U.S. patent application number 10/525345 was filed with the patent office on 2006-05-11 for dosage forms and related therapies.
Invention is credited to John Richard Baker, Nigel Robert Arnold Beelev, Garth James Smith Cooper.
Application Number | 20060100278 10/525345 |
Document ID | / |
Family ID | 31950924 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060100278 |
Kind Code |
A1 |
Cooper; Garth James Smith ;
et al. |
May 11, 2006 |
Dosage forms and related therapies
Abstract
This invention is directed in part to novel doses, dosage
formulations, and routes of administration of such doses and dose
formulations, said dose and dose formulations containing one or
more copper chelators, for example, one or more trientine active
agents, including trientine analogues, trientine salts, trientine
prodrugs, and trientine derivatives, useful in the treatment of
diseases, disorders and conditions, including in indications where
copper may play a role.
Inventors: |
Cooper; Garth James Smith;
(Auckland, NZ) ; Baker; John Richard; (Auckland,
NZ) ; Beelev; Nigel Robert Arnold; (Solano Beach,
CA) |
Correspondence
Address: |
BUCHANAN INGERSOLL, P.C.
ONE OXFORD CENTRE, 301 GRANT STREET
20TH FLOOR
PITTSBURGH
PA
15219
US
|
Family ID: |
31950924 |
Appl. No.: |
10/525345 |
Filed: |
August 20, 2003 |
PCT Filed: |
August 20, 2003 |
PCT NO: |
PCT/NZ03/00184 |
371 Date: |
August 17, 2005 |
Current U.S.
Class: |
514/554 |
Current CPC
Class: |
A61P 9/06 20180101; A61K
31/132 20130101; A61P 3/10 20180101; A61P 9/04 20180101; A61P 9/10
20180101; A61K 31/30 20130101; A61P 9/00 20180101; A61K 31/131
20130101; A61P 9/12 20180101; A61P 9/14 20180101; A61K 9/0019
20130101; A61P 39/04 20180101; A61P 43/00 20180101; A61K 31/194
20130101 |
Class at
Publication: |
514/554 |
International
Class: |
A61K 31/205 20060101
A61K031/205 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 20, 2002 |
NZ |
520896 |
Aug 20, 2002 |
NZ |
520895 |
Aug 20, 2002 |
NZ |
520897 |
Mar 17, 2003 |
NZ |
524795 |
Mar 17, 2003 |
NZ |
524794 |
Mar 17, 2003 |
NZ |
524796 |
Claims
1-128. (canceled)
129. A composition comprising a pharmaceutically acceptable carrier
and an effective amount of a pharmaceutically acceptable acid
addition salt of triethylenetetramine and succinic acid.
130. The composition of claim 129 comprising from 50 mg to 500 mg
of a pharmaceutically acceptable acid addition salt of
triethylenetetramine and succinic acid.
131. The composition of claim 129 comprising from 50 mg to 450 mg
of an acid addition salt of triethylenetetramine and succinic
acid.
132. The composition of claim 129 comprising from 50-100 mg to
about 400 mg of an acid addition salt of triethylenetetramine and
succinic acid.
133. The composition of claim 129 comprising from 50-100 mg to
about 300 mg of an acid addition salt of triethylenetetramine and
succinic acid.
134. The composition of claim 129 comprising from 110 to 290 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
135. The composition of claim 129 comprising from 120 to 280 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
136. The composition of claim 129 comprising from 130 to 270 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
137. The composition of claim 129 comprising from 140 to 260 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
138. The composition of claim 129 comprising from 150 to 250 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
139. The composition of claim 129 comprising from 160 to 240 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
140. The composition of claim 129 comprising from 170 to 230 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
141. The composition of claim 129 comprising from 180 to 220 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
142. The composition of claim 129 comprising from 190 to 210 mg of
an acid addition salt of triethylenetetramine and succinic
acid.
143. The composition of claim 129 comprising from 50 mg to 100 mg
of an acid addition salt of triethylenetetramine and succinic
acid.
144. The composition of claim 129 wherein the amount of the acid
addition salt of triethylenetetramine and succinic acid is selected
from the group consisting of 50 mg, 110 mg, 120 mg, about 130 mg,
140 mg, and 150 mg.
145. A composition comprising a pharmaceutically acceptable carrier
and a pharmaceutically acceptable acid addition salt of
triethylenetetramine and succinic acid in an amount selected from
the group consisting of 1.2 mg, 10 mg, 12 mg, 20 mg, 30 mg, and 40
mg.
146. The composition of any of claims 129-144 or 145, wherein said
acid addition salt of triethylenetetramine and succinic acid is
purified.
147. The composition of claim 146, wherein said composition is in a
form suitable for oral administration.
148. The composition of claim 147, wherein said form suitable for
oral administration is a capsule.
149. The composition of claim 147, wherein said form suitable for
oral administration is a tablet.
150. The composition of claim 149, wherein said tablet is an
enteric-coated tablet.
151. The composition of claim 149, wherein said tablet is a layered
tablet.
152. The composition of claim 147, wherein said form suitable for
oral administration is a sustained release preparation.
153. The composition of claim 152, wherein said sustained release
preparation is a delayed release preparation.
154. The composition of claim 152, wherein said sustained release
preparation is a slow release preparation.
155. The composition of claim 152, wherein said sustained release
preparation is a controlled release preparation.
156. The composition of claim 152, wherein said sustained release
preparation is an extended release preparation.
157. The composition of claim 146, wherein said composition is in a
form suitable for transdermal administration.
158. The composition of claim 146, wherein said composition is in a
form suitable for transmucosal administration.
159. The composition of claim 146, wherein said composition is in a
form suitable for administration as a suppository.
160. The composition of claim 146, further comprising at least one
delivery agent to enhance entry of the addition salt of
triethylenetetramine and succinic acid to the systemic
circulation.
161. A dosage unit of claim 160 wherein the delivery agent is
selected from the group consisting of a vasodilator and a reverse
active transport agent.
162. A method of treating a subject for diabetes, comprising
administering to said subject a composition according to claim
146.
163. The method according to claim 162, wherein said subject has
type 1 diabetes.
164. The method according to claim 162, wherein said subject has
type 2 diabetes.
165. A method of treating a subject for cardiomyopathy, comprising
administering to said subject a composition according to claim
146.
166. The method according to claim 165, wherein said cardiomyopathy
is selected from the group consisting of hypertensive
cardiomyopathy, diabetic hypertensive cardiomyopathy, hypertensive
cardiomyopathy associated with impaired glucose intolerance,
hypertensive cardiomyopathy associated with impaired fasting
glucose, ischemic cardiomyopathy associated with impaired glucose
tolerance, ischemic cardiomyopathy associated with impaired fasting
glucose, hypertensive cardiomyopathy not associated with any
abnormality of glucose metabolism, ischemic cardiomyopathy not
associated with any abnormality of glucose metabolism, ischemic
cardiomyopathy, ischemic cardiomyopathy associated with coronary
heart disease, idiopathic cardiomyopathy, metabolic cardiomyopathy,
alcoholic cardiomyopathy, drug-induced cardiomyopathy, and
hypertensive cardiomyopathy.
167. A method of treating a subject further acute coronary
syndrome, comprising administering to said subject a composition
according to claim 146.
168. The method according to claim 167, wherein said acute coronary
syndrome is diabetic acute coronary syndrome, acute coronary
syndrome associated with impaired glucose tolerance, acute coronary
syndrome associated with impaired fasting glucose, acute coronary
syndrome not associated with any abnormality of glucose
metabolism.
169. A method of treating a subject for myocardial infarction,
comprising administering to said subject a composition according to
claim 146.
170. A method of treating a subject for myocarditis, comprising
administering to said subject a composition according to claim
146.
171. A method of treatment for the prevention or amelioration of
tissue damage in a subject who does not have Wilson's disease,
which comprises administering to said subject a a composition
according to claim 146.
Description
FIELD OF THE INVENTION
[0001] The subject invention pertains to doses and dosage forms of
therapeutic agents and their use in methods for the treatment,
reversal or amelioration of diseases, disorders and/or conditions
in a mammal (hereafter "treating"). Mammals that may be treated
using the described and claimed doses and dosage forms include, for
example, a human being having, or at risk for developing,
microvascular and/or macrovascular damage, for example,
cardiovascular tissue damage and, in particular, mammals including
human beings that have or are at risk for developing undesired
copper levels, including copper levels that can cause or lead to
tissue damage, including but not limited to vessel damage.
Treatment includes but is not limited to therapies to ameliorate
and/or reverse, in whole or in part, damage resulting from
diseases, disorders or conditions that are characterized in any
part by copper-involved or mediated damage of tissue and/or
vasculature, and/or to copper-involved or mediated impairment of
normal tissue stem cell responses. The invention has application
inter alia, for example, to diabetes-related and
non-diabetes-related heart failure, macrovascular disease or
damage, microvascular disease or damage, and/or toxic (e.g.,
hypertensive) tissue and/or organ disease or damage (including such
ailments as may, for example, be characterized by heart failure,
cardiomyopathy, myocardial infarction, and related arterial and
organ diseases) by administration of an active copper-chelating
compound such as, for example, one or more of trientine, salts of
trientine, prodrugs of trientine and salts of such prodrugs,
analogs of trientine and salts and prodrugs of such analogs, and/or
active metabolites of trientine and salts and prodrugs of such
metabolites, including but not limited to N-acetyl trientine and
salts and prodrugs of N-acetyl trientine.
