Note: Descriptions are shown in the official language in which they were submitted.
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"FRUCTOSAMINE OXIDASE: ANTAGONISTS AND INHIBITORS
THE CURRENT INVENTION
The present invention relates to methods of treatment, pharmaceutical
compositions, dosage forms, uses of fructosamine oxidase enzyme inhibitors in
medicine or for manufacturing pharmaceutical compositions, treatment regimes
and
related combinations, methods and products.
Diabetes mellitus is a common disorder affecting nearly 16 million
Americans. See, Report of the Expert Committee on the Diagnosis and
Classification
of Diabetes Mellitus. Diabetes Care, 20;1183-97 (1997). Diabetic individuals
are
prone to complications which are a major threat to both the quality and the
quantity
of life. Almost half those diagnosed with diabetes before the age of 31 years
die
before they reach 50 years largely as a result of cardiovascular or renal
complications, often with many years of crippling and debilitating disease
beforehand. See, Deckert T, Poulsen J, Larsen M. Diabetologia 14:363-70
(1978). It
is estimated that diabetic individuals have a 25-fold increase in the risk of
blindness,
a 20-fold increase in the risk of renal failure, a 20-fold increase in the
risk of
amputation as a result of gangrene, and a 2- to 6-fold increased risk of
coronary heart
disease and ischaemic brain damage. See, Klein R, Klein B, Moss S, Davis M,
DeMets D. Diabetes Care 8;311-5 (1985).
Largely because of these long-term complications, the cost of diabetes in the
US was estimated as $98 billion in 1997 comprising $44 billion for direct
medical
costs such as inpatient and outpatient care plus $54 billion for indirect
costs such as
lost earnings and productivity, and premature death. Medical innovations that
can
slow the progression of diabetes have tremendous potential to mitigate the
associated
clinical and cost repercussions See, American Diabetes Association, "Economic
consequences of diabetes in the US in 1997," Diabetes Care 21:296-309(1998).
Elevated blood glucose levels are now regarded as causative of diabetic
complications based on results of the Diabetes Complications and Control Trial
(DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS). See, NEng
JMed. 379:977-85 (1993) and Lancet 352:837-53 (1998). The DCCT and the
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UKPDS have both demonstrated that the development of complications of diabetes
are related with degree of hyperglycaemia and that long-term outcome may be
ameliorated by rigorous treatment. For example, prognosis is dramatically
improved
if capillary blood glucose and glycated haemoglobin levels are maintained less
than
150mg/dL and 7.0% respectively.
The mechanism of glucose toxicity in the tissues of patients with diabetes
mellitus is unknown. Glucose condenses with free amino groups on structural
and
functional proteins to form Schiff bases which, in turn, undergo a series of
transformations to yield dark brown Maillard products. It has been proposed
that
diabetes complications are caused by the non-enzymatic cross-linking of
proteins.
See, Cerami A, Ulrich PC, Brownlee M, US Patent 4758583 (1988). However,
although increased protein cross-linking is seen in the tissues of people long-
standing diabetes, the role of Maillard products as a causative factor is
certainly not
clear. See, Wolff SP, Jiang ZY, Hunt JV. Free Rad Biol Med 10;339-52 (1991).
Amadori-rearrangement is the most important Maillard transformation
because its product, fructosamine, is the precursor of all the browning
products. We I
have isolated a novel extracellular enzyme which catalyses the elimination of
fructosamines from glycated protein. The existence of this enzyme has not
previously been recognised in the world literature. Based on its high
specificity for
glycated protein substrates and its use of oxygen as acceptor, the enzyme may
be
classified as fructosamine oxidase 1.5.3. See, Enzyme Nomenclature,
Recommendations of the Nomenclature Committee of the International Union of
Biochemistry, Academic Press, London pp. 19-22, (1979).
Fructosamine oxidase is a metalloenzyme with copper & quinone cofactors
and it belongs to the copper amine oxidase group of enzymes which have been
isolated from bacteria, fungi, yeast, and mammalian sera. Products of the
fructosamine oxidase catalysed reaction are free unglycated protein, a-
dicarbonyl
sugar, and the active oxygen species superoxide.
Increased fructosamine oxidase activity could cause many of the recognised
sequelae of diabetes by degrading fructosamines bound to basement membrane
proteins and generating reactive oxygen species as reaction products. For
example,
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superoxide anions cause an increase in intracellular calcium which modulates
the
activity of nitric oxide synthase. Nitric oxide is a potent vasodilator and it
has been
implicated in the vascular dysfunction of early diabetes. See, Ido Y, Kilo C,
Williamson JR. Nephrol Dial Transplant 11 Suppl 5:72-5 (1996). Reactive oxygen
species also cause a drastic dose-dependent decrease in de novo synthesis of
heparan
sulfate proteoglycans leading to a reduction in anionic sites on the
glomerular
basement membrane and an increase in basement membrane permeability to
cationic
plasma proteins such as albumin. See, Kashira N, Watanabe Y, Makin H, Wallner
EI, & Kanwar YS. Proc Natl Acad Sci USA 89:6309-13 (1992). Increased urinary
albumin clearance is a risk indicator in people with diabetes mellitus both
for
evolving renal disease and for early mortality mainly from coronary heart
disease.
See, Mattock MB, Barnes DJ, Viberti GC, et al. Diabetes 47:1786-92 (1998).
Once natural anti-oxidant defences are exceeded, hydroxyl radicals may be
generated from superoxide via a copper catalysed Haber-Weiss reaction. See,
Halliwell B & Gutteridge JMC "Free radicals in Biology and Medicine" Clarendon
Press, Oxford pp. 136-76 (1989). Hydroxyl radicals are extremely reactive
species
and could cause the permanent site-specific damage to basement membrane
proteins
and histopathological changes that are typical of diabetic microvascular
disease. See,
Robbins SL, Cotran RS, Kumar V. "Pathologic basis of disease" 31 ed WB
Saunders, pp. 991-1061. (1984).
