U.S. patent application number 10/948026 was filed with the patent office on 2005-03-17 for pyruvate ester composition and method of use for resuscitation after events of ischemia and reperfusion.
This patent application is currently assigned to Xanthus Life Sciences, Inc.. Invention is credited to Ajami, Alfred M., Fink, Mitchell P., Sims, Carrie A..
Application Number | 20050059738 10/948026 |
Document ID | / |
Family ID | 26814520 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059738 |
Kind Code |
A1 |
Ajami, Alfred M. ; et
al. |
March 17, 2005 |
Pyruvate ester composition and method of use for resuscitation
after events of ischemia and reperfusion
Abstract
A therapeutic composition comprising an alkyl, aralkyl,
alkoxyalkyl or carboxyalkyl ester of 2ketoalkanoic acid and a
component for inducing and stabilizing the enol resonance form of
the ester at physiological pH values is disclosed. The composition
of the invention further comprises a pharmaceutically acceptable
carier vehicle in which the enol resonance form of the ester is
stabilized at physiological pH values. Formulations containing the
compositions of the invention permit the successful use of
2-ketoalkanoic acid esters, e.g., pyruvic acid esters, to treat,
e.g., is chemic events, shock, organ reanimation, resuscitation and
other recognized pyruvate-effective treatments. The compositions of
the inventions are also useful in a process for preserving organ
parts, organs or limbs removed from a living mammal and in need of
preservation, e.g., for later transplantation to an organ
recipient.
Inventors: |
Ajami, Alfred M.;
(Brookline, MA) ; Sims, Carrie A.; (Boston,
MA) ; Fink, Mitchell P.; (Pittsburgh, PA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Xanthus Life Sciences, Inc.
Montreal
MA
Beth Israel Deaconess Medical Center, Inc.
Boston
|
Family ID: |
26814520 |
Appl. No.: |
10/948026 |
Filed: |
September 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10948026 |
Sep 23, 2004 |
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10116707 |
Apr 4, 2002 |
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6846842 |
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10116707 |
Apr 4, 2002 |
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PCT/US00/27758 |
Oct 6, 2000 |
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60158091 |
Oct 7, 1999 |
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Current U.S.
Class: |
514/546 ;
424/682 |
Current CPC
Class: |
A61K 31/235 20130101;
A61K 31/22 20130101; A61K 31/22 20130101; A61K 31/235 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 33/00 20130101; A61K 33/00 20130101 |
Class at
Publication: |
514/546 ;
424/682 |
International
Class: |
A61K 031/22; A61K
033/06 |
Goverment Interests
[0002] Part of the work leading to this invention was carried out
with United States Government support provided under a grant from
the Hational Institutes of Health, Grant No. GM3763 1. Therefore,
the U.S. Government has certain rights in this invention.
Claims
What is claimed is:
1. A method for treating a mammal suffering from a condition
characterized by ischemia or reperfusion injury, comprising
administering to said mammal a therapeutically effective amount of
a composition comprising an ester of a 2-ketoalkanoic acid selected
from the group consisting of alkyl, aralkyl, alkoxyalkyl and
carboxyalkyl ester in a pharmaceutically-acceptable carrier,
wherein said carrier further comprises a biologically safe
component for inducing and stabilizing enolization of the 2-keto
functionality of said ester at physiological pH values.
2. The method of claim 1, wherein said pharmaceutically-acceptable
carrier is a Ringer's solution of isotonic saline supplemented with
potassium ion.
3. The method of claim 1, wherein said 2-ketoalkanoic acid ester is
selected from the group consisting of ethyl pyruvate, propyl
pyruvate, butyl pyruvate, carboxymethylpyruvate,
acetoxymethylpyruvate, carbethoxymethylpyruvate and
ethoxymethylpyruvate.
4. The method of claim 1, wherein said 2-ketoalkanoic acid ester is
selected from the group consisting of ethyl 2-keto-buyrate, ethyl
2-ketopentanoate, ethyl 2-keto-3-methyl-butyrate, ethyl
2-keto-4-methyl-pentanoate and ethyl 2-keto-hexanoate.
5. The method of claim 3, wherein said 2-ketoalkanoic acid ester is
admixed in a saline solution, said solution containing a cation
selected from the group consisting of calcium and magnesium.
6. The method of claim 4, wherein said 2-ketoalkanoic acid ester is
admixed in a saline solution, said solution containing a cation
selected from the group consisting of calcium and magnesium.
7. The method of claim 1, wherein the condition characterized by
ischemia is selected from the group consisting of mesenteric
ischemia, mesenteric thrombus, mesenteric venous occlusion, aortic
aneurism repair, coronary artery bypass and surgical treatment of
arterial occlusion of limbs.
8. A process for preserving organ parts, organs or limbs removed
from a living mammal, said process comprising perfusing said organ
or limb with a solution containing an effective amount of a
composition comprising an ester of a 2-ketoalkanoic acid selected
from the group consisting of alkyl, aralkyl, alkoxyalkyl and
carboxyalkyl ester in a pharmaceutically-acceptable carrier, said
carrier further comprising a biologically safe component for
inducing and stabilizing enolization of the 2-keto functionality of
said ester at physiological pH values.
9. The process of claim 8, wherein said 2-ketoalkanoic acid ester
is selected from the group consisting of ethyl pyruvate, propyl
pyruvate, butyl pyruvate, carboxymethylpyruvate,
acetoxymethylpyruvate, carbethoxymethylpyruvate and
ethoxymethylpyruvate.