BACKGROUND
[0002] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art,
or relevant, to the presently described or claimed inventions, or
that any publication or document that is specifically or implicitly
referenced is prior art or a reference that may be used in
evaluating patentability of the described or claimed
inventions.
[0003] Diabetes mellitus is a chronic condition characterized by
the presence of fasting hyperglycemia and the development of
widespread premature atherosclerosis. Patients with diabetes have
increased morbidity and mortality due to cardiovascular diseases,
especially coronary artery disease. Vascular complications in
diabetes may be classified as microvascular, affecting the retina,
kidney and nerves and macrovascular, predominantly affecting for
example coronary, cerebrovascular and peripheral arterial
circulation.
[0004] The chronic hyperglycemia of diabetes is associated with
long-term damage, dysfunction, and failure of various organs,
especially the eyes, kidneys, nerves, heart, and blood vessels and
long-term complications of diabetes include retinopathy with
potential loss of vision; nephropathy leading to renal failure;
peripheral neuropathy with risk of foot ulcers, amputation, and
Charcot joints; and autonomic neuropathy causing gastrointestinal,
genitourinary, and cardiovascular symptoms and sexual
dysfunction.
[0005] Glycation of tissue proteins and other macromolecules and
excess production of polyol compounds from glucose are among the
mechanisms thought to produce tissue damage from chronic
hyperglycemia. Diabetic patients have an increased incidence of
atherosclerotic cardiovascular, peripheral vascular, and
cerebrovascular disease. Hypertension, abnormalities of lipoprotein
metabolism, and periodontal disease are also found in people with
diabetes.
[0006] Hyperglycemia induces a large number of alterations in
vascular tissue that potentially promote accelerated
atherosclerosis. Currently, in addition to the nonenzymatic
glycosylation of proteins and lipids, two other major mechanisms
have emerged that encompass most of the pathologic alterations
observed in the vasculature of diabetic animals and humans, namely,
oxidative stress and protein kinase C (PKC) activation. These
mechanisms are not independent. For example, hyperglycemia-induced
oxidative stress promotes the formation of AGEs and PKC activation,
and both type 1 and type 2 diabetes are independent risk factors
for coronary artery disease (CAD), stroke, and peripheral arterial
disease. Schwartz C. J., et al., "Pathogenesis of the
atherosclerotic lesion. Implications for diabetes mellitus,"
Diabetes Care 15:1156-1167 (1992); Stamler J., et al., "Diabetes,
other risk factors, and 12-yr cardiovascular mortality for men
screened in the Multiple Risk Factor Intervention Trial." Diabetes
Care 16:434-444 (1993). Atherosclerosis accounts for virtually 80%
of all deaths among North American diabetic patients, compared with
one-third of all deaths in the general North American population,
and more than 75% of all hospitalizations for diabetic
complications are attributable to cardiovascular disease. American
Diabetes Association, "Consensus statement: role of cardiovascular
risk factors in prevention and treatment of macrovascular disease
in diabetes," Diabetes Care 16:72-78 (1993).
[0007] The decline in heart disease mortality in the general U.S.
population has been attributed to the reduction in cardiovascular
risk factors and improvement in treatment of heart disease.
However, patients with diabetes have not experienced the reduction
in age-adjusted heart disease mortality that has been observed in
nondiabetics, and an increase in age-adjusted heart disease
mortality has been reported in diabetic women. Gu K, et al.,
"Diabetes and decline in heart disease mortality in U.S. adults,"
JAMA 281:1291-1297 (1999). It has also been reported that diabetic
subjects have more extensive atherosclerosis of both coronary and
cerebral vessels than age- and sex-matched nondiabetic controls.
Robertson W. B., & Strong J. P., "Atherosclerosis in persons
with hypertension and diabetes mellitus," Lab Invest 18:538-551
(1968). Additionally, it has been reported that diabetics have a
greater number of involved coronary vessels and more diffuse
distribution of atherosclerotic lesions. Waller B. F., et al.,
"Status of the coronary arteries at necropsy in diabetes mellitus
with onset after age 30 years. Analysis of 229 diabetic patients
with and without clinical evidence of coronary heart disease and
comparison to 183 control subjects," Am J Med 69:498-506
(1980).
[0008] Following large studies comparing diabetics with matched
controls, it has also been reported that diabetic patients with
established CAD undergoing cardiac catheterization for acute
myocardial infarction, angioplasty, or coronary bypass have
significantly more severe proximal and distal CAD. Granger C. B.,
et al., "Outcome of patients with diabetes mellitus and acute
myocardial infarction treated with thrombolytic agents. The
Thrombolysis and Angioplasty in Myocardial Infarction (TAMI) Study
Group," J Am Coll Cardiol 21:920-925 (1993); Stein B., et al.,
"Influence of diabetes mellitus on early and late outcome after
percutaneous transluminal coronary angioplasty," Circulation
91:979-989 (1995); Barzilay J. I., et al., "Coronary artery disease
and coronary artery bypass grafting in diabetic patients aged>
or =65 years [from the Coronary Artery Surgery Study (CASS)
Registry]," Am J Cardiol 74:334-339(1994)). Postmortem and
angioscopic evidence also shows a significant increase in plaque
ulceration and thrombosis in diabetic patients. Davies M. J., et
al., "Factors influencing the presence or absence of acute coronary
artery thrombi in sudden ischemic death," Eur Heart J 10;203-208
(1989); Silva J. A., et al. "Unstable angina. A comparison of
angioscopic findings between diabetic and nondiabetic patients,"
Circulation 92:1731-1736 (1995).
[0009] CAD is the leading cause of death in people with type 2
diabetes, regardless of duration of diabetes. Stamler I., et al.,
"Diabetes, other risk factors, and 12-yr cardiovascular mortality
for men screened in the Multiple Risk Factor Intervention Trial,"
Diabetes Care 16:434-444 (1993); Donahue R. P., & Orchard T.
J., "Diabetes mellitus and macrovascular complications. An
epidemiological perspective," Diabetes Care 15:1141-1155 (1992).
The increased cardiovascular risk is said to be particularly
striking in women. Barrett Connor E. L., et al., "Why is diabetes
mellitus a stronger risk factor for fatal ischemic heart disease in
women than in men? The Rancho Bemardo Study," JAMA 265:627-631
(1991). CAD is not confined to particular forms of diabetes,
however, and is prevalent in both type 1 and type 2 diabetes. In
type 1 diabetes, an excess of cardiovascular mortality is generally
observed after the age of 30. Krolewski A. S., et al., "Magnitude
and determinants of coronary artery disease in juvenile-onset,
insulin-dependent diabetes mellitus," Am J Cardiol 59:750-75 5
(1987). CAD risk was reported in this study to increase rapidly
after age 40, and by age 55, 35% of men and women with type 1
diabetes die of CAD, a rate of CAD mortality that far exceeded that
observed in an age-matched nondiabetic cohort. Id.
[0010] Diabetic nephropathy in type 1 diabetics also increases the
prevalence of CAD. Nephropathy leads to accelerated accumulation of
AGEs in the circulation and tissue and parallels the severity of
renal functional impairment. Makita Z., et al., "Advanced
glycosylation end products in patients with diabetic nephropathy,"
N Engl J Med 325:836-842 (1991). In diabetic patients reaching
end-stage renal disease, overall mortality has been reported to be
greater than in nondiabetic patients with end-stage renal disease.
The relative risk for age-specific death rate from myocardial
infarction among all diabetic patients during the first year of
dialysis is reportedly 89-fold higher than that of the general
population. Geerlings W., et al., "Combined report on regular
dialysis and transplantation in Europe, XXI," Nephrol Dial
Transplant 6 4]:5-29 (1991). It has also been reported that the
most common cause of death in diabetic patients who have undergone
renal transplantation is CAD, accounting for 40% of deaths in these
patients. Lemmers M. J., & Barry J. M., "Major role for
arterial disease in morbidity and mortality after kidney
transplantation in diabetic recipients," Diabetes Care 14:295-301
(1991).
[0011] It has been demonstrated that the degree and duration of
hyperglycemia are the principal risk factors for microvascular
complications in type 2 diabetes. The Diabetes Control and
Complications Trial Research Group, "The effect of intensive
treatment of diabetes on the development and progression of
long-term complications in insulin-dependent diabetes mellitus," N
Eng J Med 329:977-986 (1993). However, it has also been said that
there is no clear association between the extent or severity of
macrovascular complications and the duration or severity of the
diabetes, and an increased prevalence of CAD is apparent in newly
diagnosed type 2 diabetes subjects has been reported. Uusitupa M.,
et al., "Prevalence of coronary heart disease, left ventricular
failure and hypertension in middle-aged, newly diagnosed type 2
(non-insulin dependent) diabetic subjects," Diabetologia 28:22-27
(1985). It has also been reported that even impaired glucose
tolerance carries an increased cardiovascular risk despite minimal
hyperglycemia Fuller J. H., et al., "Coronary-heart-disease risk
and impaired glucose tolerance. The Whitehall study," Lancet
1:1373-1376 (1980).
[0012] There is also a worldwide trend towards an increasing
prevalence of diabetes. The number of cases of type 2 diabetes is
projected to increase from 135 million in 2000 to more than 300
million in 2025. This increase is related to an ageing of the
population, increasing obesity, and low socio-economic status. See,
WHO. The World Health Report 1997. As a consequence, mortality from
diabetes has increased over the last decade whereas mortality from
cardiovascular disease, stroke, and malignant diseases has remained
static or declined. See, U.S. Center for Health Studies. The causes
of premature mortality in type 2 diabetes comprise cardiovascular
disease, 58%; cerebrovascular disease, 12%; nephropathy, 3%;
diabetic coma, 1%; and malignancy, 11%.