Similarly, any prolonged increase in fructosamine oxidase activity will cause
oxidative stress which could account for the excess risk of macrovascular
disease
and the 75% increase in mortality seen in patients with diabetes mellitus
compared
with non-diabetic individuals. Recent studies have convincing demonstrated
that
oxidative modification of low density lipoprotein (LDL) is involved in the
development of atherosclerosis of coronary and peripheral arterial vessels and
elevated oxidised LDL concentrations are found in subjects with diabetes
mellitus.
See, Witztum JL Br Heart J 69 (Suppl):S 12-S 18 (1993) and Picard S, Talussot
C,
Serusclat A et. al. Diabetes & Metabolism 22:25-30 (1996). Oxidative changes
to
membrane lipids and to membrane protein SH-groups may also cause aberrations
in
cellular calcium homeostasis and contribute to the increased incidence of
cardiac
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sudden death that is typical of diabetes. See, Yucel D, Aydogdu S, Cehreli S
et al.
Clin Chem 44:148-54 (1998).
SUMMARY OF THE INVENTION
The existence of the fructosamine oxidase enzyme has not previously been
recognised in the world literature. This is a novel enzyme. I believe that
excess
fructosamine oxidase activity with glycated basement membrane protein
substrate
plays a vital role in diabetic complications by the formation of a-dicarbonyl
and
reactive oxygen free radical species.
I also believe that this damage may be ameliorated by administering specific
fructosamine oxidase inhibitors or antagonists selected from the groups: (i)
copper
chelating agents; (ii) substrate analogues; & (iii) hydrazine compounds.
As used herein (including in the claims) the terms -
= "copper chelating agents" means any agent which reduces body fructosamine
oxidase activity (e.g. by depleting body copper stores or by binding and
inactivating the copper molecule at the reactive centre of the enzyme) which
is capable of being administered in effective amounts by any appropriate
administration route (eg. orally, by injection etc). See some examples
hereafter referred to.
= "substrate analogue" means any chemically modified amino acid or peptide
substrate which inactivates fructosamine oxidase (e.g. by binding (preferably
substantially) irreversibly to the. reactive centre of the enzyme) which is
capable of being administered in effective amounts by any appropriate
administration route (eg. orally, by injection etc). See some examples
hereafter referred to.
= "hydrazine compound" means any agent containing the moiety -NH-NH2
which inactivates fructosamine oxidase (e.g. by binding and inactivating the
quinone molecule at the reactive centre of the enzyme) which is capable of
being administered in effective amounts by any appropriate administration
route (eg. orally, by injection etc). See some examples hereafter referred to.
= "at least periodically" includes from a single administration to continuous
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administration.
= "macrovascular and microvasular damage" refers to both general types of
damage but can refer to one general type of such damage provided there is a
view to minimising or ameliorating the consequences of and/or likelihood of
such category of damage.
= "comprises can mean "includes".
= "and/or" means both "and" and "or".
= "in concert with" does not necessarily mean as a result of simultaneous
administration or self administration (eg; can be serially and such serial
application can be spaced, ie; triene between meals and another agent with a
meal).
= "triethylenetetramine dihydrochloride" or "triene" includes for the target
mammalian species or for a human being any pharmaceutically acceptable
fructosamine oxidase enzyme inhibiting and/or antagonising analogue or
metabolite thereof (eg; an acetylated derivative) capable of administration or
self administration in an amount alone, or in concert with another
fructosamine oxidase enzyme inhibitor and/or antagonist (preferably not
contraindicated by toxicity concerns having regard to levels required for
effective inhibition and/or antagonism), of providing effective inhibition
and/or antagonism.
In one aspect the present invention consists in a method of treating a
mammalian patient (human or otherwise) predisposed to and/or suffering from
diabetes mellitus with a view to minimising the consequences of macrovascular
and
microvascular damage to the patient (eg. accelerated atherosclerosis,
blindness, renal
failure, neuropathy, etc.) which comprises, in addition to any treatment in
order to
control blood glucose levels, at least periodically inhibiting or antagonising
fructosamine oxidase enzyme activity in the patient.
Preferably said inhibition or antagonism occurs as a result of administration
or self-administration of at least one fructosamine oxidase reaction product
inhibitor
or antagonist.
Preferably any such inhibitor or antagonist is selected from the groups
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(i) copper chelating agents
(ii) substrate analogue
(iii) hydrazine compound
Preferably said inhibitor or antagonist is taken orally.
Preferably said inhibitor or antagonist is taken orally as part of a regime,
whether totally oral or not, which also involves the control of blood glucose
levels.
In a further aspect the present invention consists in a pharmaceutical
composition (preferably oral) suitable for use in such a method, said
composition
comprising a fructosamine oxidase inhibitor or antagonist in conjunction with
a
suitable carrier therefor.
In yet a further aspect the present invention consists in a pharmaceutical
composition for reducing macrovascular and microvascular damage in a mammalian
patient (including a human) suffering from diabetes mellitus, said composition
comprising a fructosamine oxidase inhibitor or antagonist and suitable carrier
therefor.
Preferably said carrier can be any diluent, excipient or the like and the
dosage
form of said pharmaceutical composition can be of any appropriate type whether
for
oral or other administration or self administration. Long acting release forms
are also
envisaged within the present invention.
In still a further aspect the present invention consists in the use of a
fructosamine oxidase inhibitor or antagonist in the manufacture of a
pharmaceutical
composition comprising the fructosamine oxidase inhibitor or antagonist and a
suitable pharmaceutical carrier therefor and which composition is useful in
treating a
mammalian patient (human or otherwise) which or who is suffering from diabetes
mellitus to reduce macrovascular and microvascular damage (preferably by a
method
of the present invention).
In still a further aspect the present invention consists in combination, the
treatment regimes and/or the medicaments of such regimes previously set forth
whether packed together or prescribed together or otherwise.
In still another aspect the invention consists in a method of treating a
mammalian patient (human or otherwise) predisposed to and/or suffering from
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diabetes mellitus, which includes inhibiting or antagonising fructosamine
oxidase
enzyme activity in the patient with an agent or agents preferably not
contraindicated
for the patient. Examples of inhibitor or antagonist include but are not
limited to
those listed hereinafter.
