy is also achieved
through the use of compounds that bind sphingolipid receptors that
initiate and stimulate the sphingomyelin signaling pathway. This
pathway ultimately results in increased ceramide production. An
increased level of ceramide would, in turn, be expected to result
in elevated concentrations of undesirable sphingolipids such as,
e.g., S-1-P and SPH. Such receptor-binding agents may be, by way on
non-limiting example, antibodies or antibody fragments, small
(organic) molecules, or sphingolipid derivatives that bind the
receptors but do active the SMA signaling pathway. The synthesis of
representative sphingolipid derivatives are described in, by way of
non-limiting example, PCT published patent application WO 99/12890;
U.S. Pat. Nos. 5,663,404 and 6,051,598, and U.S. Pat. Nos.
5,260,288 and 5,391,800.
[0445] In other instances, as is explained in detailed elsewhere
herein, components of the SM pathway are used to create therapeutic
proteins that retain the ability to bind sphingolipids but are
otherwise biologically inactive. Moreover, various steps in the SM
pathway are specifically inhibited by dominant-negative derivatives
of proteins involved in a particular step in the cascade, antisense
molecules and constructs, and gene therapeutics.
Example 15
Sphingolipid-Binding Protein Derivatives
[0446] Sphingolipid-binding protein derivatives are used for
sphingolipid-based cardiovascular therapy in one aspect of the
invention. Such protein derivatives retain the ability to bind
sphingolipids, even if other functions, biochemical activities
and/or characteristics of the protein are altered, compromised or
absent in the protein derivative. Protein derivatives may be
oligopeptides synthesized in vitro, proteins that have been
purified from an animal and chemically or otherwise modified,
proteins produced via recombinant DNA technology, or combinations
thereof. Non-limiting examples of sphingolipid-binding protein
derivatives include enzyme derivatives, and receptor derivatives. A
sphingolipid-binding enzyme derivative can be, for example, a
noncatalytic derivative of an enzyme involved in sphingolipid
metabolism that retains the ability to bind sphingolipids. A
sphingolipid binding receptor derivative can be, for example, a
soluble derivative of a membrane-bound sphingolipid receptor that
binds sphingolipids, e.g., soluble derivatives of a member of the
EDG or SCaMPER family of receptors. A "sphingolipid-binding protein
derivative" may also be an antibody or antibody derivative;
"antibody derivative proteins" are antibody fragments that retain
the ability to specifically bind sphingolipids. Such antibody
fragments are, by way of non-limiting example, single-chain FV
analogs (scFv's), complementarity-determining regions (CDR's), and
the like, and fusion proteins comprising such antibody fragments.
See Gavilondo et al., BioTechniques 29:128-145, 2000; and Verma et
al., Journal of Immunological Methods 216:165-181, 1998.
[0447] Such sphingolipid-binding protein derivatives can be, but
need not be, derived from an enzyme or receptor from the animal
that is intended to be treated. Such derivatives may be prepared
from homologous enzymes or receptors from a non-human mammal (e.g.,
a feline SMase derivative), or from analogous enzymes or receptors
from an organism that belongs to a different biological Family,
Order, Class or Kingdom (e.g., an arachnid or bacterial SMase).
[0448] Sphingolipid-Binding Enzyme Derivatives
[0449] Enzymes from which biologically inactive (non-catalytic)
sphingolipid-binding derivatives are obtained include but are not
limited to the following. Such derivatives of these enzymes bind
their substrate, which is a undesirable, toxic and/or cardiotoxic
sphingolipid, and thereby lower the actual or available
concentration of the sphingolipid, and/or render the sphingolipid
biologically inactive with respect to its cardiotoxic effects.
Preferably, such derivatives are soluble and may be formulated into
a pharmaceutical composition suitable for sphingolipid-based
cardiovascular therapy.
[0450] S-1-P is bound by enzymes having S-1-P as a substrate, such
as, by way of non-limiting example, S-1-P lyasc and S-1-P
phosphatase. Non-catalytic derivatives of these enzymes bind S-1-P
and interfere with its harmful effects.
[0451] SPH is bound by enzymes having SPH as a substrate, such as,
by way of non-limiting example, SPH Kinase and Ceramide synthase.
Non-catalytic derivatives of such enzymes bind SPH and interfere
with its harmful effects.
[0452] Ceramide is bound by enzymes having ceramide as a substrate,
such as, by way of non-limiting example, ceramidase, SM synthase,
ceramide kinase, and glucosylceramide synthase. Non-catalytic
derivatives of such enzymes bind ceramide and interfere with the
harmful effects of its metabolites, such as, in particular, SPH and
S-1-P.
[0453] Sphingomyelin is bound by enzymes having sphingomyelin as a
substrate, such as, by way of non-limiting example, SMase.
Non-catalytic derivatives of such enzymes bind sphingomyelin and
interfere with the harmful effects of its metabolites such as,
e.g., ceramide, SPH and S-1-P.