[0013] Diabetic heart disease is further characterized by more
severe CAD at a younger age, a 4-fold increase in frequency of
heart failure, post-acute myocardial infarction and a
disproportionate increase in left ventricular hypertrophy. See
Struthers A. D., & Morris A. D., Lancet 359:1430-2 (2002).
Subjects with type 2 diabetes also manifest a disproportionate
increase in mortality within the first 24-hours post-acute
myocardial infarction. Acute intervention can ameliorate this risk.
See, Malmberg K., Br Med J 314:1512-5 (1997).
[0014] PCT Application No. PCT/NZ99/00161 (published as WO00/18392
on 6 Apr. 2000) relates to methods of treating a mammalian subject
predisposed to and/or suffering from diabetes mellitus with a view
to minimizing the consequences of macrovascular and microvascular
damage to the patent which comprises, in addition to any treatment
in order to control blood glucose levels, at least periodically
controlling copper, for example, in the subject. An assay method is
disclosed in PCT Application No. PCT/NZ99/00160 (published as
WO00/18891 on 6 Apr. 2000). A range of different treatment agents
are disclosed in PCT/NZ99/00161. These included copper chelating
agents.
[0015] Metals are present naturally in the body and many are
essential for cells (e.g., Cu, Fe, Mn, Ni, Zn). However, all metals
are toxic at high concentrations. One reason metals may become
toxic relates to their ability to cause oxidative stress,
particularly redox active transition metals, which can take up or
give off an electron (e.g., Fe2+/3+, Cu+/2.+-.) that can give rise
to free radicals that cause damage (Jones et al., "Evidence for the
generation of hydroxyl radicals from a chromium (V) intermediate
isolated from the reaction of chromate with glutathione," Biochim.
Biophys. Acta 286:652-655 (1991); Li, Y. & Trush, M. A., DNA
damage resulting from the oxidation of hydroquinone by copper: role
for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation,"
Carcinogenes 7:1303-1311 (1993)). Metals can replace other
essential metals or enzymes, disrupting the function of these
molecules, and can be toxic for this reason as well. Some metal
ions (e.g., Hg+ and Cu+) are very reactive to thiol groups and may
interfere with protein structure and function.
[0016] As noted herein, humans subject to type 2 diabetes or
abnormalities of glucose mechanism are particularly at risk to the
precursors of heart failure, heart failure itself, and other
diseases of the arterial tree. It has been reported that more than
50% of patients with type 2 diabetes in Western countries die from
the effects of cardiovascular disease. See, Stamler, et al.,
Diabetes Care 16:434-44 (1993). It has also been reported that even
lesser degrees of glucose intolerance defined by a glucose
tolerance test (impaired glucose tolerance, or "IGT") still carry
an increased risk of sudden death. See, Balkau, et al., Lancet
354:1968-9 (1999). For a long time, it was assumed that this
reflected an increased incidence of coronary atherosclerosis and
myocardial infarction in diabetic subjects. However, evidence is
mounting that diabetes can cause a specific heart failure or
cardiomyopathy in the absence of atherosclerotic coronary artery
disease.
[0017] Cardiac function is commonly assessed by measuring the
ejection fraction. A normal left ventricle ejects at least 50% of
its end-diastolic volume each beat. A patient with systolic heart
failure commonly has a left ventricular ejection fraction less than
30% with a compensatory increase in end-diastolic volume.
Hemodynamic studies conducted on diabetic subjects without overt
congestive heart failure have observed normal left ventricular
systolic function (LV ejection fraction) but abnormal diastolic
function suggesting impaired left ventricular relaxation or
filling. See Regan, et al., J. Gun. Invest. 60:885-99 (1977). In a
recent study, 60% of men with type 2 diabetes without clinically
detectable heart disease were reported to have abnormalities of
diastolic filing as assessed by echocardiography. See Poirier, et
al., Diabetes Care 24:5-10 (2001). Diagnosis maybe made, for
example, by non-invasive measurements. In the absence of mitral
stenosis, mitral diastolic blood flow measured by Doppler
echocardiography is a direct measure of left ventricular filling.
The most commonly used measurement is the AlE ratio. Normal early
diastolic filling is rapid and is characterized by an E-wave
velocity of around 1 m/sec. Late diastolic filling due to atrial
contraction is only a minor component, and the A-wave velocity is
perhaps around 0.5 m/sec. This gives a normal AIE ratio of
approximately 0.5. With diastolic dysfunction, early diastolic
filling is impaired, atrial contraction increases to compensate,
and the AlE ratio increases to more than 2.0.
[0018] Treatment, let alone reversal or amelioration, of diabetic
cardiomyopathy is difficult and the options are limited. Tight
control of blood glucose levels might prevent or reverse myocardial
failure, although this may be true only in the early stages of
ventricular failure. Angiotensin converting enzyme inhibitors such
as captopril improve survival in heart failure particularly in
patients with severe systolic heart failure and the lowest ejection
fractions. There are, however, various therapies that are not
recommended for diabetic cardiomyopathy. For example, inotropic
drugs are designed to improve the contraction of the failing heart.
However, a heart with pure diastolic dysfunction is already
contracting normally and it is believed that inotropic drugs will
increase the risk of arrhythmias. Additionally, there appears to be
no basis for the use vasodilator drugs that reduce after-load and
improve the emptying of the ventricle because ejection fraction and
end-diastolic volume are already normal. After-load reduction may
even worsen cardiac function by creating a degree of outflow
obstruction.
[0019] Diuretics are the mainstay of therapy for heart failure by
controlling salt and water retention and reducing filling
pressures. However, they are contraindicated in diastolic
dysfunction where compromised cardiac pump function is dependent on
high filling pressures to maintain cardiac output. Venodilator
drugs such as the nitrates, which are very effective in the
management of systolic heart failure by reducing pre-load and
filling pressures, are understood to be poorly tolerated by
patients with diastolic heart failure. Ejection fraction and
end-systolic volume are often normal and any reduction in pre-load
leads to a marked fall in cardiac output. Finally, there is concern
about the use of beta-blockers in heart failure because of their
potential to worsen pump function. There is also concern regarding
the administration of beta-blockers to patients with diabetes who
are treated with sulphonylurea drugs and insulin due to a
heightened risk of severe hypoglycaemia.
[0020] Thus, it will be understood that the mechanisms underlying
various disorders of the heart, the macrovasculature, the
microvasculature, and the long-term complications of diabetes,
including associated heart diseases and conditions and long-term
complications, are complex and have long been studied without the
discovery of clear, safe and effective therapeutic interventions.
There is a need for such therapies, which are described and claimed
herein.
[0021] It is also understood there is a continuing need for
pharmaceutical compositions capable of addressing damage arising
from disease states, disorders or conditions of the cardiovascular
tree (including the heart) and dependent organs (e.g., retina,
kidney, nerves, etc.) that involve, concern or relate to, for
example, elevated or undesired copper levels such as elevated
non-intracellular free copper values levels. The described and
claimed therapies also provide low dose controlled release and/or
low dose extended release compositions useful for the reversal
and/or amelioration of structural damage in a subject whether
diabetic or not, having copper levels capable of diminishment in
order to treat, for example, the heart, the macrovasculature, the
microvasculature, and/or long-term complications of diabetes,
including cardiac structure damage. Cardiac structure damage
includes, but is not limited to, for example, atrophy, loss of
myocytes, expansion of the extracellular space and increased
deposition of extracellular matrix (and its consequences) and/or
coronary artery structure damage selected from at least media
damage (the muscle layer) and intima damage (the endothelial layer)
(and its consequences), systolic function, diastolic function,
contractility, recoil characteristics and ejection fraction.
[0022] Diseases, disorders and conditions relating to the
cardiovascular tree and/or dependent organs that may be treated by
the methods and compositions of the present invention include, for
example, any one or more of (1) disorders of the heart muscle
(cardiomyopathy or myocarditis) such as idiopathic cardiomyopathy,
metabolic cardiomyopathy which includes diabetic cardiomyopathy,
alcoholic cardiomyopathy, drug-induced cardiomyopathy, ischemic
cardiomyopathy, and hypertensive cardiomyopathy; (2) atheromatous
disorders of the major blood vessels (macrovascular disease) such
as the aorta, the coronary arteries, the carotid arteries, the
cerebrovascular arteries, the renal arteries, the iliac arteries,
the femoral arteries, and the popliteal arteries; (3) toxic,
drug-induced, and metabolic (including hypertensive and/or diabetic
disorders of small blood vessels (microvascular disease) such as
the retinal arterioles, the glomerular arterioles, the vasa
nervorum, cardiac arterioles, and associated capillary beds of the
eye, the kidney, the heart, and the central and peripheral nervous
systems; and, (4) plaque rupture of atheromatous lesions of major
blood vessels such as the aorta, the coronary arteries, the carotid
arteries, the cerebrovascular arteries, the renal arteries, the
iliac arteries, the femoral arteries and the popliteal
arteries.