Preferably in one embodiment said agent(s) is or are copper chelating
compound(s) administered or self administered to the patient.
Examples of suitable copper-chelating compounds include
triethylenetetramine dihydrochloride (triene), penicillamine, sar, diamsar,
ethylenediamine tetraacetic acid, o-phenanthroline, & histidine.
Preferably in another embodiment said agent(s) is or are substrate analogue
compound(s) administered or self administered to the patient having an amino
acid
or peptide moiety with a blocked N-terminal amine group.
Examples of a suitable substrate analogue composition is N-acetylcysteine,
captopril, lisinopril & enalapril).
Preferably in another embodiment said agent(s) is or are hydrazine
compound(s) administered or self administered to the patient ie: a compound
having
a -NHNH2 moiety.
Examples of a suitable hydrazine compound include diaminoguanidine,
hydralazine, & carbidopa.
In still another aspect, the invention consists in a dosage regimen for a
method
of the present invention and/or using dosage units of the present invention
In still a further aspect, the present invention consists in the use of a
pharmaceutical acceptable compounds being at least one of a substrate
analogue, a
hydrazine compound and a copper chelator in the manufacture of a dosage unit
or
pharmaceutical composition useful in treating a patient (human or otherwise)
which
or who is suffering from diabetes mellitus to reduce macrovascular and
microvascular damage.
In another aspect, the invention consists in a dosage unit or pharmaceutical
composition for a patient useful in a method of the present invention
comprising
(preferably in effective fructosamine oxidase reaction product inhibiting or
antagonising amounts - separately or collectively) of
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a compound (or compounds) being a substrate analogue or a hydrazine
compound having an -NHNHZ moiety, or both.
Preferably said dosage unit also includes or said pharmaceutical composition
also includes a compound (or compounds) (preferably different compound(s))
which
is (or are) a copper chelator (or copper chelators).
Preferably said dosage unit or composition is in an oral dosage form
optionally with carriers, excipients or, indeed, even other active agents
(e.g. means
to lower blood glucose levels).
In still another aspect the invention consists in a regime or dosage unit or
pharmaceutical composition for a diabetic or suspected diabetic patient of the
copper
chelator, triene, providing for the patient a sufficient fructosamine oxidase
inhibiting
and/or antagonising effect to reduce macrovascular and microvascular damage.
In still another aspect the invention consists in a regime or dosage unit or
pharmaceutical composition for a diabetic or suspected diabetic patient of
captopril [whether effective or intended to be effective in controlling blood
pressure
of the diabetic patient (at least in part) or not] providing for the patient a
sufficient
fructosamine oxidase inhibiting and/or antagonising effect to reduce
macrovascular
and microvascular damage.
In yet another aspect the invention consists in a regime or dosage unit or
pharmaceutical composition for a diabetic patient or suspected diabetic
patient of a
hydrazine compound providing for the patient a sufficient fructosamine oxidase
inhibiting and/or antagonising effect to reduce macrovascular and
microvascular
damage.
In yet another aspect the invention consists in a regime or dosage unit or
pharmaceutical composition for a diabetic patient or suspected diabetic
patient of
(i) acetylcysteine and
(ii) at lease one other fructosamine oxidase inhibitor and/or antagonist, the
mix of (i) and (ii) providing for the patient a sufficient fructosamine
oxidase inhibiting and/or antagonising effect to reduce macrovascular and
microvascular damage.
In yet another aspect the invention consists in a regime or dosage unit or
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pharmaceutical composition for a diabetic patient or suspected diabetic
patient of
(i) hydralazine and
(ii) at lease one other fructosamine oxidase inhibitor and/or antagonist, the
mix of (i) and (ii) providing for the patient a sufficient fructosamine
oxidase inhibiting and/or antagonising effect to reduce macrovascular and
microvascular damage.
In still another aspect the present invention consists in a method of treating
a
mammalian patient (human or otherwise) predisposed to and/or suffering from
diabetes mellitus which includes inhibiting and/or antagonising fructosamine
oxidase
enzyme activity in the patient with acetycysteine and hydralazine.
In still another aspect the invention consists in a regime or dosage unit or
pharmaceutical composition for a diabetic or suspected diabetic patient which
includes-acetylcysteine and hydralazine.
In still a further aspect the present invention consists in the use of co-
administration or serial administration of acetylcysteine and hydralazine for
the
purpose of reducing macrovascular and microvascular damage in a mammal.
Preferably said mammal is diabetic.
In yet another aspect the invention consists in a method of treating and/or
reducing the likelihood of diabetic cataract in a mammal which comprises at
least
periodically inhibiting and/or antagonising fructosamine oxidase enzyme
activity in
the mammal.
Preferably the method involves the administration or self administration of
effective amounts of triethylenetetramine dihydrochloride (triene).
In another aspect the invention consists in a method of treating and/or
reducing
the likelihood of diabetic cardiomyopathy in a mammal which comprises at least
periodically inhibiting and/or antagonising fructosamine oxidase enzyme
activity in
the mammal.
Preferably the method involves the administration or self administration of
effective amounts of triethylenetetramine dihydrochloride (triene).
Preferably for any of the aforesaid indications triethylenetetramine
dihydrochloride (triene) is administered and/or self administered in concert
with
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another (other) fructosamine oxidase enzyme inhibitor(s) and/or antagonist(s).
Preferably said another inhibitor and/or antagonist or said other
inhibitors and/or antagonists is or are administered or self administered to
elicit
a pharmacological effect for another indication yet together with the effect
of the
triene is or are in an amount or amounts which are effective for treating or
ameliorating macrovascular and microvascular damage of such a patient or
mammal.