10. The process of claim 8, wherein said 2-ketoalkanoic acid ester
is selected from the group consisting of ethyl 2-keto-butyrate,
ethyl 2-ketopentanoate, ethyl 2-keto-3-methyl-butyrate, ethyl
2-keto-4-methyl-pentanoate and ethyl 2-keto-hexanoate.
11. The process of claim 9, wherein said 2-ketoalkanoic acid ester
is admixed in a saline solution, said solution containing a cation
selected from the group consisting of calcium and magnesium
12. The process of claim 10, wherein said 2-ketoalkanoic acid ester
is admixed in a saline solution, said solution containing a cation
selected from the group consisting of calcium and magnesium.
13. The process of claim 9, wherein said 2-ketoalkanoic acid ester
is ethyl pyruvate.
14. The method of claim 1, wherein said 2-ketoalkanoic acid ester
is an alkyl ester.
15. The method of claim 14, wherein said alkyl ester is ethyl
pyruvate.
16. The method of claim 1, wherein said condition is characterized
by ischemia.
17. The method of claim 16, wherein said ischemia is due to an
ischemic event selected from the group consisting myocardial
infarction, cerebral infarction and intestinal infarction.
18. The method of claim 1, wherein said condition is characterized
by reperfusion injury.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/116,707, filed Apr. 4, 2002, which is a continuation of
International Application No. PCT/US00/27758, which designated the
United States and was filed on Oct. 6, 2000, published in English,
which claims the benefit of U.S. Provisional Application No.
60/158,091, filed on Oct. 7, 1999. The entire teachings of the
above applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] This invention relates to several new pyruvate compounds and
methods for resuscitation and reanimation of mammals, especially
humans, before, during and after, e.g., (1) mesenteric ischemia,
mesenteric thrombus or mesenteric venous occlusion; (2) aortic
aneurism repair, coronary artery bypass, surgical treatment of
arterial occlusion of limbs; (3) hemorrhagic shock, resulting from
either penetrating and blunt trauma; and (4) preservation and
transplantation of organs. Ischemia is defined herein as the
interruption of oxygen supply, via the blood, to an organ or to
part of an organ. Examples of ischemic events include (i)
myocardial, cerebral, or intestinal infarction following
obstruction of a branch of a coronary, cerebral, or mesenteric
artery, and (ii) removal and storage of an organ prior to
transplantation. In the case of myocardial infarction, prompt
restoration of blood flow to the ischemic myocardium, i.e. coronary
reperfusion, is a key component of the treatment. This is because
mortality is directly related to infarct size (tissue necrosed)
which is related to the severity and duration of the ischemic
event. The consequences of hemorrhagic shock are similar to those
of ischemia, although the causative event is not an interruption of
blood flow but rather the event of massive blood loss itself which
causes deprivation of the oxygen supply.
[0004] Notwithstanding the need to supply an organ cut-off from a
normal blood supply with oxygen, it has been found that reperfusion
injury may occur upon restoration of blood flow. This results from
the production of reactive oxygen species (ROS), namely, hydrogen
peroxide, hydroxyl radicals and superoxide radicals, among others,
which are formed from both extracellular and intracellular sources.
ROS are highly reactive species that, under normal conditions, are
scavenged by endogenous defense mechanisms. However, under
conditions of post-ischemic oxidative stress, ROS interact with a
variety of cellular components, causing peroxidation of lipids,
denaturation of proteins, and interstitial matrix damage and
resulting in increase of membrane permeability and release of
tissue enzymes.
[0005] In an attempt to minimize these undesirable side effects of
perfusion in the treatment of ischemia and also of shock,
researchers have demonstrated the utility of various antioxidants
in the reperfusion process.
[0006] Banda et al. (1996), together with Kurose et al. (1997),
suggested the use of an inhibitor of ROS production to protect the
reperfused myocardium and the use of agents and inhibitors that
reduce ROS levels. In a similar context, desiring to provide more
efficient resuscitation, researchers have demonstrated the additive
utility of incorporating an antioxidant and a beneficial metabolic
fuel into the reperfusion regimen. Salahudeen et al. (1991) used
solutions of pyruvate, an ROS scavenger and a metabolically
important precursor fuel for gluconeogenesis, to protect against
hydrogen peroxide induced acute renal failure. Cicalese et al.
(1996) found that pretreatment with intraluminal pyruvate
ameliorates post ischemic small bowel injury while Crestanello et
al. (1998), DeBoer et al. (1993), and O'Donnell-Tormey et al.
(1987) have substantiated this finding by examining the
ameliorative effects of both endogenously secreted pyruvate and
exogenously added material in the reperfusion and subsequent
function of organ and tissue preparations subjected to ischemia and
simulated shock. Varma et al. (1998), similarly, have shown that in
a cultured lens system, after exposure of the cultured lens to free
radical oxidant stress, pyruvate and its esters have certain
cytoprotecting and restorative effects.
[0007] In a further effort directed to protecting reperfused heart
tissue, U.S. Pat. No. 5,075,210, herein incorporated by reference,
discloses a process for reperfusing a heart for transplantation.
The patent discloses a cardioplegic solution containing sodium
chloride, potassium chloride, calcium chloride, sodium bicarbonate,
sodium EDTA, magnesium chloride, sodium pyruvate and a protein.
[0008] U.S. Pat. No. 5,294,641, herein incorporated by reference,
is directed to the use of pyruvate to prevent the adverse effects
of ischemia. The pyruvate is administered prior to a surgical
procedure to increase a patient's cardiac output and heart stroke
volume. The pyruvate is administered as a calcium or sodium salt.
The pyruvate can alternatively be an amide of pyruvic acid such as
ethylamino pyruvate. Similarly, U.S. Pat. No. 5,508,308, herein
incorporated by reference, discloses the use of pyruvyl glycine to
treat reperfusion injury following myocardial infarction.