SUMMARY OF THE INVENTION
[0023] The present invention is based, in part, on new doses and
dosage forms for treatments aimed at reduction in available free
copper that are useful, for example, in treating and preventing
macrovascular, microvascular and/or toxic/metabolic diseases of the
kind referenced herein and in tissue repair processes. This is
irrespective of the glucose metabolism of the subject and
irrespective of whether or not fructosamine oxidase is involved in
any such disease. The invention also relates to doses and dosage
forms of treatments relating to the cardiovascular accumulation of
redox-active transition metal ions in diabetes.
[0024] Under physiological conditions, injury to a target organ is
sensed by distant stem cells that migrate to the site of damage and
undergo alternate stem cell differentiation to assist in structural
and functional repair. The doses and dosage forms of treatments
described herein will also alleviate the accumulation of
redox-active transition metals, particularly copper, in cardiac or
vascular tissues in subjects with diabetes that is believed,
without wishing to be bound by any particular theory or mechanism,
to be accompanied by a suppression of the normal tissue
regeneration effected by the migration of stem cells. Elevated
tissue levels of copper that suppress the normal biological
behaviors of such undifferentiated cells exist irrespective of
diabetic status, although the condition may be more prevalent in
mammals, including humans, with diabetes.
[0025] Conditions occurring in the context of diabetes and/or
impaired glucose tolerance in which the suppression of normal stem
cell responses can cause impairment of normal tissue responses, and
that would be improved with therapy to lower copper values using
the doses and dosage forms of treatments described herein, include
the following:
[0026] 1. Heart failure. A significant regeneration of cardiac
tissues can occur within a few days of cardiac transplantation. The
likely mechanism is migration of stem cells from extra-cardiac
sites to the heart, with subsequent differentiation of such cells
into various specialized cardiac cells, including myocardial,
endothelial and coronary vascular cells. We have determined that
copper accumulation in cardiac tissues is likely to severely impair
these regenerative responses and that, for example, there is a role
for acute intravenous therapy with a copper chelator in the
treatment of heart failure, including but not limited to, diabetic
heart failure.
[0027] 2. Acute Myocardial infarction (AMI). AMI is accompanied by
proliferation of cells in the ventricular myocardium when, for
example, AMI occurs in the context of diabetes. The presence of
elevated tissue levels of redox-active transition metals suppresses
normal stem cell responses, resulting in impaired structural and
functional repair of damaged tissues. The mechanism of the
impairment of cardiac function in, for example, diabetes, is
believed to be a toxic effect of accumulated transition metals on
tissue dynamics, resulting in impaired tissue regeneration caused
in turn by suppression of normal stem cell responses, which mediate
physiological tissue regeneration by migration to damaged tissue
from external sites. Treatment of AMI, for example, in the context
of diabetes, will be improved by acute (if necessary, parenteral)
as well as by subsequent chronic administration of a copper
chelator as described herein.
[0028] 3. Wound healing and ulceration. The processes of normal
tissue repair require intervention of mobilizing stem cells, which
effect repair of the various layers of blood vessels, for example.
An accumulation of transition metals (particularly copper) in
vascular tissues causes the impaired tissue behavior characteristic
of diabetes, including impaired wound repair following surgery or
trauma, and the exaggerated tendency to ulceration and poor healing
of established ulcers. Treatment of diabetics with copper chelators
before they undergo surgery, or in the context of traumatic tissue
damage, may also be beneficially carried out using the doses and
dosage forms of treatments described herein. Surgery in diabetics
would have a better outcome if excess transition metals were
removed from blood vessels prior to surgery. This may be
accomplished on either an acute basis (with parenteral therapy) or
on a more chronic basis (with oral therapy) prior to actual surgery
or both.
[0029] 4. Tissue damage resulting from infection. Processes of
normal tissue repair following infection require intervention of
mobilized stem cells that migrate to sites of tissue damage to
effect tissue regeneration and repair of, for example, the various
layers of blood vessels. Such tissue damage repair will be impaired
by suppressed stem cell responses, such as those caused by the
build up of redox-active transition metals (particularly copper) in
tissues, for examples the walls of blood vessels. Tissue damage
repair, including repair following infection, will be improved, for
example, in people with diabetes by use of the doses and dosage
forms of treatments described herein.
[0030] 5. Diabetic kidney damage. Treatment of diabetics and others
having kidney failure by administration of a copper chelator
according to the doses and dosage forms of treatments described
herein will improve organ regeneration by restoring normal tissue
healing by allowing stem cells to migrate and differentiate
normally.
[0031] However, even in the non-diabetic mammal and even in a
mammal without a glucose mechanism abnormality, a reduction in
extra-cellular copper values is advantageous in that such lower
levels will lead to either a reduction in copper mediated tissue
damage and/or to improvement in tissue repair by restoration of
normal tissue stem cell responses.
[0032] In the studies described herein using streptozocin-diabetic
(STZ) rat model, a high frequency of tissue damage in both heart
and coronary artery tissues in severely diabetic animals has been
found, which reflects what is found in man. In one aspect, this
invention features a method of diminishment of available free
copper values in any at risk subject, whether diabetic or not, and
particularly a subject not suffering from Wilson's Disease and who
has copper levels capable of diminishment by the administration of
an effective amount of an agent capable of lowering copper levels
in a subject.
[0033] A preferred copper chelator is trientine, including
trientine acid addition salts and active metabolites including, for
example, N-acetyl trientine, and analogs, derivatives, and prodrugs
thereof. Alternative names for trientine include
N,N'-Bis(2-aminoethyl)-1,2-ethanedi-amine; triethylenetetramine
("TETA"); 1,8-diamino-3,6-diazaoctane; 3,6-diazaoctane-1,8-diamine;
1,4,7,10-tetraazadecane; trien; TECZA; and, triene. In one
embodiment, the trientine is rendered less basic (e.g., as a acid
addition salt).
[0034] In another embodiment, trientine is modified, i.e., it may
be as an analogue or derivative of trientine (or an analogue or
derivative of a copper-chelating metabolite of trientine, for
example, N-acetyl trientine). Derivatives of trientine or trientine
salts or analogues include those modified with polyethylene glycol
(PEG). The structure of PEG is
HO--(--CH.sub.2--CH.sub.2--O--).sub.n--H. It is a linear or
branched, neutral polyether available in a variety of molecular
weights. Analogues of trientine include, for example, compounds in
which one or more sulfur molecules is substituted for one or more
of the NH groups in trientine. Other analogues include, for
example, compounds in which trientine has been modified to include
one or more additional --CH.sub.2 groups. The chemical formula of
trientine is
NH.sub.2--CH.sub.2--CH.sub.2--NH--CH.sub.2--CH.sub.2--NH--CH.sub.2--CH.su-
b.2--NH.sub.2. The empirical formula is C.sub.6N.sub.4H.sub.18.
Analogues of trientine include, for example: TABLE-US-00001 1.
SH-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-NH.sub.2-
, 2.
SH-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-NH.sub.2,
3. NH2-CH2-CH2-NH-CH2-CH2-S-CH2-CH2-SH, 4.
NH.sub.2-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-SH,
5. SH-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-SH,
6.
NH.sub.2-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH-
.sub.2-NH.sub.2, 7.
SH-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-
-NH.sub.2, 8.
SH-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2--
NH.sub.2, 9.
NH.sub.2-CH.sub.2-CH.sub.2-NH-CH.sub.2-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.-
sub.2-SH, 10.
NH.sub.2-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.-
sub.2-SH, 11.
SH-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2-CH.sub.2-S-CH.sub.2-CH.sub.2--
SH, 12. and so on.
One or more hydroxyl groups may also be substituted for one or more
amine groups to create an analogue of trientine (with or without
the substitution of one or more sulfurs for one or more nitrogens).
Additional analogues, including acyclic and cyclic analogues, are
provided below in reference to Formula I and Formula II.
[0035] In another embodiment, trientine is delivered as a prodrug
of trientine or a copper chelating metabolite of trientine.
[0036] Salts of trientine (which optionally can be salts of a
prodrug of trientine or a copper chelating metabolite of trientine)
include, in one embodiment, acid addition salts such as, for
example, those of suitable mineral or organic acids. Salts of
trientine (such as acid addition salts, e.g., trientine
dihydrochloride) act as copper-chelating agents that aid in the
elimination of copper from the body by forming a stable soluble
complex that is readily excreted by the kidney.
[0037] In another aspect the present invention consists in a method
of (1) improvement or reversal, in whole or in part, of at least
one or more of cardiac structure damage in the subject (for
example, atrophy, loss of myocytes, expansion of the extracellular
space, and/or increased deposition of extracellular matrix (and its
consequences), and/or, (2) improvement, in whole or in part, of any
one or more of systolic function, diastolic function,
contractility, recoil characteristics, and ejection fraction (as
determined, for example, by ultrasound, MRI or other imaging),
and/or (3) improvement or reversal, in whole or in part, of any
damage from disorders of the heart muscle, macrovascular disease,
microvascular disease, and/or plaque rupture of athereomatous
lesions of major blood vessels (and the consequences thereof),
and/or (4) Improvement or reversal, in whole or in part, of damage
resulting from diabetic kidney disease, diabetic nephropathy,
copper accumulation in the kidney, and/or damage to the renal
arteries. This method may comprise: [0038] (i) Diagnosing the
mammal as being at risk and at least likely to be subject to some
damage capable of being ameliorated and/or reversed, and [0039]
(ii) Providing to the mammal, for example, a trientine active agent
composition as described herein.
[0040] In one embodiment the composition is provided to the subject
in a dosage form(s) capable of providing a lower effective dose,
and a less pulsile exposure to trientine than has hitherto been the
case with "QID" Wilson's disease regimens.