Reference is drawn to PCT/NZ99/00160, published as WO 00/18891. It
discloses methods of monitoring fructosamine oxidase inhibition and/or
antagonism
of patients, screening and/or determining patients at risk to vascular
(particularly
microvascular) damage and identifying those individuals who will benefit by
treatment with fructosamine oxidase inhibitors and/or antagonists, methods of
determining fructosamine oxidase levels in a mammal, methods of determining
blood plasma fructosamine oxidase levels in a diabetic individual or a
suspected
individual, methods of assaying blood serum or blood plasma in vitro for
fructosamine oxidase, methods of identifying or testing candidate substances
and to
related methods and procedures.
Preferably the measurement conducted in vitro is of the superoxide
reaction product (or any other oxygen free radical product) of fructosamine
oxidase.
In another aspect, the present invention provides use of a pharmaceutically
acceptable amount of a copper binding triene compound for treating a human for
diabetes mellitus.
In another aspect, the present invention provides use of a triene that binds
copper in an amount ranging from 1.2 to 2.4 grams per day for treating a human
for
diabetes mellitus.
In another aspect, the present invention provides use of a triene that binds
copper in an amount ranging from 17 mg/kg to 35 mg/kg per day for treating a
human for diabetes mellitus.
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In another aspect, the present invention provides use of an effective amount
of fructosamine oxidase inhibitor for treating an individual with diabetes
mellitus.
In another aspect, the present invention provides use of an effective amount
of a combination of fructosamine oxidase inhibitors to inhibit fructosamine
oxidase
activity associated with the diabetic state for treating an individual with
diabetes
mellitus.
In another aspect, the present invention provides use of an effective amount
of copper chelator for treating a human patient with diabetes mellitus.
In another aspect, the present invention provides use of triene in an amount
effective to ameliorate macrovascular and microvascular damage in the patient
for
treating a human patient with diabetes mellitus.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a detailed reaction mechanism for the formation of
fructosamine and Maillard products from glucose and protein. Fructosamine
oxidase
degrades fructosamine by a two-step reaction with initial release of an a-
dicarbonyl
sugar and subsequent oxidation of the enzyme/protein complex to release free
unglycated protein. The reduced copper cofactor is oxidised in vivo by
molecular
oxygen and the oxidation product is superoxide.
Figure 2 shows absorbance spectra of the fructosamine oxidase enzymes
extracted from pooled human sera (A) and from the microbial organism,
Enterhacter
aerogenes (B).
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In another aspect, the present invention provides use of an effective amount
of a combination of fructosamine oxidase inhibitors to inhibit fructosamine
oxidase
activity associated with the diabetic state for treating an individual with
diabetes
mellitus.
In another aspect, the present invention provides use of an effective amount
of
copper chelator for treating a human patient with diabetes mellitus.
In another aspect, the present invention provides use of triene in an amount
effective to ameliorate macrovascular and microvascular damage in the patient
for
treating a human patient with diabetes mellitus.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a detailed reaction mechanism for the formation of
fructosamine and Maillard products from glucose and protein. Fructosamine
oxidase
degrades fructosamine by a two-step reaction with initial release of an a-
dicarbonyl
sugar and subsequent oxidation of the enzyme/protein complex to release free
unglycated protein. The reduced copper cofactor is oxidised in vivo by
molecular
oxygen and the oxidation product is superoxide.
Figure 2 shows absorbance spectra of the fructosamine oxidase enzymes
extracted from pooled human sera (A) and from the microbial organism,
Enterbacter
aerogenes (B).
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Figure 3 shows spectra ofp-nitrophenylhydrazine (NPH) adduct of the
Enterbacter aerogenes enzyme (A) and a red absorbance shift when the NPH-
enzyme adduct is diluted in 2M KOH.
Figure 4 shows survival curve for non-treated STZ-diabetic rats compared
with diabetic animals treated with fructosamine oxidase inhibitors.
Figure 5 shows monthly growth of treated and untreated STZ-diabetic rats
compared with non-diabetic animals.
DETAILED DESCRIPTION OF THE INVENTION
(i) Extraction of holoenzyme
Fructosamine oxidase in blood plasma is largely found as an enzyme-substrate
conjugate, bound to peptides and proteins (Fig 1). To obtain a maximal yield
of
active holenzyme, it is necessary to make the pH of the media alkaline
preferably
with phosphate buffer, to add sulphydryl reagents, and to incubate the mixture
with
pro-oxidant so that glycated species are released. Most effective activation
is found
with cupric salts.
Fructosamine oxidase holenzyme is separated from inactive apoenzyme by
affinity adsorption chromatography. A suitable glycated affinity support is
prepared
from alkylamine beads or beaded cross-linked agarose with amino terminal
residues
attached by 6-10 atom spacer arms (available from PierceTM, Bio-RadTM, &
PharmaciaTM). Affinity support is glycated by incubating with 400mM potassium
phosphate buffer pH 7.4 containing 50mM glucose and 0.01% sodium azide at 37
C
for 7 days. Holoenzyme binds tightly to glycated amino residues and residual
copper
is readily removed by washing with water. Active holoenzyme is eluted with
800mM NaC1 in 50mM sodium acetate buffer pH 4.8. Active fractions are pooled
and protein is precipitated with 50% cold acetone solvent. The protein pellet
is
reconstituted with a minimal volume of water or physiological saline and
lyophilised
for long term storage.
Extraction of 35mL pooled diabetic and non-diabetic human sera yielded a clear
colourless preparation with absorbance peaks at 196nm & 264nm typical of the
absorbance spectra of fructosamine oxidase (Fig 2). A fructosamine oxidase
enzyme
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from Enterobacter aerogenes showing absorbance peaks at 196nm & 255nm is
included for comparison. Enzyme activity and relative activity is as follows.
TABLE 1.
Sample Protein Cytochrome c activity* Sp activityt
(pglmL) (U/L) (Ulg)
human 32.9 4.58 139.4
E aerogenes 541.5 66.32 115.11
* Enzyme extract was preincubated in 0.05M TES buffer pH 7.4 containing 1mM
DMF substrate at 37
C for 5 minutes. Enzyme activity was measured with 10 M ferricytochrome C. The
reaction was
started with 50 M fructosamine substrate as g-BSA and AA550nm was determined
over 5 minutes.
t Protein concentration determined from A oõm A22Dnm. compared with BSA
standards.