[0454] Sphingolipid-Binding Receptor Derivatives
[0455] Receptors from which biologically inactive (e.g., non-signal
transducing) sphingolipid-binding derivatives are obtained include
but are not limited to the following. Such derivatives of these
receptors bind their ligand, which is a cardiotoxic sphingolipid,
and thereby lower the actual or available concentration of the
sphingolipid, and/or render the sphingolipid biologically inactive
with respect to its cardiotoxic effects. Preferably, such
derivatives are soluble and may be formulated into a pharmaceutical
composition suitable for sphingolipid-based cardiovascular
therapy.
[0456] EDG Receptors
[0457] Receptors that bind S-1-P are used in this aspect of the
invention (for a review of some S-1-P-binding receptors, see
Spiegel et al., Biochim. Biophys. Acta 1484:107-116, 2000).
[0458] EDG-1 was the first identified member of a class of G
protein-coupled endothelial-derived receptors (EDG). Such receptors
include but are not limited to members of the EDG family of
receptors (a.k.a. IpA receptors, Chun, Crit. Rev. Neuro.
13:151-168, 1999), and isoforms and homologs thereof such as NRG 1
and AGR16.
[0459] For reviews, see Goctzl et al., Adv. Exp. Med. Biol.
469:259-264, 1999; Chun et al., Cell. Biochem. Biophys. 30:213-242,
1999); Sato, "A new role of lipid receptors in vascular and cardiac
morphogenesis", The Journal of Clinical Investigation, 6:939-940,
2000.
[0460] EDG1 is described by Lee et al, (Ann. NY Acad. Sci.
845:19-31, 1998). Human EDG-1c genes and proteins are described in
published PCT application WO 99/46277 to Bergsma et al. Described
in Au-Young, et al., U.S. Pat. No. 5,912,144, issued Jun. 15, 1999,
"EDG-1 Receptor Homolog"; Bergsma et al., WO 97/46277, published
Sep. 16, 1999, "Human EDG-1c Polynucleotides and Polypeptides and
Methods of Use"; and Okamoto et al., "EDG1 Is a Functional
Sphingosine-1-Phosphate Receptor That Is Linked via a G.sub.i/o to
Multiple Signaling Pathways, Including Phospolipase C Activation,
Ca.sup.2+ Mobilization, Ras-Mitogen-activated Protein Kinase
Activation, and Adenylate Cyclase Inhibition", The Journal of
Biological Chemistry, 273:27104-27110, 1998.
[0461] EDG-3 is described by Okamoto et al. (Biochem. Biophys. Res.
Commun. 260:203-208, 1999) and An et al. (FEBS Letts. 417:279-282,
1997). See also An et al., J. Biol. Chem. 275:288-296, 2000; EDG-3
(a.k.a. LP(B4)); Tsui, U.S. Pat. No. 6,130,067, issued Oct. 10,
2000, "Human EDG3SB Gene"; and Siehler et al., "Sphingosine
1-Phosphate Activates Nuclear Factor-.eta.B through Edg Receptors:
Activation Through Edg-3 and Edg-5, but not Edg-1, in Human
Embryonic Kidney 293 Cells," JBC Papers in Press Published Oct. 22,
2001 as Manuscript M01107220.
[0462] EDG-5 human and mammalian genes are described in U.S. Pat.
No. 6,057,126 to Munroe el at. and published PCT application WO
99/33972 to Munroe et al. The rat homolog, H218 (a.k.a. ARG16) is
described in U.S. Pat. No. 5,585,476 to MacLennan et al. Van
Brocklyn et al., J. Biol. Chem. 274:4626-4632, 1999; and Gonda et
al., Biochem. J. 337:67-75, 1999. See also An et al., J. Biol.
Chem. 275:288-296, 2000.
[0463] EDG-6 is described by Graler et al. (Genomics 53:164-169,
1998) and Yamazaki et al. (Biochem. Biophys. Res. Commun.
268:583-589, 2000).
[0464] EDG-8 from rat brain is described by Im et al., (J. Biol.
Chem. 275:14281-14286, 2000). Homologs of EDG-8 from other species,
including humans, may also be used.
[0465] The Mil Receptor (Mil is an abbreviation for "miles apart")
binds S-1-P and regulates cell migration during vertebrate heart
development. The Mil receptor of Zebrafish is described by Mohler
et al. (J. Immunol. 151:1548-1561, 1993). Another S-1-P receptor is
NRG1 (nerve growth factor regulated gene-1), the rat version of
which has been identified (Glickman et al., Mol. Cel. Neurosci.
14:141-152, 1999).
[0466] Receptors that bind SPC are also used in this aspect of the
invention. Such receptors include but are not limited to members of
the SCaMPER family of receptors (Mao et al., Proc. Natl. Acad. Sci.
U.S.A. 93:1993-1996, 1996; Betto et al., Biochem. J. 322:327-333,
1997), and ovarian cancer G-protein-coupled receptor 1; Xu et al.,
"Sphingosylphopsphorylcoline is a ligand for ovarian cancer
G-protein-coupled receptor 1", Nature Cell Biology, 2:261-267,
2000.
[0467] Some evidence suggests that EDG-3 may bind SPC in addition
to S-1-P (Okamoto et al., Biochem. Biophys. Res. Commun.
260:203-208, 1999). Derivatives of EDG-3 that bind both S-1-P and
SPC are used in one aspect of the invention. Also usuable is the
receptor described in Ames et al., WO 01/04139 A2, published Jan.