[0041] In another aspect the present invention consists in a method
of ameliorating or reversing, in whole or in part, in (I) a
diabetic human being or other diabetic mammal or (II) a human being
or other mammal with copper levels capable of diminishment ("the
subject") one or more of atrophy, loss of myocytes, expansion of
the extracellular space, and/or increased deposition of
extracellular matrix (and its consequences) and/or coronary artery
structure damage, including media damage (the muscle layer) and
intima damage (the endothelial layer) (and its consequences). The
method comprises or includes the step of administration and/or self
administration to the subject a slow or sustained release dosage
form sufficient to provide effective chelation of copper for an
overall diminishment thereof in the subject, said dosage form
having as the or an active agent trientine, at least one salt of
trientine, at least one trientine prodrug or a salt of such a
prodrug, at least one trientine analog or a salt or prodrug of such
an analog, and/or at least one active metabolite of trientine or a
salt or prodrug of such a metabolite, including but not limited to
N-acetyl trientine and salts and prodrugs of N-acetyl trientine
("trientine active agents").
[0042] In one embodiment the subject has been identified prior to
treatment as being at risk.
[0043] In another aspect the present invention consists in a method
of ameliorating or reversing, in whole or in part, any one or more
of systolic dysfunction, diastolic dysfunction, contractility, lack
of desired recoil characteristics and/or desired ejection fraction
function (as determined, for example, by ultrasound, MRI or other
imaging), disorders of the heart muscle, macrovascular disease,
microvascular disease and plaque rupture of athereomatous lesions
of major blood vessels (and consequences thereof), in a subject at
risk who is either (I) a diabetic subject or (II) a subject with
copper levels capable of diminishment, said method comprising the
step of administration and/or self administration of a low, slow,
and/or controlled release dosage form sufficient to provide
effective treatment, for example, by chelation of copper, for an
overall diminishment thereof in the subject, said dosage form
having one or more copper chelators, for example, one or more
trientine active agents.
[0044] Diseases, disorders and conditions that are usefully be
targeted by the compositions and procedures of the present
invention include, but are not limited to, any one or more of the
following: diabetic cardiomyopathy, diabetic acute coronary
syndrome (e.g.; myocardial infarction (MI), diabetic hypertensive
cardiomyopathy, acute coronary syndrome associated with impaired
glucose tolerance (IGT), acute coronary syndrome associated with
impaired fasting glucose (IFG), hypertensive cardiomyopathy
associated with IGT, hypertensive cardiomyopathy associated with
IFG, ischemic cardiomyopathy associated with IGT, ischemic
cardiomyopathy associated with IFG, ischemic cardiomyopathy
associated with coronary heart disease (CHD), acute coronary
syndrome not associated with any abnormality of the glucose
metabolism, hypertensive cardiomyopathy not associated with any
abnormality of the glucose metabolism, ischemic cardiomyopathy not
associated with any abnormality of the glucose metabolism
(irrespective of whether or not such ischemic cardiomyopathy is
associated with coronary heart disease or not), and any one or more
disease of the vascular tree including, by way of example, disease
states of the aorta, carotid, cerebrovascular, coronary, renal,
retinal, vasa nervorum, iliac, femoral, popliteal, arteriolar tree
and capillary bed.
[0045] In a further aspect the present invention consists in the
use of at least one trientine active agent together with other
material(s) appropriate for the dosage form, in the manufacture of
a sustained release dosage form useful for ameliorating or
reversing, in whole or in part, in a subject who is either (I) a
diabetic subject or (II) a subject with copper levels capable of
diminishment, damage associated with, or irregularity of, any one
or more of systolic function, diastolic function, contractility,
recoil characteristics and ejection fraction (e.g., as determined
clinically, by ultrasound, MRI or other imaging), and/or any one or
more of at least some of any damage arising from diabetic kidney
disease, diabetic nephropathy and/or copper accumulation in the
kidney and/or at least some of any damage to the renal arteries,
and/or_cardiac structure damage selected from one or more of
atrophy, loss of myocytes, expansion of the extracellular space and
increased deposition of extracellular matrix (and its
consequences), and/or coronary artery structure damage selected
from at least media damage (the muscle layer) and intima damage
(the endothelial layer) (and its consequences).
[0046] The present invention in another aspect provides a method
for treating a subject having, for example, any one or more of the
indications as defined herein comprising the parenteral
administration of a composition having a therapeutically effective
amount of a copper chelator wherein said therapeutically effective
amount administered is from about 5 mg to about 1100 mg per does
and/or per day.
[0047] In one embodiment the copper chelator is a trientine active
agent. Trientine active agents include, for example, salt(s) of
trientine, a trientine prodrug or a salt of such a prodrug, a
trientine analogue or a salt or prodrug of such an analog, and/or
at least one active metabolite of trientine or a salt or prodrug of
such a metabolite, including but not limited to N-acetyl trientine
and salts and prodrugs of N-acetyl trientine. Trientine active
agents also include the analogues of Formulae I and II.
[0048] In one embodiment other therapeutically effective dose
ranges of trientine active agents, including but not limited to
trientine, trientine salts, trientine analogues of formulae I and
II, and so on, for example, include from 10 mg to 1100 mg, 10 mg to
1000 mg, 10 mg to 900 mg, 20 mg to 800 mg, 30 mg to 700 mg, 40 mg
to 600 mg, 50 mg to 500 mg, 50 mg to 450 mg, from 50-100 mg to
about 400 mg, 50-100 mg to about 300 mg, 110 to 290 mg, 120 to 280
mg, 130 to 270 mg, 140 to 260 mg, 150 to 250 mg, 160 to 240 mg, 170
to 230 mg, 180 to 220 mg, 190 to 210 mg, and/or any other amount
within the ranges as set forth.
[0049] The composition may include, depending on the rate of
parenteral administration, for example, solutions, suspensions,
emulsions that can be administered by subcutaneous, intravenous,
intramuscular, intradermal, intrastemal injection or infusion
techniques.
[0050] The formulation can further include, for example, any one or
more of the following a buffer, for example, an acetate, phosphate,
citrate or glutamate buffer to obtain a pH of the final formulation
from approximately 5.0 to 9.5, a carbohydrate or polyhydric alcohol
tonicifier, an antimicrobial preservative that may be selected from
the group of, for example, m-cresol, benzyl alcohol, methyl, ethyl,
propyl and butyl parabens and phenol and a stabilizer.
[0051] A sufficient amount of water for injection is used to obtain
the desired concentration of solution. Sodium chloride, as well as
other excipients, may also be present, if desired. Such excipients,
however, must maintain the overall stability of the trientine
active form.
[0052] The formulation of the invention should be substantially
isotonic. An isotonic solution may be defined as a solution that
has a concentration of electrolytes, non-electrolytes, or a
combination of the two that will exert an equivalent osmotic
pressure as that into which it is being introduced, in this case,
mammalian tissue. By "substantially isotonic" is meant within
.+-.20% of isotonicity, preferably within .+-.10%. The formulated
product may be included within a container, typically, for example,
a vial, cartridge, prefilled syringe or disposable pen.
[0053] In another aspect the present invention provides a
parenteral composition comprising a therapeutically effective
amount of a copper chelator to be administered to a subject having
any one or more of the indications as defined herein.
[0054] The indications include, for example, diabetic
cardiomyopathy, diabetic acute coronary syndrome (e.g.; myocardial
infarction--MI), diabetic hypertensive cardiomyopathy, acute
coronary syndrome associated with impaired glucose tolerance (IGT),
acute coronary syndrome associated with impaired fasting glucose
(IFG), hypertensive cardiomyopathy associated with IGT,
hypertensive cardiomyopathy associated with IFG, ischemic
cardiomyopathy associated with IGT, ischemic cardiomyopathy
associated with IFG, ischemic cardiomyopathy associated with
coronary heart disease (CHD), disorders of the heart muscle
(cardiomyopathy or myocarditis) that include, for example,
idiopathic cardiomyopathy, metabolic cardiomyopathy which includes
diabetic cardiomyopathy, alcoholic cardiomyopathy, drug-induced
cardiomyopathy, ischemic cardiomyopathy, and hypertensive
cardiomyopathy, acute coronary syndrome not associated with any
abnormality of glucose metabolism, hypertensive cardiomyopathy not
associated with any abnormality of glucose metabolism, ischemic
cardiomyopathy not associated with any abnormality of glucose
metabolism (irrespective of whether or not such ischemic
cardiomyopathy is associated with coronary heart disease or not),
and any one or more diseases of the vascular tree including, by way
of example, disease states of the aorta, carotid, and of the
arteries including cerebrovascular, coronary, renal, retinal,
iliac, femoral, popliteal, vasa nervorum, arteriolar tree and
capillary bed, atheromatous disorders of the major blood vessels
(macrovascular disease) such as the aorta, the coronary arteries,
the carotid arteries, the cerebrovascular arteries, the renal
arteries, the iliac arteries, the femoral arteries, and the
popliteal arteries, cardiac structure damage which includes, but is
not limited to, for example, atrophy, loss of myocytes, expansion
of the extracellular space and increased deposition of
extracellular matrix (and its consequences) and/or coronary artery
structure damage selected from at least media (the muscle layer)
and/or intima (the endothelial layer) damage (and its
consequences), plaque rupture of atheromatous lesions of major
blood vessels such as the aorta, the coronary arteries, the carotid
arteries, the cerebrovascular arteries, the renal arteries, the
iliac arteries, the femoral arteries and the popliteal arteries,
systolic dysfunction, diastolic dysfunction, aberrant
contractility, recoil characteristics and ejection fraction, toxic,
drug-induced, and metabolic (including hypertensive and/or diabetic
disorders of small blood vessels (microvascular disease) such as
the retinal arterioles, the glomerular arterioles, the vasa
nervorum, cardiac arterioles, and associated capillary beds of the
eye, the kidney, the heart, and the central and peripheral nervous
systems.