[0009] U.S. Pat. Nos. 4,988,515 and 5,075,210, herein incorporated
by reference, use pyruvate salts in cardioplegic solutions and in
preservation solutions for the heart before transplantation. U.S.
Pat. No. 4,970,143, herein incorporated by reference, discloses the
use of acetoacetate for preserving living tissue, including
addition of the pyruvate to the preservation solution.
[0010] U.S. Pat. No. 5,100,677 herein incorporated by reference,
discloses the composition of various parenteral solutions. Of
interest is a recommendation to include pyruvate anions (apparently
from metal salts) in intravenous solutions.
[0011] U.S. Pat. No. 5,798,388, herein incorporated by reference,
further describes the utility of pyruvate salts and of various
complex derivatives, such as amides, for the treatment of ROS in
the context of airway inflammation. The patent discloses a pyruvate
compound in the form of a covalently linked pyruvoyl-amino acid. By
utilizing this type of a pyruvate delivery system, the negative
effect of pyruvate salt is avoided. However, administration of
large amounts of pyruvate-amino acid may result in nitrogen
overload which could harm patients with liver and/or kidney
pathology.
[0012] In a similar context and based on a similar rationale for
pyruvate delivery, U.S. Pat. No. 5,876,916 pertains to the utility
of pyruvate thiolesters and polyol esters for the treatment or
prevention of reperfusion injury following ischemia, diabetic
effects, cholesterol levels, injured organs, ethanol intoxication
or as a foodstuff; and U.S. Pat. Nos. 5,633,285; 5,648,380;
5,652,274; and 5,658,957, each herein incorporated by reference,
disclose various compositions, salts, prodrugs and derivatives of
pyruvate in mixtures with other antioxidants, fatty acids as
anti-inflammatory and immunostimulating wound healing compositions.
However, administration of large amounts of complex pyruvate-amino
acid and other pro-drug derivatives requiring enzymatic hydrolysis
prior to liberation of their antioxidant effects may result in
nitrogen and/or other xenobiotic overload, which could harm
patients directly, interfere with normal detoxifying processes, or
cause toxic effects through by-products of limited shelf life.
[0013] Notwithstanding the acceptance of pyruvate as an effective
component of a reperfusion solution or other varied applications,
pyruvic acid is a strong and unstable acid which cannot be infused
as such. On standing in solution, pyruvic acid and its salts at
various pH values, including in the physiological range, are known
to form both a stable hydrate and a dimer (para-pyruvate), neither
of which reach with ROS as antioxidants and both of which are known
inhibitors of pyruvate utilization as a metabolic fuel, thereby
abrogating any of the beneficial effects which might have accured
from pyruvate administration in accordance with the prior art just
described.
[0014] Furthermore, it has been recognized that traditional
pharmacological pyruvate compounds, such as salts of pyruvic acid,
are not particularly physiologically suitable. For example, these
compounds lead to the accumulation of large concentrations of ions
(e.g., calcium or sodium) in the patient's body fluids. Similarly,
amino acid compounds containing pyruvate can lead to excessive
nitrogen loads. It has also been proposed to infuse pyruvylglycine,
the amide function of which is presumably hydrolyzed in plasma
and/or tissues, thus liberating pyruvate.
[0015] However, at the high rates of pyruvoylglycine infusion
required to achieve 1 mM pyruvate in plasma, the glycine load may
be harmful to patients suffering from hepatic or renal pathologies.
Also, flooding plasma with glycine may interfere with the transport
of some amino acids across the blood-brain barrier. Accordingly,
while potentially suitable to organ preservation, these pyruvate
compounds are less suited to treating an organ in vivo, and it is
recognized that a need exists to provide a pyruvate delivery
compound that is more physiologically acceptable.
[0016] There is also a recognized need to provide a pyruvate
delivery system that is cost effective, simple, and devoid of
opportunities for contamination because of 1) limited shelf-life,
2) complexity of formulation, 3) reactivity and co-reactivity with
excipients and other formulation materials, 4) adverse biochemical
reactivity during transport, translocation, and uptake into
tissues, and 5) the requirement for metabolic activation via
enzymatic hydrolysis by amidases or peptidases. Therefore, it would
be desirable to have available an alternate physiologically
compatible therapeutic pyruvate compound.
SUMMARY OF THE INVENTION
[0017] The invention described herein provides a new and improved,
accessible composition for the above-indicated uses.
[0018] In one aspect, the invention is directed to a composition
comprising an alkyl, aralkyl, alkoxyalkyl or carboxyalkyl ester of
2-ketoalkanoic acid and a component for inducing and stabilizing
the enol resonance form of the ester at physiological pH values.
The composition of the invention further comprises a
pharmceutically acceptable carier vehicle in which the enol
resonance form of the ester is stabilized at physiological pH
values.
[0019] Preferably, the ester in the composition of the invention is
an alkyl ester of 2-ketopropionic acid (pyruvic acid), most
preferably the ethyl ester, and the stabilizing component is a
cationic material, preferably a divalent cation, and most
preferably calcium or magnesium. The pharmaceutically acceptable
carrier in the composition of the invention can be any carrier
vehicle generally recognized as safe for administering a
therapeutic agent to a mammal, e.g., a buffer solution for
infusion, a tablet for oral administration or in gel, micelle or
liposome form for on-site delivery. A preferred buffer solution is
isotonic or hypertonic saline; or a bicarbonate, phosphate, plasma
extender, microcolloid or microcrystalline solution. Particularly
preferred is Ringer's solution of isotonic saline supplemented with
potassium ion. In a particularly preferred aspect, the composition
of the invention comprises ethyl pyruvate admixed with calcium ion
in a Ringer's solution at a pH in the range of 7-8.