18, 2001, "Polynucleotide and Polypeptide Sequences of Human AXOR29
Receptor and Methods of Screening for Agonists and Antagonists of
the Interaction Between Human AXOR29 Receptor and its Ligands".
[0468] Receptors that bind LPA (lysophosphatic acid) are alos used
in this aspect of the invention. LPA receptors are described by Im
et al., "Molecular Cloning and Characterization of a
Lysophosphatidic Acid Recetpor, Edg-7, Expressed in Prostate",
Molecular Pharmacology, 57:753-759, 2000; An et al.,
"Characterization of a Novel Subtype of Human G Protein-coupled
Receptor for Lysophosphotatidic Acid", The Journal of Biological
Chemistry, 273:7906-7910, 1998; Fukushima et al., "A single
receptor encoded by vzg-1/lp.sub.A/edg-2 couples to G proteins and
mediates multiple cellular responses to lysophosphatidic acid",
Proc. Natl. Acad. Sci., 95:6151-6156, 1998; Chun et al., "U.S. Pat.
No. 6,140,060 issued Oct. 31, 2000, "Closed Lysophosphatidic Acid
Receptors"; and Kimura et al., "Two Novel Xenopus Homologs of
Mammalian LPEDG-2 Function as Lysophosphatidic Acid Receptor
Xenopus Oocytes and Mammalian Cells", JBC Papers in Press Published
on-line Feb. 5, 2001 as Manuscript M011588200.
[0469] Soluble receptor fragments are derivatives of membrane-bound
receptors in which the transmembrane portions of the receptor have
been removed. Receptor-derived polypeptides that are soluble, i.e.,
have lost their transmembrane portion, and which retain their
ability to bind the receptor substrate, are solube receptor
fragments that may have therapeutic value as agents that bind
undesirabel sphingolipids. Studies that have identified portions of
Edg receptors that bind to S-1-P are helpful as guides in designing
soluble receptor fragments (Parrill et al., Identification of Edg1
Receptor Residues That Recognize Sphingosine 1-Phosphate, The
Journal of Biological Chemistry, 275:39379-39384, 2000; and Wang et
al., A Single Amino Acid Determines Lysophospholipid Specificity of
the S1 P.sub.1(EDG1) and LPA, (EDG2) Phospholipid Growth Factor
Receptors, JBC Papers in Press Published on-line Oct. 16, 2001 as
Manuscript M107301200.
[0470] Sphingolipid-Binding Proteins from Natural Sources
[0471] Sphingolipid-binding proteins that may be used in the
invention include those isolated from natural sources. A
non-limiting example of such a protein is lysenin, a protein that
binds sphingomyelin, which has been prepared from the earthworm
Eisenia foetida (Yamaji et al., J. Biol. Chem. 273:5300-5306,
1998).
Example 16
Cloning of Rat and Human Scamper Genes
[0472] The rat and human SCaMPER genes were obtained using a
combination of reverse transcription and polymerase chain reactions
(RT-PCR) as is known in the art (see, for example, PCR Technology:
Principles and Applications for DNA Amplification, H. A. Erlich,
cd., 1989).
[0473] Tissue was prepared from various human and rat sources as
follows. Human heart tissue from an expired heart failure patient
was collected, frozen in liquid nitrogen and stored at -70.degree.
C. Rat heart and skeletal tissue was freshly obtained from
sacrificed adult Sprague-Dawley rats. RNA from these tissues was
isolated using the commercially available Qiagen Rneasy Mini Kit
according to the manufacturer's protocols (Qiagen, Valencia,
Calif.).
[0474] PCR amplification of the target sequences was performed
using the commercially available Qiagen One-Step RT-PCR kit
according to the manufacturer's protocols. Primers sets
included:
[0475] 5'-CCAGGATTCATCATATGTTAAAAG-3' (upper) (SEQ ID NO.:1);
and
[0476] 5'-ATCAGTGGGTGCATCAGTAGC-3' (lower) (SEQ ID NO.:2) for the
open reading frame (ORF) and the 3'-sequence of SCaMPER (designed
from GenBank accession number U33628).
[0477] Amplification utilized cycling regimens according to the
manufacturer's recommendations. Briefly, reactions were optimized
using 40 PCR cycles, each cycle consisting of 45 seconds
denaturation at 95.degree. C., 45 seconds of annealing at
50.degree. C., and 1 minute of polymerization at 72.degree. C. The
resulting PCR products were subcloned into pCR3.1 TOPO or
pcDNA3.1-V5/HIS TOPO (Invitrogen, Carlsbad, Calif.) using standard
techniques. Sequencing with a T7 primer was performed at the San
Diego State University DNA Core Facility (San Diego, Calif.). The
resultant SCaMPER sequences are presented as SEQ ID NO.: 3, the
open reading frame of the rat SCaMPER gene and SEQ ID NO.: 4, the
open reading frame of the the human SCaMPER gene.