[0055] In one embodiment the copper chelator is a trientine active
agent Trientine active agents include, for example, salt(s) of
trientine, a trientine prodrug or a salt of such a prodrug, a
trientine analog or a salt or prodrug of such an analog, and/or at
least one active metabolite of trientine or a salt or prodrug of
such a metabolite, including but not limited to N-acetyl trientine
and salts and prodrugs of N-acetyl trientine.
[0056] A therapeutically effective amount of a copper chelator, for
example, one or more trientine active agents, including but not
limited to trientine, trientine salts, trientine analogues of
formulae I and II, and so on, is from about 5 mg to 1200 mg per
day. Other therapeutically effective dose ranges, for example,
include from 10 mg to 1100 mg, 10 mg to 1000 mg, 10 mg to 900 mg,
20 mg to 800 mg, 30 mg to 700 mg, 40 mg to 600 mg, 50 mg to 500 mg,
50 mg to 450 mg, from 50-100 mg to about 400 mg, 50-100 mg to about
300 mg, 110 to 290 mg, 120 to 280 mg, 130 to 270 mg, 140 to 260 mg,
150 to 250 mg, 160 to 240 mg, 170 to 230 mg, 180 to 220 mg, 190 to
210 mg, and/or any other amount within the ranges as set forth.
[0057] The composition may include, depending on the rate of
parenteral administration, for example, solutions, suspensions,
emulsions that can be administered by subcutaneous, intravenous,
intramuscular, intradermal, intrastemal injection or infusion
techniques.
[0058] The formulation can further include, for example, any one or
more of the following a buffer, for example, an acetate, phosphate,
citrate or glutamate buffer to obtain a pH of the final formulation
from approximately 5.0 to 9.5, a carbohydrate or polyhydric alcohol
tonicifier, an antimicrobial preservative that may be selected from
the group of, for example, m-cresol, benzyl alcohol, methyl, ethyl,
propyl and butyl parabens and phenol and a stabilizer.
[0059] A sufficient amount of water for injection is used to obtain
the desired concentration of solution. Sodium chloride, as well as
other excipients, may also be present, if desired. Such excipients,
however, must maintain the overall stability of the trientine
active form.
[0060] The formulation of the invention should be substantially
isotonic. An isotonic solution may be defined as a solution that
has a concentration of electrolytes, non-electrolytes, or a
combination of the two that will exert an equivalent osmotic
pressure as that into which it is being introduced, in this case,
mammalian tissue. By "substantially isotonic" is meant within
.+-.20% of isotonicity, preferably within .+-.10%. The formulated
product may be included within a container, typically, for example,
a vial, cartridge, prefilled syringe or disposable pen.
[0061] In a further aspect the present invention provides the use
of a therapeutically effective amount of a copper chelator in the
manufacture of a medicament for the treatment of a subject having
any one or more of the following indications: diabetic
cardiomyopathy, diabetic acute coronary syndrome (e.g.; myocardial
infarction--MI), diabetic hypertensive cardiomyopathy, acute
coronary syndrome associated with impaired glucose tolerance (IGT),
acute coronary syndrome associated with impaired fasting glucose
(IFG), hypertensive cardiomyopathy associated with IGT,
hypertensive cardiomyopathy associated with IFG, ischemic
cardiomyopathy associated with IGT, ischemic cardiomyopathy
associated with IFG, ischemic cardiomyopathy associated with
coronary heart disease (CHD), disorders of the heart muscle
(cardiomyopathy or myocarditis) that include, for example,
idiopathic cardiomyopathy, metabolic cardiomyopathy which includes
diabetic cardiomyopathy, alcoholic cardiomyopathy, drug-induced
cardiomyopathy, ischemic cardiomyopathy, and hypertensive
cardiomyopathy, acute coronary syndrome not associated with any
abnormality of glucose metabolism, hypertensive cardiomyopathy not
associated with any abnormality of glucose metabolism, ischemic
cardiomyopathy not associated with any abnormality of glucose
metabolism (irrespective of whether or not such ischemic
cardiomyopathy is associated with coronary heart disease or not),
and any one or more diseases of the vascular tree including, by way
of example, disease states of the aorta, carotid, and of the
arteries including cerebrovascular, coronary, renal, retinal,
iliac, femoral, popliteal, vasa nervorum, arteriolar tree and
capillary bed, atheromatous disorders of the major blood vessels
(macrovascular disease) such as the aorta, the coronary arteries,
the carotid arteries, the cerebrovascular arteries, the renal
arteries, the iliac arteries, the femoral arteries, and the
popliteal arteries, cardiac structure damage which includes, but is
not limited to, for example, atrophy, loss of myocytes, expansion
of the extracellular space and increased deposition of
extracellular matrix (and its consequences) and/or coronary artery
structure damage selected from at least media (the muscle layer)
and/or intima (the endothelial layer) damage (and its
consequences), plaque rupture of atheromatous lesions of major
blood vessels such as the aorta, the coronary arteries, the carotid
arteries, the cerebrovascular arteries, the renal arteries, the
iliac arteries, the femoral arteries and the popliteal arteries,
systolic dysfunction, diastolic dysfunction, aberrant
contractility, recoil characteristics and ejection fraction, toxic,
drug-induced, and metabolic (including hypertensive and/or diabetic
disorders of small blood vessels (microvascular disease) such as
the retinal arterioles, the glomerular arterioles, the vasa
nervorum, cardiac arterioles, and associated capillary beds of the
eye, the kidney, the heart, and the central and peripheral nervous
systems.
[0062] In one embodiment, the copper chelator is a trientine active
agent Trientine active agents include, for example, salt(s) of
trientine, a trientine prodrug or a salt of such a prodrug, a
trientine analog or a salt or prodrug of such an analog, and/or at
least one active metabolite of trientine or a salt or prodrug of
such a metabolite, including but not limited to N-acetyl trientine
and salts and prodrugs of N-acetyl trientine.
[0063] The therapeutically effective amount of a copper chelator,
for example, a trientine active agents, including but not limited
to trientine, trientine salts, trientine analogues of formulae I
and II, and so on, is from about 5 mg to 1200 mg per day. Other
therapeutically effective dose ranges, for example, include from 10
mg to 1100 mg, 10 mg to 1000 mg, 10 mg to 900 mg, 20 mg to 800 mg,
30 mg to 700 mg, 40 mg to 600 mg, 50 mg to 500 mg, 50 mg to 450 mg,
from 50-100 mg to about 400 mg, 50-100 mg to about 300 mg, 110 to
290 mg, 120 to 280 mg, 130 to 270 mg, 140 to 260 mg, 150 to 250 mg,
160 to 240 mg, 170 to 230 mg, 180 to 220 mg, 190 to 210 mg, and/or
any other amount within the ranges as set forth.
[0064] The composition may include, depending on the rate of
parenteral administration, for example, solutions, suspensions,
emulsions that can be administered by subcutaneous, intravenous,
intramuscular, intradermal, intrastemal injection or infusion
techniques.
[0065] The formulation can further include, for example, any one or
more of the following a buffer, for example, an acetate, phosphate,
citrate or glutamate buffer to obtain a pH of the final formulation
from approximately 5.0 to 9.5, a carbohydrate or polyhydric alcohol
tonicifier, an antimicrobial preservative that may be selected from
the group of, for example, m-cresol, benzyl alcohol, methyl, ethyl,
propyl and butyl parabens and phenol and a stabilizer.
[0066] A sufficient amount of water for injection is used to obtain
the desired concentration of solution. Sodium chloride, as well as
other excipients, may also be present, if desired. Such excipients,
however, must maintain the overall stability of the trientine
active form.
[0067] The formulation of the invention should be substantially
isotonic. An isotonic solution may be defined as a solution that
has a concentration of electrolytes, non-electrolytes, or a
combination of the two that will exert an equivalent osmotic
pressure as that into which it is being introduced, in this case,
mammalian tissue. By "substantially isotonic" is meant within
.+-.20% of isotonicity, preferably within .+-.10%. The formulated
product may be included within a container, typically, for example,
a vial, cartridge, prefilled syringe or disposable pen.
[0068] As used herein, parenteral administration, includes, but is
not limited, to any one or more of the following administration
routes; subcutaneous, intravenous, intramuscular, intraperitoneal,
intrasternal, intraarticular or intrastemal injection or infusion
techniques (e.g., as sterile injectable aqueous or non-aqueous
solutions or suspensions); nasally such as by inhalation spray;
topically, such as in the form of a cream or ointment; or
vaginally.
[0069] Therapy may be monitored with a 24-hour urinary copper
analysis periodically. Urine must be collected in copper-free
glassware. It is expected that the patient probably will be in the
desired state of negative copper balance if 0.5 to 1.0 milligram of
copper is present in a 24-hour collection of urine.
[0070] The present invention in one aspect provides a method for
treating a subject having, for example, any one or more of the
indications as defined herein comprising the parenteral
administration of a composition having a therapeutically effective
amount of a copper chelator wherein said therapeutically effective
amount administered parenterally per dose rate is in the range of
about 0.1 mg/kg to about 40 mg/kg based on the body weight of the
subject.