(ii) Cofactor identification
The p-nitrophenylhydrazine (NPH) adduct of Enterbacter aerogenes enzyme
with A,,õx 399nm was obtained as described previously. See, Palcic MM, Janes
SM.
Meth Enzymol 258:34-8 (1995). A red absorbance shift to ARõX 438nm was
observed
when the NPH-enzyme adduct was diluted in 2M KOH. Such an absorbance shift is
typical of the quinone cofactors of copper amine oxidase.
EXAMPLE 1: Identifying fructosamine oxidase inhibitors
The purpose of this example is to demonstrate how the fructosamine oxidase
assay, the subject of PCT/NZ99/00160, published as WO 00/18891, may be
used in identifying and grading candidate fructosamine oxidase
inhibitors. This approach takes into account the activity of the drug in a
human
plasma matrix in vitro. Enzyme inhibitors find wide application in clinical
medicine
as treatments for a range of metabolic disorders. For example, angiotensin
converting enzyme inhibitors have been used in the treatment of hypertension.
See,
Harris EE, Patchett AA, Tristram EW, & Wyvratt MJ. Aminoacid derivatives as
antihypertensives. US Patent 4374829 (1983). Similarly, 3-hydroxy-3-
methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme inhibitors have been
used in the treatment of hypercholesterolaemia. See, Hoffman WF, Smith RL, Lee
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TJ. Novel HMG-CoA reductase inhibitors. US Patent 4866090 (1989). Fructosamine
oxidase inhibitors may be selected from those substances which bind and block
the
quinone co-factor (hydrazine compounds), the copper co-factor (copper
chelators),
or which mimic the normal substrate of the enzyme (substrate analogue).
Method:
Potential fructosamine oxidase inhibitors were tested on human serum or
plasma (individually and in combination) using the method of assaying
fructosamine
oxidase activity described in detail in PCT/NZ99/00160, published as WO
00/18891.
Irreversible enzyme inhibition is characterised by a progressive decrease in
activity with time ultimately reaching complete inhibition even with very
dilute
inhibitor concentrations provided that the inhibitor is in excess of the
amount of
enzyme present.
Results:
The relative activity of a selection of hydrazine, copper chelator, and
substrate
analogue Fructosamine oxidase inhibitors are shown in TABLE 2. In some
instances, there was a degree of overlap between classes i.e. some hydrazine
compounds are also copper chelators. To clarify this point, copper chelating
potential for some compounds is indicated (P). The effectiveness of the
inhibitor is
expressed not by an equilibrium constant but by a velocity constant (K) which
determines the fraction of the enzyme inhibited in a given period of time by a
certain
concentration of inhibitor. The specificity of the inhibitor for the active
centre of the
enzyme is indicated by the concentration of inhibitor CAUSING 50% inactivation
of
the enzyme (ICS.).
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TABLE 2
Inhibitor: Hydrazine compounds ICS,' K(min'')z 33
aminoguanidine 231.2pM 0.0067* -
semicarbazide 45.1 pM 0.0276* +++
benserazide 13.6pM 0.0095*
oxalic dihydrazide 1.59pM 0.0542
hydralazine 1.52pM 0.0029 ++
phenylhydrazine 0.81 pM 0.1160
carbidopa 0.50pM 0.1496
diaminoguanidine 0.36pM 0.1340
Inhibitor: Substrate analogues
lisinopril 216.9pM 0.0174
enalapril 3.95pM 0.0326 +++
captopril 1.78pM 0.0259
acetylpenicillamine 1.06pM 0.0811
acetylcysteine 0.83pM 0.1677
Inhibitor: Copper chelators
desferrioxamine 40.6pM 0.0109*
EDTA 15.7pM 0.0755*
Sodium azide 9.48pM 0.0004
Potassium cyanide 6.36pM 0.0116
triene. 5.4OpM 0.0196
o-phenanthroline 4.25pM 0.0385
histidine 2.29pM 0.0554
Inhibitor: Combined agents
acetylcysteine + hydralazine 0.57pM 0.1654
acetylcysteine + diaminoguanidine 1.07pM 0.0795
acetylcysteine + histidine 1.11 PM 0.0722
acetylcysteine + carbidopa 0.27pM 0.2000
1 fresh human sera was incubated with 0-1,000pM inhibitor in 0.05M TES buffer
pH 7.4 at 37 C for 5
minutes. Enzyme activity was measured with I OpM ferricytochrome c. The
reaction was started with
5OpM fructosamine substrate as g-BSA and AA550nm was determined over 5
minutes.
2 rate constants were calculated from the reaction of fructosamine oxidase
either with 1.OpM inhibitor or
with 10.OpM inhibitor (*).
3 copper chelating potential (0) was determined from ability of agent to
remove copper under dialysis
from copper-saturated BSA substrate.
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CONCLUSION:
1. Irreversible inhibition of fructosamine oxidase is feasible.
2. Inhibitors may be broadly categorised in three classes of compound:
hydrazines; substrate analogues; & copper chelators.
3. Fructosamine oxidase activity in human blood plasma may be eliminated by
micromolar concentrations of inhibitors.
4. Many of the active inhibitors are drugs which have already been
administered
as medicines in humans to treat other disorders (not diabetes).
EXAMPLE 2: Clinical Utility of fructosamine oxidase inhibition:
First Preclinical study
The purpose of this example is to demonstrate how the clinical usefulness of
candidate fructosamine oxidase inhibitors may be assessed using a standard
animal
model of diabetes mellitus, the streptozocin-diabetic rat (STZ rat). This
approach
takes into account drug bioavailability, the activity of the drug and its
metabolites,
and any drug adverse effects or toxicity factors.
Method:
48 WISTAR rats aged 6-8 weeks & weighing 200-300g were randomized:
Group 1 Non-diabetic control
= Group 2 Diabetic control
= Group 3 Diabetic treated with hydralazine
= Group 4 Diabetic treated with EDTA
= Group 5 Diabetic treated with hydralazine &
acetylcysteine
= Group 6 Diabetic treated with acetylcysteine.