6 Rat SCaMPER ORF (SEQ ID NO.: 3) 001 ATGTTAAAAG TGAGCAGGGT
CTCAAGTGAA GGTTTAATAT CACTTTCTAT CACTGAGGCA 061 CCTGATCTTA
AGATCAGGGA TCCTAAGATA GAGAAACTCT ACCTTCCAGT TTTTTATTTA 121
AATGCACACA TCTACTTAAA TCCACTCAGT ACTCTCCTGA ACTCTCATTG TGGCGAGAAC
181 TGTTTTCAT GGTTATGAAC AATTACAGAA TGCCACTTTT CCAGTTTGGA
GAAATATATT 241 CATTTATATA AACAGGGTCA GGAACACCAA GAGGCAAGGA
GGAGGGGGTG GTGTGACTGG 301 GAAAGGTGAG ATGAAGCAGT GCTTCCTCTC TTAA
Human SCaMPER ORF (SEQ ID NO.: 4)
ATGTTAAAAGTGAGCAGGGTCTCAAGTGAAGGTTTAATAT- CACTTTCTATCACTGA
GGCACCTGATCTTAAGATCAGGGATCCTAAGATAGAGAAAC- TCTACCTTCCAGTTT
TTTATTTAAATGCACACATCTACTTAAATGCACTCAGTACTC- TCCTGAACTCTCAT
TGTGGCGAGAACTGTTTTCATGGTTATGAACAATTACAGAATG- CCACTTTTCCAGT
TTGGAGAAATATATTCATTTATATAAACAGGGTCAGGAACATCA- AGAGGCAAGGAG
GAGCGGGTGGTGTGAGTGGGAAAGGTGAGATGAAGCAGTGCTTCC- TCTCTTAA
Example 17
Cloning of Rat EDC-3 Genes
[0478] The rat Edg-3 gene was obtained using a combination of
reverse transcription and polymerase chain reactions (RT-PCR) as is
known in the art (see, for example, PCR Technology: Principles and
Applications for DNA Amplification, H. A. Erlich, ed., 1989).
[0479] Rat heart, liver and skeletal muscle tissue was freshly
obtained from sacrificed rats. RNA from these tissues was isolated
using the commercially available Qiagen Rneasy Mini Kit according
to the manufacturer's protocols (Qiagen, Valencia, Calif.).
[0480] PCR amplification of the target sequences was performed
using the commercially available Qiagen One-Step RT-PCR kit
according to the manufacturer's protocols. Primers sets
included:
7 (SEQ ID NO. :5) 5'-TTATGGCAACCACGCACGCGCAGG-3'(upper) (SEQ ID NO.
:6) 5'-AGACCGTCACTTGCAGAGGAC-3'(lower- )
[0481] Amplification utilized cycling regimens according to the
manufacturer's recommendations. Briefly, reactions were started at
50.degree. C. for 30 minutes, followed by 95.degree. C. for 15
minutes; and then optimized using 40 PCR cycles, each cycle
consisting of 45 seconds denaturation at 95.degree. C., 45 seconds
of annealing at 63+5.degree. C., and 1 minute of polymerization at
72.degree. C. The resulting PCR products were subcloned into pCR3.1
vector using the Invitrogen TA cloning kit (Invitrogen, Carlsbad,
Calif.) using standard techniques. Briefly, after RT-PCR, a 1%
agarose gel was used to separate the PRC products from unused
primers and dNTPs electrophoretically. The approximately 1200 bp
fragment was then excised quickly under UV light and the Bio101
Geneclean kit was then used to purify the DNA. The purified DNA was
then ligated into the PCR3.1 vector. The ligation mix was then
transformed into Invitrogen Top10 chemically competent cells with
heat shock. Following a 1 hour incubation shaking at 37.degree. C.,
the cells were spread on a LB plate containing ampicillin and
allowed to grow overnight at 37.degree. C. Several individual
colonies were chosen and used to inoculate culture tubes containing
3 mls of LB-ampicillin media. After 8-12 hours of shaking at
37.degree. C., 1.5 mls of the culture was used in the Qiaspin
miniprep kit to isolate plasmid DNA. The restriction enzyme EcoR1
was used to confirm that the plasmid contained a piece of DNA that
was approximately 1200 bp.
[0482] Sequencing with a T7 primer was performed at the San Diego
State University DNA Core Facility (San Diego, Calif.). The
resultant rat Edg-3 sequence are presented as SEQ ID NO.: 7.