[0071] In another embodiment the therapeutically effective amount
of copper chelator, for example, one or more trientine active
agents, including but not limited to trientine, trientine salts,
trientine analogues of formulae I and II, and so on, is from about
5 mg to 1200 mg per day. Other therapeutically effective dose
ranges, for example, include from 10 mg to 1100 mg, 10 mg to 1000
mg, 10 mg to 900 mg, 20 mg to 800 mg, 30 mg to 700 mg, 40 mg to 600
mg, 50 mg to 500 mg, 50 mg to 450 mg, from 50-100 mg to about 400
mg, 50-100 mg to about 300 mg, 110 to 290 mg, 120 to 280 mg, 130 to
270 mg, 140 to 260 mg, 150 to 250 mg, 160 to 240 mg, 170 to 230 mg,
180 to 220 mg, 190 to 210 mg, and/or any other amount within the
ranges as set forth.
[0072] The composition may include, depending on the rate of
parenteral administration, for example, solutions, suspensions,
emulsions that can be administered by subcutaneous, intravenous,
intramuscular, intradermal, intrastemal injection or infusion
techniques.
[0073] The formulation can further include, for example, any one or
more of the following a buffer, for example, an acetate, phosphate,
citrate or glutamate buffer to obtain a pH of the final formulation
from approximately 5.0 to 9.5, a carbohydrate or polyhydric alcohol
tonicifier, an antimicrobial preservative that may be selected from
the group of, for example, m-cresol, benzyl alcohol, methyl, ethyl,
propyl and butyl parabens and phenol and a stabilizer.
[0074] A sufficient amount of water for injection is used to obtain
the desired concentration of solution. Sodium chloride, as well as
other excipients, may also be present, if desired. Such excipients,
however, must maintain the overall stability of the trientine
active form.
[0075] The formulation of the invention should be substantially
isotonic. An isotonic solution may be defined as a solution that
has a concentration of electrolytes, non-electrolytes, or a
combination of the two that will exert an equivalent osmotic
pressure as that into which it is being introduced, in this case,
mammalian tissue. By "substantially isotonic" is meant within
.+-.20% of isotonicity, preferably within .+-.10%. The formulated
product may be included within a container, typically, for example,
a vial, cartridge, prefilled syringe or disposable pen.
[0076] In a further aspect the present invention consists in a
transdermal patch, pad, wrap or bandage ("patch") capable of being
adhered or otherwise associated with the skin of a subject, said
patch being capable of delivering an effective amount of one or
more trientine active agents when so applied to a subject who is
either (I) a diabetic subject or (II) a subject with copper levels
capable of diminishment to ameliorate or reverse, in whole or in
part, any one or more of systolic dysfunction, diastolic
dysfunction, contractility dysfunction, recoil dysfunction and
ejection fraction dysfunction (as determined, for example, by
ultrasound, MRI or other imaging) and/or any one or more of at
least some of any damage arising from diabetic kidney disease,
diabetic nephropathy and/or copper accumulation in the kidney
and/or at least some of any damage to the renal arteries and/or
cardiac structure damage selected from one or more of atrophy, loss
of myocytes, expansion of the extracellular space and increased
deposition of extracellular matrix (and its consequences), and/or
coronary artery structure damage selected from at least media
damage (the muscle layer) and intima damage (the endothelial layer)
(and its consequences).
[0077] In another aspect the present invention consists in an
article of manufacturing comprising a vessel containing as a CR, SR
and/or ER dosage form or one or more active agents, or containing
in CR, SR and/or ER dosage forms one or more pharmaceutically
copper chelators, including but not limited to one or more
acceptable trientine active agents; and instructions for use for
ameliorating and/or reversing, in whole or in part, in subject who
is either (I) a diabetic subject or (II) a subject with copper
levels capable of diminishment any one or more of the above-listed
indications.
[0078] In another aspect the present invention consists in an
article of manufacture comprising packaging material; and contained
within the packaging material one or more pharmaceutically
acceptable trientine active agents in a CR, SR and/or ER dosage
form, wherein the packaging material has a label that indicates
that the dosage form can be used for ameliorating, reversing and/or
improving in a subject who is either (I) a diabetic subject or (II)
a subject with copper levels capable of diminishment, any one or
more of the above-listed indications.
[0079] In one embodiment the dosage form, effective amount and/or
dosage regimen as herein referred to is able to provide an
effective daily dosage to the subject of a trientine active agent
(when expressed, for example, as the dihydrochloride salt of
trientine, irrespective of whether or not the dosage unit includes
that salt) of 4 g per day or below although if given orally the
dosage is from 1 mg to 4 g per day.
[0080] In another embodiment the oral dose delivery (cumulative or
otherwise) is in the range of from 200 mg to 4 g per day if given
orally. In a further embodiment the daily dosage is such as to
deliver 1.2 g per day or below.
[0081] In another aspect the dosage delivery is to provide, for
example, when expressed as trientine dihydrochloride or other
compound herein, a delivery into the subject (irrespective of the
dosage included in the dosage unit or units) being administered of
from 1 mg to 1.2 g per day. If orally administered the dosage is
from 200 mg to 1.2 g per day.
[0082] In a further embodiment the dosage is such as to deliver,
for example, the trientine active agent in a dosage unit that
administers the trientine active agent at a pH of from 7.2 to 7.6
(preferably a pH of 7.4.+-.0.1).
[0083] In another embodiment the dosage of, for example, the
trientine active agent, for example, trientine dihydrochloride in
sustained release is such that there is always less of the active
ingredient in a subject's body than results from the 250 mg plus
oral dosage forms for Wilson's disease.
[0084] In another embodiment a sustained release dosage form or
forms of, for example, the trientine active agent, for example,
trientine dihydrochloride is provided that are suitable for once
daily administration and that provide sustained or controlled and
long-lasting in vivo release. The form may deliver, for example,
not more than 10% trientine dihydrochloride in about 5 hours at an
acid pH of about <4.5 and delivers greater than 50% of trientine
dihydrochloride in 12 hrs at a pH of about <6.5 in a controlled
manner during in vivo and in vitro dissolution.
[0085] In yet a further aspect the present invention provides a
method of administering an effective amount of, for example, one or
more trientine active agents formulated in a delayed release
preparation (DR), a slow release preparation (SR), an extended
release preparation (ER), a controlled release preparation (CR)
and/or in a repeat action preparation (RA). In one embodiment the
formulations of DR, SR, ER, RA, or CR are suitable for use in the
treatment of any of the indications listed herein, including but
not limited to, heart failure, diabetic heart disease, acute
coronary syndrome, hypertensive heart disease, ischemic heart
disease, coronary artery disease, peripheral arterial disease,
Wilson's disease, or any form of cancer. Formulations of DR, SR,
ER, RA, or CR may contain an effective dosage unit for delivery to
the subject of from about 1 mg to abut 600 mg per unit of at-least
one trientine active agent, although in a further embodiment the
total daily dose rate is from between 5 grams to 1 mg and may work
to maintain a desired blood plasma concentration of the trientine
active agent for a desired period of time, preferably at least
about from between 18 to 24 hours.
[0086] In another aspect the present invention consists in a
formulation of, for example, at least one trientine active agent
that maintains constant plasma concentrations of the trientine
active agent for extended periods and is effective in removing
copper from the body of subjects with any one or more of the
indications listed herein, including but not limited to, heart
failure, diabetic heart disease, acute coronary syndrome,
hypertensive heart disease, ischemic heart disease, coronary artery
disease, peripheral arterial disease, Wilson's disease, or any form
of cancer.
[0087] In another aspect of the present invention consists in a
device containing, for example, one or more trientine active agents
in a monolithic matrix device and employed for the treatment of any
one or more of the indications listed herein, including but not
limited to, heart failure, diabetic heart disease, acute coronary
syndrome, hypertensive heart disease, ischemic heart disease,
coronary artery disease, peripheral arterial disease, Wilson's
disease, or any form of cancer.
[0088] In one embodiment the monolithic matrix device contains said
one or more trientine active agents in a dispersed soluble matrix,
in which said one or more trientine active agents becomes
increasingly available as the matrix dissolves or swells. The
monolithic matrix device, may include but is not limited to one or
more of the following excipients: hydroxypropylcellulose (BP) or
hydroxypropyl cellulose (USP); hydroxypropyl methylcellulose (BP,
USP); methylcellulose (BP, USP); calcium carboxymethylcellulose
(BP, USP); acrylic acid polymer or carboxy polymethylene (Carbopol)
or Carbomer (BP, USP); or linear glycuronan polymers such as
alginic acid (BP, USP), for example those formulated into
microparticles from alginic acid (alginate)-gelatin hydrocolloid
coacervate systems, or those in which liposomes have been
encapsulated by coatings of alginic acid with poly-L-lysine
membranes. Alternatively, said monolithic matrix includes one or
more trientine active agents dissolved in an insoluble matrix, from
which said one or more trientine active agents becomes available as
an aqueous solvent enters the matrix through micro-channels and
dissolves the trientine particles.
[0089] In a further embodiment the monolithic matrix contains, for
example, said one or more trientine active agents particles in a
lipid matrix or insoluble polymer matrix, including but not limited
to preparations formed from Carnauba wax (BP; USP); medium-chain
triglyceride such as fractionated coconut oil (BP) or triglycerida
saturata media (PhEur); or cellulose ethyl ether or ethylcellulose
(BP, USP). The lipids can be present in said monolithic matrix from
between 20-40% hydrophobic solids w/w. The lipids may remain intact
during the release process.