Streptozotocin (60mg per kg) was administered into a lateral tail vein. Non-
diabetic controls received a sham injection of buffer. Diabetes was confirmed
by
venous blood glucose measurement >15mmol/L after 1 week & diabetic animals
were treated with subcutaneous injections of ultralente insulin (4U/injection)
3-5
days per week to maintain body growth. Medications were administered 50mg/L in
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the drinking water over an 8 month period. Timed urine collects and venous
plasma
samples were obtained at monthly intervals.
Results:
(a) Blood glucose control: Rate of conversion to diabetes with intravenous STZ
administration was >95%. Intravenous STZ induced a severe form of insulin-
dependent diabetes which was sustained over the entire 8 month duration of the
study. Despite insulin replacement therapy, glycaemia control was poor as
evidenced
by mean SD glucose (week 4) and HbA,C (week 32) levels in TABLE 3.
TABLE 3.
Group I Group 2 Group 3 Group 4 Group 5 Group 6
Glucose (mmol/L) 9.1 1.5 30.1 9.7 35.7 9.5 39.0 6.4 30.4 8.8 37.8 5.2
HbAj. (%) 3.92 0.11 10.85 0.05 8.65 1.18 9.30 0.63 8.72 0.55 9.47 1.23
(b) Survival: Mortality rate amongst untreated STZ rats was extremely high.
Survival was improved significantly by the administration offructosamine
oxidase
inhibitors (TABLE 4).
TABLE 4.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Survivors at week 32 8 2 6 5 8 7
Significance* - - ns ns P <0.025 P <0.05
= Chi-square test compared with untreated STZ rats (Group 2)
The survival curve for STZ rats compared with non-diabetic controls is shown
in FIGURE 4. Death was presumed secondary to a cardiovascular event. In
general,
renal function remained normal.
(c) Weight gain: There was a progressive weight gain amongst non-diabetic
controls over the 32 weeks of the study which was abolished in the STZ
diabetic
animals. At the end of the 32 week study period, mean weight change amongst
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surviving study animals was: Group 1, +74.6%; Group 2, -21.0%, Group 3, -
11.0%%; Group 4, +1.2%, Group 5,+16.0%; & Group 6, -8.1% (FIGURE 5).
Compared with untreated diabetic controls, fructosamine oxidase inhibitors
caused
an improvement in weight gain roughly in proportion to the activity of the
inhibitor
(TABLE 2) i.e. acetylcysteine/hydralazine > EDTA > acetylcysteine >
hydralazine.
(d) Clinical pharmacokinetics:
- Hydralazine The bioavailability of hydralazine in man after oral
administration is 26-55%. However, only 2.0-3.6% of the drug is excreted in
the urine unchanged over 24 hours after oral administration. Most of the drug
is
recovered as an inactive acetylated product. See, Talseth T, Eur J Clin
Pharmacol 10:395-401 (1976) and Talseth T, Clin Pharmacol Ther 21:715-20
(1977). This could account for the reduced efficacy of hydralazine as a
fructosamine oxidase inhibitor in the current study. Furthermore, drug doses
administered to each STZ rat were calculated as 12.5mg hydralazine/day or
35mg/kg, based on an average consumption 250mL water per day and
assuming a mean body mass 350g. This rat dose far exceeds the maximum
recommended human dose of 200mg hydralazine per day (3mg/kg assuming a
mean body mass 70kg).
- EDTA The bioavailability of EDTA after oral administration is very low (less
than 5%) because of poor absorption from the gut limiting its usefulness in
humans to parenteral administration or irrigation techniques. See, Wynn JE et
al Toxicol Appl Pharmacol 16:807-17 (1970).
- Acetylcysteine Acetylcysteine is rapidly absorbed from the gut with an
bioavailability in man varying between 6 and 10%. See, Borgstrom L et al Eur
J Clin Pharmacol 31:217-22 (1986). However, the drug is rapidly degraded in
the liver by elimination of the acetyl moiety. See, Holdiness MR. Clin
Pharmacokinet 20:123-34 (1991). Induction of liver enzymes could account for
the progressive loss of drug efficacy seen after week 12 in the current study.
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CONCLUSION:
5. Streptozocin induces a severe form of insulin dependent diabetes in the rat
with
a high morbidity and mortality.
6. Survival of STZ rats was enhanced by treating with fructosamine oxidase
inhibitors in proportion to their activity in an in vitro assay.
7. Weight gain of STZ rats was enhanced by treating with fructosamine oxidase
inhibitors.
8. There was benefit in co-administering acetylcysteine and hydralazine
suggesting a synergy effect between classes offructosamine oxidase
inhibitors.
9. Based on these in vivo studies in the rat, the efficacy of a candidate of
fructosamine oxidase inhibitor in a human is likely to be influenced by
bioavailablity of the drug, degradation of the active compound in vivo, and
maximum oral tolerated dose of the drug.
EXAMPLE 3: Clinical utility offructosamine oxidase inhibition:
Second Preclinical study
The purpose of this example is to demonstrate how the clinical usefulness of
candidate fructosamine oxidase inhibitors, alone and in combination, may be
assessed using a standard animal model of diabetes mellitus, the streptozocin-
diabetic rat (STZ rat). This approach takes into account drug bioavailability,
the
activity of the drugs and their metabolites, interactions between drugs, and
any drug
adverse effects or toxicity factors.
Method:
80 Wistar rats weighing 200-300g and aged of 6-8 weeks were randomized:
Group I Non-diabetic control
Group 2 Diabetic control
Group 3 Diabetic treated with captopril (substrate analogue)
Group 4 Diabetic treated with carbidopa (hydrazine)
Group 5 Diabetic treated with triene (copper chelator)
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Group 6 Diabetic treated with captopril & triene.
Group 7 Diabetic treated with captopril & carbidopa
Group 8 Diabetic treated with triene & cardidopa.