8 Rat Edg-3 Sequence (SEQ ID NO.: 7) 0001 ATGGCAACCA CGCACGCGCA
GGGGCACCCG CCAGTCTTGG GGAATGATAC TCTCCGGGAA 0061 CATTATGATT
ACGTGGGGAA CCTGGCAGGC AGGCTGCGGG ATCCCCCTGA GGGTAGCACC 0121
CTCATCACCA CCATCCTCTT CTTGGTCACC TGTAGCTTCA TCGTCTTGGA GAACCTGATG
0181 GTTTTGATTG CCATCTGGAA AAACAATAAA TTTCATAACC GCATGTACTT
TTTCATCGGC 0241 AACTTGGCTC TCTGCGACCT GCTGGCCGGC ATAGCCTACA
AGGTCAACAT TCTGATGTCC 0301 GGTAGGAAGA CGTTCAGCCT GTCTCCAACA
GTGTGGTTCC TCAGGGAGGG CAGTATGTTC 0361 GTAGCCCTGG GCGCATCCAC
ATGCAGCTTA TTGGCCATTG CCATTGAGCG GCACCTGACC 0421 ATCATCAAGA
TGAGGCCGTA CGACGCCAAC AAGAAGCACC GCGTGTTCCT TCTGATTGGG 0481
ATGTGCTGGC TAATTGCCTT CTCGCTGGGT GCCCTGCCCA TCCTGGGCTG GAACTGCCTG
0541 GAGAACTTTC CCGACTGCTC TACCATCTTG CCCCTCTACT CCAAGAAATA
CATTGCCTTT 0601 CTCATCAGCA TCTTCACAGC CATTCTGGTG ACCATCGTCA
TCTTGTACGC GCGCATCTAC 0661 TTCCTGGTCA AGTCCAGCAO CCGCAGGGTG
GCCAACCACA ACTCCGAGAG ATCCATGGCC 0721 CTTCTGCGGA CCGTAGTGAT
CGTGGTGAGC GTGTTCATCG CCTGTTGGTC CCCCCTTTTC 0781 ATCCTCTTCC
TCATCGATGT CGCCTGCAGG GCGAAGCAGT GCTCCATCCT CTICAAGAGT 0841
CAGTGGTTCA TCATGCTGGC TGTCCTCAAC TCGGCCATGA ACCCTGTCAT CTACACGCTG
0901 GCCAGCAAAG AGATGCGGCG TGCTTTCTTC CGGTTGGTGT GCGGCTGTCT
GGTCAAGGGC 0961 AAGGCGACCC AGGCCTCCCC GATGCAGCCT GCTCTTGACC
CGAGCAGAAG TAAATCAAGC 1021 TCCACTAACA ACAGCAGCAG CCACTCTCCA
AAGGTCAAGG AAGACCTGCC CCATGTGGCT 1081 ACCTCTTCCT GCGTCACTGA
CAAAACGAGG TCGCTTCAGA ATCGGGTCCT CTGCAAGTGA 1141 CGGTCT
Example 18
Molecular Genetic Approaches to Sphingolipid-Based Cardiovascular
Therapy
[0483] In addition to traditional approaches to therapeutic agents,
approaches based on molecular genetics and recombinant DNA
technology are used to produce agents for sphingolipid-based
cardiovascular therapy.
[0484] Dominant Negative Mutant Proteins
[0485] Dominant negative mutant proteins of enzymes that catalyze
reactions leading to the production of undesirable, toxic and/or
cardiotoxic sphingolipids, or of sphingolipid receptors, are used
as agents for sphingolipid-based cardiovascular therapy. Such
proteins are delivered to cardiac or other tissues to decrease
sphingolipid production and/or to minimize the cardiotoxic effects
of circulating sphingolipids. Enzymes of particular interest
include ceramidase, sphingomyelinase and SPH kinase. Receptors of
particular interest include members of the EDG family of receptors,
especially those presently known to bind S-1-P, i.e., EDG-1, EDG-3
and EDG-5.
[0486] Dominant negative mutants are prepared in a variety of ways.
In general, dominant negative mutants of proteins retain their
ability to interact with other molecules but have lost some other
function present in the wildtype protein. For example, a dominant
negative mutant of a multimeric enzyme involved in sphingolipid
metabolism would be one that retains the ability to form multimers
but has a catalytic domain that has been inactivated by deletion or
mutation of amino acid residues in the catalytic domain. Such
deletions and mutations are created by site-directed mutagenesis,
by random mutagenesis, or by any other suitable procedure.
[0487] One non-limiting example of a dominant negative mutant that
may be used in the invention of the disclosure is a dominant
negative mutant of human SPH kinase. This mutation (Gly82Asp),
which was created by site-directed mutagenesis of the presumed
catalytic domain of the enzyme, is stated to block the activation
of endogenous SPH kinase by TNF-alpha and IL-1-beta (Pitson et al.,
J. Biol. Chem. 275:33945-33950, 2000).
[0488] Antisense
[0489] Antisense oligonucleotides against mRNAs of key sphingolipid
production enzymes (e.g., sphingomyelinase, ceramidase, sphingosine
kinase) are delivered to cardiac and/or other tissues to inhibit or
completely block the production and/or harmful effects of
undesirable, toxic and/or cardiotoxic sphingolipids. Antisense
oligonucleotides are designed to decrease the expression of key
sphingolipid binding proteins or receptors such as EDG and SCaMPER
are delivered to cardiac and/or other tissues to minimize the
cardiotoxic effects of circulating sphingolipids. Ribozymes that
degrade such mRNAs are also used. Additionally or alternatively,
anti sense expression constructs, which transcribe RNA molecules
antisense in vivo, may be introduced into an animal, which may be a
human, in need thereof.