[0020] In other aspects, the ester portion of the 2-ketoalkanoic
acid ester compound in the composition of the invention is selected
preferably from the group consisting of ethyl, propyl, butyl,
carboxymethyl, acetoxymethyl, carbethoxymethyl and ethoxymethyl
esters. The 2-ketoalkanoic acid portion is selected preferably from
the group consisting of 2-keto-butyrate, 2-ketopentanoate,
2-keto-3-methyl-butyrate- , 2-keto-4-methyl-pentanoate and
2-keto-hexanoate.
[0021] In another aspect, the invention is directed to methods for
treating injuries, conditions or disorders associated with events
such as ischemic events or reperfusion. Formulations containing the
novel compositions of the invention permit the successful use of
2-ketoalkanoic acid esters, e.g., pyruvic acid esters, to treat,
e.g., ischemic events, shock, organ reanimation, resuscitation and
other recognized pyruvate-effective treatments as sufficiently high
loads of pyruvate can be administered without a toxic constituent.
Moreover, use of the compositions of the invention provides a
direct replacement for traditional lactated Ringer's solutions
uncomplicated by the addition of co-active ingredients or complex
excipients, such as those comprised of multiple compounds or
molecular derivatives of pyruvate itself. The compositions of the
inventions are also useful in a process for preserving organ parts,
organs or limbs removed from a living mammal and in need of
preservation, e.g., for later transplantation to an organ
recipient. Such processes are well known to those of skill in the
art, e.g., as described in U.S. Pat. No. 5,066,578, hereby
incorporated by reference herein.
[0022] A further practical advantage of the methods of the
invention is the formulation of the active 2-ketoalkanoic acid
ingredient as a biologically safe, readily hydrolyzable ester which
can be taken up into tissues and cells by diffussive processes
through membranes, owing to said ester's greater lipophilicity over
the corresponding salt, while retaining the ability to be
hydrolyzed intracellularly by means of non-specific esterases
and/or non-specific, marginally alkaline solvolysis catalyzed by
organic acids or bases such as amino acid residues at physiological
pH values.
[0023] More importantly, the method of this invention provides
2-ketoalkanoic acids, e.g., pyruvic acid, in a stabilized ester
form that inactivates reactive oxygen species by more than one
mechanism of reaction and whose reaction products with reactive,
hypervalent oxygen, such as hydrogen peroxide, affords degradation
products that themselves are metabolic fuels instead of potentially
harmful excretory products or metabolites.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof and from the claims, taken in conjunction with
the accompanying drawings, in which:
[0025] FIG. 1 shows the structures of the preferred 2-ketoalkanoic
acid esters in the composition of the invention;
[0026] FIG. 2 shows the structures of certain preferred esters in
the composition of the invention, their enol resonance structures
and the structures of certain prior art compounds;
[0027] FIG. 3 shows the system and computational parameters used
for the measurement of mucosal-to-serosal intestinal permeability
following practice of the method of the invention;
[0028] FIG. 4 shows the intestinal permeability results achieved
for a control composition relative to compositions of the
invention; and
[0029] FIG. 5 shows the results obtained for mucosal injury scores
for compositions of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Accordingly, it is a primary object of this invention to
provide new and improved compositions containing 2-ketoalkanoic
acid esters and methods of using them to treat certain conditions
as described above.
[0031] To achieve the foregoing objects and in accordance with the
purpose of the invention, as embodied and broadly described herein,
one novel composition of this invention comprises a 2-ketoalkanoic
acid ester, in accordance with the molecular structures shown in
FIG. 1, admixed with a sufficient concentration of biologically
safe organic or inorganic cations to induce enolization of the
2-keto functionality of the ester at physiological pH values. In a
preferred embodiment, the composition comprises an alkyl ester of
2-ketopropionic acid (pyruvic acid), the ester is the ethyl analog
and the cation is a divalent cation, particularly either calcium or
magnesium. In a particularly preferred formulation of the
composition of the invention, the ester compound is ethyl pyruvate
admixed with calcium ion in a Ringer's solution at a pH of about
7-8.
[0032] The therapeutic compositions of the invention may be
administered orally, topically, or parenterally, (e.g.,
intranasally, subcutaneously, intramuscularly, intravenously,
intraluminally, intra-arterially, intravaginally, transurethrally
or rectally) by routine methods in pharmaceutically acceptable
inert carrier substances. For example, the therapeutic compositions
of the invention may be administered in a sustained release
formulation using a biodegradable biocompatible polymer, or by
on-site delivery using micelles, gels, liposomes, or a buffer
solution. The active ester agent in the composition of the
invention can be administered, as an infusate, at a concentration
of, e.g., 20-200 mM, at a rate of, preferably, 10-100 mg/kg/hr, in
a buffer solution as described herein. In bolus form, the active
ester agent can be administered at a dosage of, e.g., 10-200 mg/kg
from 1-4 times daily. The cation in the composition of the
invention is at an appropriate concentration to induce enolization
of the 2-keto functionality of the amount of active ester agent in
the administered composition. Optimal dosage and modes of
administration can readily be determined by conventional
protocols.