Diabetes was induced by administering streptozotocin (60mg per kg) by
intraperitoneal injection. Non-diabetic controls received a sham injection of
buffer.
Diabetes was confirmed by venous blood glucose measurement >15mmol/L after 1
week & diabetic animals were treated with subcutaneous injections of
ultralente
insulin (4U/injection) 3 days per week to maintain body growth. Medications
were
administered 50mg/L in the drinking water over an 6 month period. Timed urine
collects and venous plasma samples were obtained at monthly intervals. Animals
were sacrificed and subjected to post-mortem at the end of the study.
Results:
(a) Blood glucose control: Rate of conversion to diabetes with intraperitoneal
STZ administration was z 80%. Poor glycaemic control was sustained over the 6
month duration of the study as evidenced by mean SD HbA, c (week 4, 12, &
24)
levels (TABLE 5).
TABLE 5.
HbA,C (%) Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
Week 4 4.1 0.1 8.3 0.9 8.5 0.9 9.0 1.0 8.0 1.0 9.0 5.2 9.1 1.5 9.1 1.5
Week 12 4.1 0.1 9.2 0.6 9.2 1.1 9.6 0.7 8.8 0.9 9.5 0.8 9.5 1.0 9.3 0.9
Week 24 3.7 0.1 9.4 1.3 9.6 1.3 9.9 1.1 9.0 1.4 9.5 1.3 9.8 1.2 9.1 1.2
(b) Survival: Compared with intravenous administration of STZ, intraperitoneal
adminstration of STZ induced a less severe form of diabetes with lesser
mortaility
rate. At the end of the 24 week study period, mortality rate amongst study
animals
was: Group 1, 0%; Group 2, 14.3%, Group 3, 0%; Group 4, 0%, Group 5, 0%;
Group 6, 12.5%, Group 7, 0%, & Group 6, 0%. There was no significant
difference
between groups because of the low frequency of events.
(c) Weight gain: STZ diabetes causes a profound weight loss in diabetic rats
compared with non-diabetic controls. Mean weight gain of study animals from
the
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beginning to the end of the 24 week period are indicated in TABLE 6.
TABLE 6.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
Mean t SEM 342.8:t 54.4 t 60.7 t 33.7 t 123.6 t 56.1 t 55.1 t 75.8 t
weight gain 13.7 12.5 20.7 20.4 20.5 21.3 17.1 25.4
P ns ns ns 0.0138 ns ns ns
= Student's t test compared with untreated STZ rats (Group 2)
In terms of general well-being, triene (Group 5) appears more effective than
captopril (Group 3) and carbidopa (Group 4). The usefulness of triene was
reduced
when the drug was co-administered with captopril (Group 6) or carbidopa (Group
8).
There is no evidence of synergy between classes of fructosamine oxidase
inhibitors.
(d) Cataract formation Cataract is a recognised long-term complication of
poorly
controlled diabetes. Gross cataract formation in STZ rats compared with
diabetic
control animals by the end of the study at week 24 is shown (TABLE 7)
TABLE 7.
Group I Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
No (%) with 0(0%) 8 (40%) 2 (25%) 2(25%) 0(0%) 2(28%) 5(62%) 1(12%)
cataract
P - - ns ns < 0.10 ns ns ns
= Chi-square test compared with diabetic control rats (Group 2)
Although not significant at the P = 0.05 level, triene appears more effective
than captopril and carbidopa in inhibiting gross cataract formation. There is
no
evidence of synergy between classes of fructosamine oxidase inhibitors.
(e) Diabetic cardiomyopathy Cardiomyopathy is a recognised long-term
complication of poorly controlled diabetes. Macroscopically, hearts of STZ
rats were
dilated with thinning of the ventricular wall. Sections stained with
haematoxylin and
eosin and Masson's Trichrome showed focal pallor with a loss of normal
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architecture in the myocardium of both ventricles that began at the sub-
endocardial
and sub-epicardial regions and spread to encompass the whole ventricular wall
in
severely affected animals. There was also marked infiltration by fibrous
connective
tissue between myocytes and increased fibrous connective tissue in the walls
of
intramural arteries. These appearance are consistent with dilated
cardiomyopathy.
Gross myocardial fibrosis in STZ rats compared with non-diabetic control
animals
by the end of the study at week 24 is shown (TABLE 8).
TABLE 8.
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8
No (%) rats 0 10 6 2 0 6 8 7
with severe (0%) (50%) (75%) (25%) (0%) (75%) (100%) (87%)
fibrosis
P - - ns ns < 0.005 ns <0.005 <0.05
= Chi-square test compared with diabetic control rats (Group 2)
Triene appears highly effective in inhibiting the development of diabetic
cardiomyopathy.. There is no evidence of synergy between classes
offructosamine
oxidase inhibitors.
(f) Clinical pharmacokinetics:
- Triene The bioavailability of triene is less than 10%. Most of the unchanged
drug is cleared in the urine within the first 6 hours of oral dosing mainly as
an
acetyl derivative indicating that a three or four times daily drug regimen or
a
sustained release preparation will be required. See, Kodama H et al Life Sci
6 1 :899-907 (1997). In addition, plasma levels in non-fasted rats are
significantly lower than those observed in fasted animals and the uptake of
triene from the intestinal brush border is competitively inhibited by other
amine
compounds. See, Tanabe R et al J Pharm Pharmacol 48:517-21 (1996). This
implies that triene is best administered in the fasting state. Interference in
the
absorption of drug from the intestinal brush border could account for
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discrepancies between triene treatment groups (Groups 5, 6, & 8). Finally, in
the current study, each STZ rat consumed approximately 250mL water per day
(12.5mg triene/rat/day). Assuming a mean body mass 350g, this dose of triene
equates to 35mg/kg. The dose of triene previously used in treating humans with
another condition (not diabetes) ranges 1.2-2.4g (17-35mg/kg assuming a
mean body mass 70kg). See, Walshe JM Lancet 8273:643-7 (1982). This
implies that humans may be safely treated with comparable doses of trienes to
those administered to rats in the current study to thereby elicit the
fructosamine
oxidase inhibition and/or antagonism advantages in a diabetic patient referred
to herein.