[0490] Gene Therapy
[0491] Various forms of gene therapy are used to carry out
sphingolipid-based cardiovascular therapy according to the
invention. The therapeutic nucleic acid molecule ("gene therapy
construct") that is introduced into a patient in need thereof
generally comprises nucleic acids having the following genetic
elements: (i) an "open reading frame (ORF)," i.e., a
protein-encoding nucleotide sequence that is sought to be
expressed, and (ii) a "gene therapy vector," which provides the
genetic sequences needed for expression of an ORF introduced
thereinto, replication of the vector and/or recombination with
other nucleic acids. A gene or protein is said to be "expressed"
when it is actively creating mRNA molecules having the nucleotide
sequence of the ORF of a gene (transcription); and, using these
mRNA molecules as blueprints, producing proteins having specific
amino acid sequences (translation). It is understood in the art
that transcription and translation need not occur
contemporaneously. It is also understood in the art that a protein
is one type of gene product, but other gene products that may be
expressed by gene therapy constructs may be nucleic acids, e.g.,
antisense transcripts, or structural or enzymatic RNA
molecules.
[0492] Expression of proteins that influence sphingolipid
concentration and/or activity via the administration of gene
therapy constructs comprising an ORF that encodes a protein of
interest is one type of gene therapy. Such proteins may be
expressed at a level that is approximately equal to what is
normally found in healthy individuals, "over-expressed" (i.e.,
expressed at a level that is greater than the amount that is
normally found in healthy individuals), or constitutively expressed
(i.e., expressed at a constant level).
[0493] Another type of gene therapy, in which a genetic deficiency
(loss of function of a protein due to mutation) is compensated for
by virtue of the in vivo expression of a wildtype protein from a
gene therapy construct, is generally called "replacement therapy."
This type of gene therapy may be used to treat patients having
genetic deficiencies that reduce the amount, activity, or
distribution in enzymes involved in sphingolipid metabolism, or in
sphingolipid receptors.
[0494] In another type of gene therapy, proteins that degrade
undesirable sphingolipids, or stimulators thereof, are
overexpressed for the immediate and/or long-term treatment of
cardiac disorders. In order to achieve gene therapy of this type,
gene therapy constructs are designed, by way of non-limiting
example, to encode and overexpress an enzyme that degrades an
undesirable sphingolipid or metabolite thereof. Such enzymes
include, e.g., S-1-P lyase, sphingomyelin synthase, SM deacylase,
ceramide synthase, glucosylceramide synthase, ceramide kinase, and
S-1-P phosphatase. An indirect way of realize this type of gene
therapy involves the administration of gene therapy constructs that
encode proteins that are inhibitors or activators of enzymes
involved in sphingolipid metabolism or sphingolipid receptors (see
Examples 7 to 10).
[0495] In another type of gene therapy, proteins that bind
undesirable sphingolipids are overexpressed for the immediate
and/or long-term treatment of cardiac disorders. In order to
achieve gene therapy of this type, gene therapy constructs are
designed that encode, by way of non-limiting example, antibodies or
antibody derivatives directed to an undesirable sphingolipid
(Example 6); naturally occurring proteins, such as lysenin, that
bind an undesirable sphingolipid; or polypeptide derivatives of
receptors and enzymes that bind undesirable sphingolipids (Example
15).
[0496] Another type of gene therapy involves the administration of
gene therapy constructs that express a RNA having a nucleotide
sequence that is the antisense (hybridizing) of all or a portion of
an mRNA that encodes a sphingolipid enzyme that produces, or a
receptor that facilitates the cellular uptake of, an undesirable
sphingolipid. Binding of the antisense transcript reduces or
prevents the expression of the mRNA encoding the enzyme or
receptor. Such enzymes include but are not limited to SMase, SPH
kinase, ceramidase, cerebrosidase, desaturase, ceramide synthase,
ceramide-1-phosphatase, serine palmitoyl transferase, and
NADPH-dependent reductase. Such receptors include but are not
limited to members of the EDG family of receptors (Example 15).
[0497] Another type of gene therapy involves the use of gene
therapy constructs that encode and express dominant negative
mutants of key sphingolipid production enzymes (e.g., SPH kinase,
sphingomyelinase, etc.) or sphingolipid receptors (e.g., the EDG
family of receptors). These expression constructs are delivered to
cardiac or other tissues in order to decrease sphingolipid
production and/or to minimize the effects of circulating
undesirable, toxic and/or cardiotoxic sphingolipids.
[0498] Gene Therapy Vectors
[0499] Gene therapy constructs for the in vivo production of
antisense transcripts and dominant negative mutants, and for gene
therapy, may be contained in any suitable expression vector known
in the art, such as a plasmid, cosmid, or viral vector. Viral
vectors such as retroviral vectors, adenovirus vectors, herpes
simplex virus vectors, vaccinia virus and the like are particularly
useful for the administration of these expression constructs. The
choice of vector and route of administering the vector will depend,
for example, on the particular target cells, tissues and animal
(including a human) that are targeted for drug delivery, and can be
determined by those skilled in the art.
[0500] Non-limiting examples of vectors for gene therapy include,
in general, those that are nonviral (Li et al., Gene Ther. 7:31-34,
2000; episomal (Van Craenenbroeck et al., Eur. J. Biochem.
267:5665-5678, 2000); viral (Walther et al., Drugs 60:249-271,
2000), including, in particular, those derived from retroviruses
Kurian et al., Mol. Pathol. 53:173-176, 2000; Takeuchi et al., Adv.