[0033] It is believed that pyruvate, and other 2-ketoalkanoic
acids, when liberated intracellularly from the esters delivered,
e.g., by the reanimation perfusate, acts as a NADH trap and a trap
for ROS generated upon reperfusion. In the first instance, a
2-ketoalkanoic acid reacts to afford lactate, oxidizing excess NADH
and thereby protecting against the "reductant stress" generated
during the physiological insult caused by hypoxia. In the latter
instance, a 2-ketoalkanoic acid reacts with hypervalent oxygen, as
demostrated in the prior art, to form a transient peracid which
decomposes spontaneously, and eventually, to acetate and carbon
dioxide. The resulting acetate is a waste product, which may be
salvaged by re-entry into the acetylCoA pool and harvested
biochemically via intermediary metabolism in the Krebs cycle or via
gluconeogenesis.
[0034] However, and more significantly for the purposes of this
invention, the 2-ketoalkanoic acid ester itself serves as an
antioxidant by a different mechanism, namely, via reaction with
hypervalent oxygen at the enol methylene group. ROS is a membrane
associated process, since hypervalent oxygen is generated by a
redox cascade mediated by cytochromes in the microsomes or the
mitochodria. It is also an intracellular process that takes place
in lipophilic environment rather than in cytosol, and the
thermodynamic properties of a 2-ketoalkanoic acid ester are such
that its reactivity towards redox reaction in a lipophilic phase is
putatively favored by the cation mediated keto-enol equilibrium. Ab
initio and semi-empirical thermodynamic analyses on ethyl pyruvate
as a representative enolizable molecule in the presence of calcium
are discussed in greater detail as part of Example I below.
[0035] For example, using pyruvate as the exemplary 2-ketoalkanoic
acid, formation of transient epoxides and subsequent rearrangement
affords the corresponding hydroxylated pyruvate esters at the
3-carbon, by a mechanism similar to that of 3-hydroxy-pyruvate
formation in intermediary metabolism as well as that of carbon
additions to the phosphoenolpyruvate congener. Hydroxylation alpha
to keto groups is also a recognized cytochrome mediated process in
steroid metabolism and in microsomal hydroxylation of drugs. The
resulting hydroxypyruvates, in turn, when solvolyzed into the
carboxylic anions, can then react once again with hypervalent
oxygen to afford hydroxyacetic acid (glycolic acid), the net result
being that pyruvate esters can ultimately quench two equivalents of
ROS while pyruvates are limited thermodynamically to quenching only
one. As mentioned above, 2-ketoalkanoic acid esters other than
pyruvate esters are also appropriate for use in compositions of the
invention as long as the active compound is metabolizable as
described above for the pyruvate ester.
[0036] The following examples are presented to illustrate the
advantages of the present invention and to assist one of ordinary
skill in making and using the same. These examples are not intended
in any way otherwise to limit the scope of the disclosure.
EXAMPLE 1
Thermodynamic Modeling of Pyruvate Esters
[0037] Semiempirical quantum chemistry permits the comparative
evaluation of various pyruvate analogs with regard to the
properties that determine each molecule's reactivity. As one can
note a marked difference in the biological effect of ethyl pyruvate
versus sodium pyruvate as antioxidants, the hypothesis that these
two molecules are thermodynamically different can be tested by
Huckel Molecular Orbital (HMO) analysis followed by Complete
Neglect of Differential Overlap Analysis (CNDO), using Molecular
Modeling Pro/MOPAC software (ChemSW, Inc. Fairfield, Calif.). The
following results were obtained for the structures shown in FIG. 2,
after their conformations were set by energy minimization to the
optimal conformation:
1TABLE 1 Comparison of Thermodynamic Properties Compound Energy
Dipole LogP H-Acceptor H-Donor Na Pyruvate (1) -31.7 355.6 -85.9
17.8 2.9 Na Pyruvate -16.5 462.8 -71.2 23.9 4.8 Hydrate (2) Na
Enol-pyruvate -30.7 358.0 -72.1 17.7 2.8 (3) Ethyl pyruvate -86.5
2.8 -.21 .73 8.5 (4) Ethyl -84.1 2.5 -.37 .71 7.3 enol-pyruvate (5)
Ca enol ethyl -82.3 2.7 -.41 .85 7.2 pyruvate (6)
[0038] From the trend in minimization energies, the lower and,
therefore, the more stable configurations are those associated with
the pyruvate esters, although the differences all fall within an
order magnitude. On the other hand, the esters show markedly lower
dipole moments, reflecting their relatively weak ionization and
dissociation potentials, a fact that is further supported by the
higher LogP values, which are a measure of relative lipophilicity.
Also, the esters are poorer hydrogen bonding acceptors and better
hydrogen bonding donors, consistent with their dipolar and
lipophilic properties.
[0039] Thus, on an ab initio thermodynamic basis, one would predict
that ethyl pyruvate, and its putative partition enol tautomers, are
more likely to partition between a polar aqueous phase and a lipid
phase, while retaining conformational stability of the same order
as the pyruvate sodium salts. Further, it should be noted that the
coordination complex of the pyruvate enolate ester with a divalent
cation, such as calcium, shown in FIG. 2 as structure 6, affords
the most pronounced change in properties over pyruvate itself,
substantiating the utility of these cation-enolate-ester complexes
as promoters of heretofore unexploited reactivities of the pyruvate
carbon skeleton conformation.
EXAMPLE 2
Reactivity Modeling of Pyruvate Esters
[0040] Searches of the Chemical Abstracts and the ISIS databases
(MDL Information Systems, Inc.) were conducted to uncover actual
examples of the reactivity of pyruvates and their enolates. While
numerous precedents for the reactions of pyruvate salts have been
recorded, far fewer examples of the molecular interactions between
pyruvate esters and hypervalent oxygen are reported in the organic
and biochemical literature. The principal reactions of pyruvates at
physiological pH values are hydrate formation (FIG. 2, structure 2)
and dimerization to para-pyruvate (FIG. 2, structure 7).