- Captopril The bioavailability of captopril is approximately 65% after an
oral
dose. However, the drug is almost completely bound in vivo to albumin and
other plasma proteins, and forms inactive mixed disulphides with endogenous
thiols so that plasma levels of active drug may be very low. The elimination
half life of unchanged captopril is approximately 2 hours. See, Duchin KL et
al
Clin Pharmacokinet 14:241-59 (1988). These observations might explain the
reduced efficacy of captopril in the STZ rat compared with in vitro studies.
Furthermore, each STZ rat consumed approximately 12.5mg captopril/day
which equates to 35mg/kg assuming a mean body mass 350g. This dose far
exceeds the maximum recommended human dose of 150mg captopril per day
(2mg/kg assuming a mean body mass 70kg).
- Carbidopa In a study of beagle dogs, the oral absorption of carbidopa was
almost complete and the absolute bioavailability was 88%. The biological half-
life was 5 hours. See, Obach R et al JPharm Pharmacol 36:415-6 (1984).
However, carbidopa is an unstable compound and it degrades naturally in a
short period. Solutions left to stand exposed to light at room temperature
will
undergo 50% oxidative degradation in 24 hours. See, Pappert EJ et al
Movement Disorders 12:608-23 (1997). Reduced bioavailability due to
oxidative degradation of the active drug both prior to its consumption and
post-
ingestion in the rat could explain (in part) the reduced efficacy of carbidopa
in
the current study. Finally, each STZ rat consumed approximately 12.5mg
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carbidopa/day which equates to 35mg/kg assuming a mean body mass 350g.
This dose far exceeds the maximum recommended human dose of 200mg
carbidopa per day (3mg/kg assuming a mean body mass 70kg).
CONCLUSION:
10. Intraperitoneal streptozocin is associated with a lower mortality rate
than
intravenous streptozocin in the rat.
11. Weight gain was enhanced in STZ rats treated with the copper chelator,
triene.
Captopril and carbidopa were ineffective.
12. Cataract development may be inhibited by triene. Efficacy of triene is
diminished when the drug is co-administered with either captopril or
carbidopa.
13. The development of diabetic cardiomyopathy was prevented by treatment with
triene. Efficacy of triene is diminished when the drug is co-administered with
either captopril or carbidopa.
14. Oral doses of triene which inhibit the development of complications in the
rat
(cataract, cardiomyopathy, and early death) are equivalent on a body mass
basis to doses of triene which have previously been used to treat human beings
with another condition (not diabetes).
15. When administered to humans on a three or four times daily basis or as a
sustained release preparation in previously tolerated doses1.2-2.4g/day,
triene
may provide an effective means of treating the long-term complications of
diabetes mellitus.
EXAMPLE 4: Clinical Utility of fructosamine oxidase inhibition:
double-blind, placebo-controlled clinical trial
The purpose of this example is to demonstrate how the clinical usefulness of
candidate fructosamine oxidase inhibitors will be assessed in diabetic human
subjects. A detailed protocol based on this proposal has been approved by the
Auckland Regional Ethics Committee. This approach takes into account drug
bioavailability, the activity of the drugs and their metabolites, interactions
between
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drugs, any drug adverse effects or toxicity factors and the "scale-up" factor
from rat
to human treatment..
Objective: A pilot study to determine whether triene will reduce the rate
of progression of renal disease and associated microvascular complications in
patients with diabetic nephropathy due to NIDDM.
Patient population: 60 men and women aged between 40 years and 70 years of
age with poor blood glucose control and diabetic nephropathy due to NIDDM.
Study design and duration: Randomised double-blind, placebo-controlled
study design consisting of five periods:
= screening period (detecting possible candidates who meet study criteria);
= enrolment period (securing informed consent & baseline measurements);
= run-in period (trial of acceptability of study protocols & study
medication);
= maintenance period (treatment with drug/placebo, monitoring
efficacy/safety);
= follow-up period (detect any untoward effect when medication is
discontinued).
Blinded therapy (triene 400mg or placebo) will be administered three time
daily `/2
hour before meals in addition to current antihypertensive and hypoglycaemic
therapies. The study will terminate when all patients are randomised and have
been
in the study (maintenance period) for a minimum of 6 months. All randomised
patients who discontinue study drug for any reason other than death will be
followed
for the entire duration of the study; patients who undergo renal
transplantation or
dialysis will be followed for vital status only.
Outcomes Efficacy:
= The primary outcome measure will consist of rate of decline in renal
function
as measured by glomerular filtration rate (creatinine clearance).
= The secondary outcome measures to be evaluated are development of diabetic
retinopathy, diabetic peripheral neuropathy, and diabetic autonomic
neuropathy.
Safe :
= Safety parameters evaluated will be adverse events and clinical laboratory
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abnormalities which will be assessed at time points by medical history,
physical examination, and laboratory analyses and compared between groups.
Statistical considerations: The sample size estimate for this trial is
determined for the primary hypothesis that the projected rate of decay of
creatinine
clearance (lmL.min-1) in NIDDM patients with diabetic nephropathy (creatinine
clearance <90 mL.min-1) will be reduced by treating with triene. The study is
powered to detect (80%) a 6mL.min-1 change in creatinine clearance over 6
months
with four 2-monthly readings (i.e. 0, 2, 4, & 6 months) assuming a 10% rate of
loss
to follow-up at the 5% significance level.
CONCLUSION:
16. The efficacy of triene as a treatment of microvascular complications in
patients
with NIDDM will be confirmed.
17. The safety of long-term administration of triene in patients with poor
blood
glucose control and diabetic nephropathy due to NIDDM will be confirmed.
18. It also provides a means to determine the clinical usefulness of
alternative
fructosamine oxidase inhibitors such as the copper chelating compounds D-
penicillamine, sar, and diamsar (ie; triene could be used in place of placebo
in
ensuing clinical trials).