Exp. Med. Biol. 465:23-35, 2000) and other RNA viruses (Hewson,
Mol. Med. Today 6:28-35, 2000). Non-limiting examples of particular
gene therapy vectors include those derived from adenovirus (see
Danthinne et al., Gene Ther. 7:1707-1704, 2000); AAV,
adeno-associated virus (Athanasopoulus et al., Int. J. Mol. Med.
6:363-375, 2000; Tal, J. Biomed. Sci. 7:279-291, 2000; Monahan et
al., Gene Ther. 7:24-30, 2000); HSV-1, herpes simplex virus
(Sean-Esteves et al., Mol. Ther. 2:9-15, 2000; Latchman, Mol. Med.
Today 6:28-35, 2000); and lentiviral vectors (Vigna et al., J. Gene
Med. 2:308-316, 2000; Buchschacher et al., Blood 95:2499-2504,
2000; Trono, Gene Ther. 7:20-23, 2000).
[0501] Gene therapy vectors and constructs may be designed to be
targeted to specific cells or tissues (Hallenbeck et al., Adv. Exp.
Med. Biol. 465:37-46, 2000). Gene therapy vectors designed to be
targeted to myocardial cells and tissues, and strategies for the
use thereof, are preferably used in certain modalities of gene
therapy. See U.S. Pat. No. 6,121,246; Allen, Ann. Thorac. Surg.
68:1924-1928, 1999; Ponder, Trends Cardiovasc. Med. 9:158-162,
1999; Yla-Herttuala et al., Lancet 355:213-222, 2000; Duckers et
al., Med. Clin. North Am. 84:199-213, 2000; Sinnaeve et al.,
Cardiovasc. Res. 44:498-506, 1999; Hiltunen et al., Vasc. Med.
5:41-48, 2000; Hajjar et al., Circ. Res. 86:616-621, 2000; Stephan
et al., Ann. Endocrinol. (Paris) 61:85-90, 2000; O'Brien et al.,
Mayo Clin. Proc. 75:831-834, 2000; Dedieu et al., Curr. Cardiol.
Rep. 2:39-47, 2000.
Example 19
Combination Therapies
[0502] The therapeutic compositions and methods of the invention
may be used in combination with each other and/or with other agents
for cardiovascular therapy, i.e., non-sphingolipid-based
therapeutic agents. In such instances, pharmaceutical compositions,
medical devices, emergency kits and the like contain two or more of
the therapeutic agents of the invention; or at least one of the
therapeutic agents of the invention and at least one
non-sphingolipid-based therapeutic agent; or two or more of the
therapeutic agents of the invention and two or more other
therapeutic agents. That is, compositions, devices, methods and the
like that are used in combination therapies may be described as
those having or using at least one member from two or more of a, b
and c, wherein "a" is a first therapeutic agent of the invention;
"b" is a second (i.e., other than "a") therapeutic agent of the
invention; and "c" is a non-sphingolipid-based therapeutic
agent.
[0503] In general, any therapeutic agent may be combined with the
therapeutic agents of the invention so long as neither agent has a
negative impact on the activity of the other agent. Therapeutic
agents that may be combined with the therapeutic agents of the
invention includes presently known agents for cardiovascular
therapy, as well as agents that are discovered or created
subsequent to the filing of the present application.
[0504] Presently known agents for cardiovascular therapy include
but are not limited to alpha and beta adrenergic blocking drugs;
parasympathetic drugs; calcium channel blockers; drugs that affect
the renin-angiotensin system; diuretic therapy; magnesium,
potassium and calcium; digitalis and other inotropic agents;
organic nitrates and nitroprusside; antiadrenergic drugs with
central action; ganglionic blockers and neuron depletors;
nonspecific antihypertensive vasodilators; antiarrhythmic drugs;
antiplatelet and other antithrombotic drugs; thrombolytic agents;
lipid-lowering drugs; selective dopamine receptor agonists; and
prostacyclin. See Cardiovascular Pharmacotherapeutics: Companion
Handbook, Wm. H. Frishman and Edmund. H Sonnenblick, McGraw Hill,
N.Y., 1998.
[0505] As is noted in the preceding Examples, certain therapeutic
agents of the invention are preferably used in combination with a
second therapeutic agent designed to ameriolate potential undesired
side-effects that may occur as a result of treatment with the first
therapeutic agent.
[0506] By way of non-limiting example, inhibition of SPH kinase
will result in decreased production of the harmful sphingolipid
S-1-P, but may lead to an accumulation of SPH, which is also an
undesirable sphingolipid, albeit generally less harmful than S-1-P.
In order to avoid or mitigate this effect should it occur,
additional agents are administered that lower the concentration of
available SPH. Such agents include but are not limited to ones that
(i) stimulate or are enzymes having SPH as a substrate, with the
proviso that such enzymes should not be ones that produce S-1-P;
(ii) inhibit enzymes having SPH as a product; (iii) are SPH
receptor derivatives, or antibodies to SPH, that bind molecules of
SPH, thus sequestering them from locations in the body where they
exert their toxic effects; (iii) inhibit the action of inflammatory
cytokines and chemokines; (iv) are antisense molecules, or genetic
constructs that express antisense transcripts, that act to reduce
the expression of a protein that increases concentrations of SPH,
e.g., an enzyme that catalyzes reactions that produce SPH; (v) a
dominant negative derivative of a protein that increases
concentrations of SPH, e.g., an enzyme that catalyzes reactions
that produce SPH; and (vi) a gene therapy construct that encodes
and expresses a protein that leads to decreased function and/or
concentration of SPH including, by way of non-limiting example, a
protein that is characterized as being of the type encompassed by
the classes defined above in (i), (ii), (iii) and/or (v).