[0041] As reported by Margolis et al. (1986), sodium pyruvate at
concentrations of 1 Mol/liter or less forms varying amounts of the
hydrate and the linear dimer, 4-hydroxy-4-methyl-2-ketoglutaric
acid. The hydrate can reach 6-10% and the dimer 20-25% on standing
for 48 hrs. This reactivity pattern is an important consideration
in the evaluation of sodium pyruvate-containing infusates and
perfusates, since the hydrate is unreactive towards hypervalent
oxygen and the dimer is an inhibitor of 2-ketoglutarate
dehydrogenase, a mitochondrial respiratory enzyme, as well as an
inhibitor of glutamate transaminases and lactic acid dehydrogenase.
By contrast, neither hydrate formation nor dimerization of pyruvate
esters have been reported in the chemical literature.
[0042] While the enol forms of pyruvate are thermodynamically
stable in principle, their occurrence in aqueous media is unfavored
and half-lives of enolates are measurable only in the 3-5 sec range
(Kuo et al. (1979)). As the polarity of the solvent decreases,
exemplified by the solvation environment provided by
dimethylsulfoxide or dimethylformamide, the half life of the enol
increases by at least two orders of magnitude (Chiang et al.
(1993); Peliska et al. (1991); Sawyer et al. (1983).
[0043] As to reactivity toward hypervalent oxygen, both pyruvate
salts and pyruvate esters react to form an initial hydroperoxide
intermediate at the carbonyl site, which rearranges by
disproportionation to afford acetic acid and carbon dioxide or
ethoxycarbonic acid, which undergoes subsequent aqueous solvolysis
into carbon dioxide and ethanol (Constantopoulos et al (1984);
Sawyer et al. (1983); Starostin et al. (1980)).
[0044] However, enolpyruvates can also react by an alternate
mechanism that involves addition to the exo-methylene group, as in
the case of enolpyruvate C-bromination at the 3-carbon (Sekine et
al. (1980)), the chelation controlled addition to allylic compounds
(Muderawan et al. (1998)), and the biological addition of carbon
dioxide to form oxaloacetate via phosphoenolpyruvate carboxylase
(Ausenhus et al. (1992)). Enols of biological ketones in general,
as exemplified by D-ring acetyl steroids, react with activated
oxygen via the cytochrome P-450 oxidase system to afford
hydroxyketones via a transient exomethylene epoxide intermediate
(Yamazaki et al. (1997)).
[0045] When evaluated on the grounds of thermodynamic likelihood
and chemical precedent, pyruvate salts can be predicted, via the
REACCS software database correlation system, to react with
hypervalent oxygen to afford only decarboxylation to acetate and
carbon dioxide. Pyruvate esters, on the other hand, can be expected
to afford not only the paired decarboxylation products, acetate and
alcohol, but also hydroxylated adducts at the 3-carbon, most
probably a 3-hydroxypyruvate. These latter species can again react
with hypervalent oxygen to yield glycolic acid and carbon dioxide
(Perera et al. (1997)), thereby consuming two equivalents of
oxidant.
EXAMPLE 3
Stability and Reactivity of Pyruvate Esters in Solution
[0046] Based on the foregoing modeling exercises, the following
hypothesis driven experiments provide verification in chemical and
biological systems and further differentiate the method of this
invention from prior art.
[0047] Ethyl pyruvate affords a more stable aqueous solution than
sodium pyruvate in the presence of calcium salts (Ringer's
solution), and this observation can be extended to the study of
other pyruvate analogs, as shown in FIG. 1, by dissolving them in
Ringer's solution containing at least 0.2 equivalent of calcium per
molar equivalent of pyruvate analog titrated with sodium hydroxide,
or other suitable inorganic alkali, to physiological pH values.
Specifically, the preferred embodiment of this "pyruvated" Ringer's
solution for use in NMR, stability, and subsequent biological
studies is shown in Table 2. It is to be understood that the
pyruvate analog in the instant example may be substituted with any
of the analogs shown in FIG. 1 at any concentration sufficient to
afford a homogenous solution or substituted by control substances
for comparative purposes, such as pyruvic acid, lactic acid (as
would be the case in "lactated" Ringer's solution and other
reference or inactive ketoacid analogs. The calcium cation may also
be substituted, e.g., with magnesium or any other biologically safe
cation capable of substituting for calcium and stabilizing the
formation of transient coordination complexes with pyruvate ester
enolates in aqueous solution.
2TABLE 2 Constituents of Pyruvated Ringer's Solution Component
Composition Range Isotonic saline 75 cc (fixed) KCl 11.25 (fixed)
CaCl.sub.2 7.5 mg 5-20 mg Ethyl pyruvate 0.781 ml 0.5-1.5 ml NaOH
To pH 7.5 7.35-7.55 (pH)
[0048] Following the procedural recommendations for analysis of
Margolis et al. (1986) with respect to scanning times and
frequencies on a 400 MHz spectrometer operating in pulse-Fourier
transform mode, both proton and carbon shifts in the characteristic
resonances for each carbon and proton cluster at the enolizable
carbon were monitored as a function of time and demonstrated that a
greater proportion of pyruvate esters showed a propensity to
enolize in Ringer's solutions, especially those containing calcium
or magnesium, while pyruvate acid anions showed preponderant
hydration and dimerization under similar conditions. The
ultraviolet absorptions of these solutions were also measured
periodically over the 230-260 nm range and 300-340 nm span, where
changes in enol formation become evident, and provided confirmatory
evidence about the distinctly different solvation properties of
pyruvate ester analogs in comparison to pyruvate salts applied in
the various methods of prior art.
[0049] The experimental sequence in which to establish the greater
utility of the pyruvate derivatives in this invention follows along
the same lines as the comparative spectral experiments just
described. The same solutions of test substances used to
demonstrate enolization and related phenomena were also used in the
comparison of basal values for each candidate pyruvate to the
effects of oxidants on the disappearance of characteristic pyruvate
resonances and the appearance of acetate or other degradants of the
initial test preparation as a function of exposure to these
oxidants.
[0050] For example, 1 mMolar solutions of pyruvic acid and ethyl
pyruvate showed average absorption values, corrected for blanks, of
0.15 and 0.2 respectively at 230-260 nm in the absense of calcium
at pH 7.2; addition of calcium had no effect on pyruvate, which
showed only a marginal increase in absorption to 0.16, while ethyl
pyruvate rose twofold to 0.41 in 3 replicate experiments with a
coefficient of variation of less than 15%. When 28 mM solutions
were examined in a similar manner at 300-340 nm, the absorbance of
pyruvate remained unchanged before and after calcium addition at a
value of 0.03, while the ethyl pyruvate solutions become noticeably
straw colored to the naked eye, rising in absorbance from 0.07 to
0.85. The yellow coloration and increases in spectrophotometric
absorption in the ultraviolet region confirms the formation of a
1,3-conjugated ketone system, as would result from the enolization
of ethylpyruvate under conditions which appear not to enolize
pyruvic acid.
[0051] Thus, applications of hypervalent oxygen mimics, whose redox
potential is known to be a model for ROS, such as hydrogen
peroxide, Fenton's reagent, and meta-chloroperbenzoic acid, were
dispensed into the test solutions at concentrations ranging from 1
to 50 mMolar and their degradative effects noted. It was shown that
pyruvate esters consume a greater proportion of oxidant per molar
equivalent than their congeneric free acid analogs.
EXAMPLE 4
Stability and Reactivity of Pyruvate Esters in Tissue Culture
[0052] Pyruvate esters, and in particular ethyl pyruvate, in the
presence of calcium ion are sufficiently lipophilic to be taken up
by cells at a faster rate than equimolar amounts of pyruvate in the
cell preparation perfusate. Moreover, the compounds of this
invention serve as prodrugs for intracellular pyruvate delivery and
are, therefore, utilized as antioxidants in part by direct
decarboxylation of the pyruvate moiety that is delivered
intracellularly and made bioavailable after non-specific ester
solvolysis by ubiquitous cytosolic carboxylesterases. Prior to
hydrolysis intracellularly, these pyruvate beneficially via
enol-mediated, transient epoxidation mediated by hypervalent
oxygen, and related toxic oxidants, to form 3-hydropyruvates.
[0053] The resulting hydroxypyruvate esters, especially in the case
of ethyl pyruvate and its analogs which are depicted in FIG. 1, are
then taken up as a metabolic fuel by anapleurotic incorporation,
after solvolysis, or subjected to further decarboxylative oxidation
by additional equivalents of reactive oxygen species to form the
corresponding hydroxyacetates (glyoxylic acids). Thus, it is to be
understood that pyruvate esters can quench twice as many reactive
oxygen species than the non-enolyzing forms of the corresponding
unesterified ketoacid anion; that is, first by the formation of
3-hydroxypyruvates and then by the latter's decarboxylative
degradation into a smaller metabolite, which like acetate can be
readily incorporated into intermediary metabolism. These outcomes
in which the compounds of this invention prove more effective
antioxidants, as well as metabolic fuels, after exposure to ROS are
demonstrable by combinations of NMR and spectral (UV) analytical
procedures that follow, for example, the fate of stable isotope
labeled pyruvate [3-.sup.13C] species under various experimental
conditions.
[0054] Accordingly, cell and tissue cultures present a effective
means for comparing the relative rates of uptake and subsequent
disposition of pyruvate analogs dispensed into the culture or
perfusion medium and then monitored for incorporation into cells by
means of a stable isotopic tracer that is amenable to proton and
carbon magnetic resonance analysis in real time or by mean of mass
spectral analysis of suitable extracts of the test biomass after a
suitable period of incubation or perfusion.
[0055] In particular, since bowel ischemia is one of the more
damaging conditions for which pyruvates are known to provide rescue
and resuscitation, the use of enterocyte cell cultures provides a
appropriate test model. This model consists of exposing enterocytes
after a basal period under various conditions of anoxia and then
hyperoxia to a perfusate containing Ringer's solution supplemented
with calcium as control and then various tests compositions of
pyruvates, including sodium pyruvate, all labeled at the 3-methyl
position with .sup.13C. For the carbon MR experiments, cells are
seeded on the surface of polystyrene microcarrier beads in
bacteriological Petri dishes and grown for 3 days to confluency
before harvesting and spectroscopic analysis, following the method
of Artemov et al. (1998) and modeling rubrics of Yu et al. (1997)
and of Vogt et al. (1997). The test perfusates during the study
period are also monitored for purposes of background subtraction
from the acquisition of carbon resonances characteristic of the
Krebs cycle.
[0056] Thus, the rate of carbon flux of exogenously added pyruvate
can be followed throughout the process of conversion into citrate
and ketoglutarate/glutamic acid. The 3-carbon of pyruvate and the
2-carbon of acetate, derived from pyruvate, are expected to provide
differential enrichments at the 2 versus the 4 position of citrate
and ketoglutarate. Direct incorporation of the pyruvate carbon
skeleton into citrate and ketoglutarate should be expressed as a
faster increase in label at the 2 position versus the 4 position,
since the latter is more likely to diluted by the larger
acetate-acetyl-CoA pool.