[0507] One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. The methods, procedures, treatments, devices, and
compositions described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Upon reading this
specification, changes therein and other uses will occur to those
skilled in the art, each of which is encompassed within the spirit
of the invention as defined by the attached claims.
[0508] All patents and publications referred to above are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0509] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, or
limitation or limitations, which is not specifically disclosed
herein.
[0510] Thus, for example, in each instance herein any of the terms
"comprising," "consisting essentially of," and "consisting of" may
be replaced with either of the other two terms. The terms and
expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by preferred embodiments and optional
features, modification and variation of the concepts herein
disclosed may be resorted to by those skilled in the art, and that
such modifications and variations are considered to be within the
scope of this invention as defined by the appended claims. Other
embodiments are within the following claims.
Sequence CWU 1
1
7 1 24 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 ccaggattca tcatatgtta aaag 24 2 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 atcagtgggt gcatcagtag c
21 3 333 DNA Rattus sp. 3 atgttaaaag tgagcagggt ctcaagtgaa
ggtttaatat cactttctat cactgaggca 60 cctgatctta agatcaggga
tcctaagata gagaaactct accttccagt tttttattta 120 aatgcacaca
tctacttaaa tgcactcagt actctcctga actctcattg tggcgagaac 180
tgttttcatg gttatgaaca attacagaat gccacttttc cagtttggag aaatatattc
240 atttatataa acagggtcag gaacaccaag aggcaaggag gagggggtgg
tgtgagtggg 300 aaaggtgaga tgaagcagtg cttcctctct taa 333 4 333 DNA
Homo sapiens 4 atgttaaaag tgagcagggt ctcaagtgaa ggtttaatat
cactttctat cactgaggca 60 cctgatctta agatcaggga tcctaagata
gagaaactct accttccagt tttttattta 120 aatgcacaca tctacttaaa
tgcactcagt actctcctga actctcattg tggcgagaac 180 tgttttcatg
gttatgaaca attacagaat gccacttttc cagtttggag aaatatattc 240
atttatataa acagggtcag gaacatcaag aggcaaggag gagggggtgg tgtgagtggg
300 aaaggtgaga tgaagcagtg cttcctctct taa 333 5 24 DNA Artificial
Sequence Description of Artificial Sequence Primer 5 ttatggcaac
cacgcacgcg cagg 24 6 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 6 agaccgtcac ttgcagagga c 21 7 1146 DNA
Rattus sp. 7 atggcaacca cgcacgcgca ggggcacccg ccagtcttgg ggaatgatac
tctccgggaa 60 cattatgatt acgtggggaa gctggcaggc aggctgcggg
atccccctga gggtagcacc 120 ctcatcacca ccatcctctt cttggtcacc
tgtagcttca tcgtcttgga gaacctgatg 180 gttttgattg ccatctggaa
aaacaataaa tttcataacc gcatgtactt tttcatcggc 240 aacttggctc
tctgcgacct gctggccggc atagcctaca aggtcaacat tctgatgtcc 300
ggtaggaaga cgttcagcct gtctccaaca gtgtggttcc tcagggaggg cagtatgttc
360 gtagccctgg gcgcatccac atgcagctta ttggccattg ccattgagcg
gcacctgacc 420 atgatcaaga tgaggccgta cgacgccaac aagaagcacc
gcgtgttcct tctgattggg 480 atgtgctggc taattgcctt ctcgctgggt
gccctgccca tcctgggctg gaactgcctg 540 gagaactttc ccgactgctc
taccatcttg cccctctact ccaagaaata cattgccttt 600 ctcatcagca
tcttcacagc cattctggtg accatcgtca tcttgtacgc gcgcatctac 660
ttcctggtca agtccagcag ccgcagggtg gccaaccaca actccgagag atccatggcc
720 cttctgcgga ccgtagtgat cgtggtgagc gtgttcatcg cctgttggtc
cccccttttc 780 atcctcttcc tcatcgatgt ggcctgcagg gcgaaggagt
gctccatcct cttcaagagt 840 cagtggttca tcatgctggc tgtcctcaac
tcggccatga accctgtcat ctacacgctg 900 gccagcaaag agatgcggcg
tgctttcttc cggttggtgt gcggctgtct ggtcaagggc 960 aaggggaccc
aggcctcccc gatgcagcct gctcttgacc cgagcagaag taaatcaagc 1020
tccagtaaca acagcagcag ccactctcca aaggtcaagg aagacctgcc ccatgtggct
1080 acctcttcct gcgtcactga caaaacgagg tcgcttcaga atggggtcct
ctgcaagtga 1140 cggtct 1146
* * * * *