[0057] If hydroxypyruvate is formed in the reaction, not only can
the methyl group resonance be detected directly, but the subsequent
utilization of hydroxypyruvate via decarboxylation into glyoxylate
and homologation to malate can also be traced by the same scheme of
differential labeling analysis. Experiments of this nature confirm
that pyruvate esters act differently as a carbon source from
pyruvate salts. Furthermore, such experiments confirm that lactate,
acetoacetate and related esters, when substituted for pyruvate
esters, do not show enolization and are not incorporated into cells
and/or processed via oxidative metabolism in a manner similar to,
and to the extent of, the pyruvate esters used in the method of
this invention.
EXAMPLE 5
Application of the Invention in Ischemia Rescue
[0058] The utility of ethyl pyruvate in a Ringer's solution
infusate as a resuscitation fluid in ischemia/reperfusion mucosal
injury and barrier dysfunction is demonstrated in this illustrative
experiment using a rat model of superior mesenteric artery
occlusion. The model system and calculation parameters are
illustrated in FIG. 3.
[0059] After induction of general anesthesia using intraperitoneal
ketamine and pentobarbital, male Sprague-Dawley rats (250-350 g)
were subjected to 60 minutes of superior mesenteric artery
occlusion followed by 60 minutes of reperfusion. Heart rate and
mean arterial blood pressure were measured via a right carotid
arterial catheter. The left internal jugular vein was cannulated
for intravenous infusions.
[0060] Controls (n=6) received lactated Ringer's solution (lactate,
28 nM, 111.5 ml/kg/hr infusion, 1.5 ml/kg bolus prior to ischemia,
and 3.0 ml/kg bolus prior to reperfusion). Experimental groups (n=6
each) received similar volumes (3 ml) of either pyruvate Na salt
(28 mM) or pyruvate ethyl ester (28 mM), prepared in accordance
with the method of this invention as shown in Table 2 and at a
dosage rate equivalent to 10 mg/kg/hr. Small intestinal
mucosal-to-serosal permeability (CMS, nl/min/cm.sup.2) of
FITC-dextran (mw=4 kDA) was evaluated using an everted gut sac
technique as previously described by Wattanasirichaigoon (1999).
Permeability was measured at baseline, after 30 and 60 minutes of
ischemia (130 and 160) respectively, and after 30 and 60 minutes of
reperfusion (R30 and R60, respectively). Histologic samples at
baseline, 160 and R60 were evaluated for villous height (VH, .mu.)
and mucosal thickness (MT, .mu.). Mucosal injury grade was
determined according to the method described by Chiu et al. (1970),
scored as in Table 3, as follows:
3TABLE 3 Mucosal Injury Grade Grade 0 Normal Mucosa Grade 1
Subepithelial space formation Grade 2 Moderate epithelial lifting
confined to the tip of the villi Grade 3 Extensive epithelial
lifting, a few tips are denuded Grade 4 Denuded villi, dilated
exposed capillaries, increased cellularity in the lamina propria
Grade 5 Hemorrhagic ulceration
[0061] Data were summarized as means.+-.standard error of the mean.
Significances of differences were determined using Student's
t-test. Differences were considered significant for p<0.05.
[0062] The results of these experiments on the utility of the
method of invention revealed that both pyruvate compositions, as
free acid as well as ethyl ester, significantly decreased mucosal
permeability during reperfusion, as shown in FIG. 4. The ester
showed a significant trend towards effecting earlier and greater
cytoprotection as judged by the extent of permeability increase,
which is a sign of irreversible tissue damage and in terms of the
significant diminution in mucosal injury score, shown graphically
in FIG. 5. Pyruvate ethyl ester, moreover, significantly maintained
villous height and mucosal thickness during both ischemia and
reperfusion (p<0.01) as shown in Table 4:
4TABLE 4 Histological Findings on Beneficial Effects of "Pyruvated"
Ringer's Solution Pyruvate Lactate Pyruvate Ester VH MT VH MT VH MT
Baseline 470 .+-. 30 553 .+-. 34 461 .+-. 25 524 .+-. 28 486 .+-.
12 583 .+-. 8 I60 244 .+-. 20 298 .+-. 32 290 .+-. 30 372 .+-. 36
381 .+-. 24.sctn. 466 .+-. 25.sctn. R60 130 .+-. 25 141 .+-. 22 201
.+-. 44 266 .+-. 50 296 .+-. 26.sctn. 352 .+-. 34.sctn. Note:
Lactate vs Pyruvate and Lactate vs Pyruvate Ester, p < 0.05 and
.sctn.p < 0.01
[0063] Taken as a whole, these findings confirm the utility of
pyruvate esters in the method of this invention in compositions for
the treatment of ischemia and related conditions caused by hypoxia
and then reperfusion, with its attendant reactive oxygen damage.
The model system described above, a rat model of superior
mesenteric artery occlusion, is a standard model system familiar to
those of ordinary skill who wish to provide therapeutic treatment
of the kind described, and the results reported above are easily
extrapolatable for human use.
[0064] Thus, it is apparent that there has been provided, in
accordance with the invention, novel 2-ketoalkanoic acid ester
compounds and compositions and methods of treating the deleterious
effects of hypervalent oxidants resulting from hypoxic damage,
followed by reperfusion, that fully satisfies the objects, aims and
advantages set forth above.
[0065] While the invention has been described in conjunction with
specific embodiments thereof, it is evident that many alternatives,
modifications, and variations will be apparent to those skilled in
the art in light of the foregoing description. Accordingly, it is
intended that the invention shall be directed to all such
alternatives, modifications and variations as fall within the
spirit and broad scope of the appended claims.
* * * * *