[0090] In another embodiment the device contains in addition to,
for example, said one or more trientine active agents, one or more
of the following, for example: a channeling agent, such as sodium
chloride or one or more sugars, which leaches from the formulation,
forming aqueous micro-channels (capillaries) through which solvent
enters, and through which drug is released.
[0091] Alternatively the device is any hydrophilic polymer matrix,
in which said one or more, for example, trientine active agents
is/are compressed as a mixture with any water-swellable hydrophilic
polymer.
[0092] The trientine active agent(s), for example, contained in the
hydrophilic polymer matrix may be between 20-80% (w/w).
[0093] In one embodiment the hydrophilic polymer matrix contains in
addition to said one or more, for example, trientine active agents
any one or more of the following, for example: a gel modifier such
as one or more of a sugar, counter ions, a pH buffer, a surfactant,
a lubricant such as a magnesium stearate and/or a glidant such as
colloidal silicon dioxide.
[0094] In another aspect the present invention consists in any
device containing an effective amount of, for example, said one or
more tritentine active agents comprising or including a
rate-controlling membrane surrounding a drug reservoir and
containing lactulose mixed with microcrystalline cellulose. The
ratio of lactulose to microcrystalline cellulose may be, for
example, about 60:40.
[0095] Clinical trials referred to hereinafter revealed that a
divided dose of 1.2 g/day of trientine is effective for and yet
(insofar as an instantaneous body level is concerned) in excess of
dosage levels to be required chronically in practice for the
purpose of amelioration and/or reversal of cardiac structure damage
and/or coronary artery structure damage. Such a dose rate of 1.2 g
per day is capable of being provided by the use of capsules of 300
mg trientine hydrochloride given half an hour before meals two
being given in the morning and two being given at night.
[0096] A measurement of free copper [which equals total plasma
copper minus ceruloplasmin-bound copper] can be made using the
procedure disclosed in the Merck & Co Inc datasheet
(www.Merck.com) for SYPRINE.RTM. (trientine dihydrochloride)
capsules: It states, "The most reliable index for monitoring
treatment is the determination of free cooper in the serum, which
equals the difference between quantitatively determined total
copper and ceruloplasmin-copper. Adequately treated subjects will
usually have less than 10 mcg free copper/dL of serum. Therapy may
be monitored with a 24-hour urinary copper analysis periodically.
Urine must be collected in copper-free glassware. Since a low
copper diet should keep copper absorption down to less than one
milligram a day, the subject probably will be in the desired state
of negative copper balance if 0.5 to 1.0 milligram of copper is
present in a 24-hour collection of urine".
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] We have conducted studies reliant on trientine
dihydrochloride in the STZ rat model as well in humans and wish to
describe the invention further by reference to the accompanying
drawings in which:
[0098] FIG. 1 shows the urine excretion in diabetic and
non-diabetic animals in response to increasing doses of trientine
or equivalent volume of saline, wherein urine excretion in diabetic
and nondiabetic animals in response to increasing doses of
trientine (bottom; 0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l
saline followed by 125 .mu.l saline flush injected at time shown by
arrow) or an equivalent volume of saline (top), and each point
represents a 15 min urine collection period (see Example 2 Methods
for details); error bars show SEM and P values are stated if
significant (P<0.05).
[0099] FIG. 2 shows urine excretion in non-diabetic and diabetic
animals receiving increasing doses of trientine or an equivalent
volume of saline, wherein urine excretion in diabetic (top) and
nondiabetic (bottom) rats receiving increasing doses of trientine
(0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l saline followed by 125
.mu.l saline flush injected at time shown by arrow) or an
equivalent volume of saline, and each point represents a 15 min
urine collection period (see Example 2 Methods for details); error
bars show SEM and P values are stated if significant
(P<0.05).
[0100] FIG. 3 shows copper excretion in the urine of diabetic and
non-diabetic animals receiving increasing doses of trientine or an
equivalent volume of saline, wherein copper excretion in urine of
diabetic (top) and nondiabetic (bottom) rats receiving increasing
doses of trientine (0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l
saline followed by 125 .mu.l saline flush injected at time shown by
arrow) or an equivalent volume of saline, and each point represents
a 15 min urine collection period (see Example 2 methods for
details); error bars show SEM and P values are stated if
significant (P<0.05).
[0101] FIG. 4 shows the same information in FIG. 18 with
presentation of urinary copper excretion per gram of bodyweight,
wherein urinary copper excretion per gram of bodyweight in diabetic
and nondiabetic animals in response to increasing doses of
trientine (bottom; 0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l
saline followed by 125 .mu.l saline flush injected at time shown by
arrow) or an equivalent volume of saline (top), and each point
represents a 15 min urine collection period (see Example 2 methods
for details); error bars show SEM and P values are stated if
significant (P<0.05).
[0102] FIG. 5 shows the total amount of copper excreted in
non-diabetic and diabetic animals administered saline or drug,
wherein total urinary copper excretion (.mu.mol) in nondiabetic
animals administered saline (black bar, n=7) or trientine (hatched
bar, n=7) and in diabetic animals administered saline (grey bar,
n=7) or trientine (white bar, n=7); error bars show SEM and P
values are stated if significant (P<0.05).
[0103] FIG. 6 shows the total amount of copper excreted per gram of
bodyweight in animals receiving trientine or saline, wherein total
urinary copper excretion per gram of bodyweight
(.mu.mol.gBW.sup.-1) in animals receiving trientine (nondiabetic:
hatched bar, n=7; diabetic: white bar, n=7) or saline (nondiabetic:
black bar, n=7; diabetic: grey bar, n=7); error bars show SEM and P
values are stated if significant (P<0.05).
[0104] FIG. 7 shows the iron excretion in urine of diabetic and
non-diabetic animals receiving increasing doses of trientine or an
equivalent volume of saline, wherein iron excretion in urine of
diabetic (top) and nondiabetic (bottom) rats receiving increasing
doses of trientine (0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l
saline followed by 125 .mu.l saline flush injected at time shown by
arrow) or an equivalent volume of saline, and each point represents
a 15 min urine collection period (see Example 2 methods for
details); error bars show SEM and P values are stated if
significant (P<0.05).
[0105] FIG. 8 shows the urinary iron excretion per gram of
bodyweight in diabetic and non-diabetic animals receiving trientine
or saline, wherein urinary iron excretion per gram of bodyweight in
diabetic and nondiabetic animals in response to increasing doses of
trientine (bottom; 0.1, 1.0, 10, 100 mg.kg.sup.-1 in 75 .mu.l
saline followed by 125 .mu.l saline flush injected at time shown by
arrow) or an equivalent volume of saline (top), and each point
represents a 15 min urine collection period (see Example 2 methods
for details); error bars show SEM and P values are stated if
significant (P<0.05).
[0106] FIG. 9 shows the total urinary iron excretion in
non-diabetic and diabetic animals administered saline or drug,
wherein total urinary iron excretion (.mu.mol) in nondiabetic
animals administered saline (black bar, n=7) or trientine (hatched
bar, n=7) and in diabetic animals administered saline (grey bar,
n=7) or trientine (white bar, n=7); error bars show SEM and P
values are stated if significant (P<0.05).
[0107] FIG. 10 shows the total urinary iron excretion per gram of
bodyweight in animals receiving trientine or saline, wherein total
urinary iron excretion per gram of bodyweight (.mu.mol.gBW.sup.-1)
in animals receiving trientine (nondiabetic: hatched bar, n=7;
diabetic: white bar, n=7) or saline (nondiabetic: black bar, n=7;
diabetic: gray bar, n=7); error bars show SEM and P values are
stated if significant (P.ltoreq.0.05).
[0108] FIG. 11 shows urinary [Cu] by AAS (.DELTA.) and EPR
(.tangle-solidup.) following sequential 10 mg.kg.sup.-1 (A) and 100
(B) trientine boluses, as in FIG. 19; (inset) background-corrected
EPR signal from 75-min urine indicating presence of
Cu.sup.II-trientine; *, P<0.05, **, P<0.01 vs. control.
[0109] FIG. 12 is a table comparing the copper and iron excretion
in the animals receiving trientine or saline, which is a
statistical analysis using a mixed linear model.
[0110] FIG. 13 shows the body weight of animals changing over the
time period of experiment in Example 5.
[0111] FIG. 14 shows the glucose levels of animals changing over
the time period of the experiment in Example 5.
[0112] FIG. 15 is a diagram showing cardiac output in animals as
measured in Example 5.
[0113] FIG. 16 is a diagram showing coronary flow in animals as
measured in Example 5.
[0114] FIG. 17 is a diagram showing coronary flows normalized to
final cardiac weight in animals as measured in Example 5./
[0115] FIG. 18 is a diagram showing aortic flow in animals as
measured in Example 5.
[0116] FIG. 19 is a diagram showing the maximum rate of positive
change in pressure development in the ventricle with each cardiac
cycle (contraction) in animals as measured in Example 5.
[0117] FIG. 20 is a diagram showing the maximum rate of decrease in
pressure in the ventricle with each cardiac cycle (relaxation) in
animals as measured in Example 5.
[0118] FIG. 21 shows the percentage of functional surviving hearts
at each after-load in animals as measured in Example 5.
[0119] FIG. 22 shows the structure of LV-myocardium from
STZ-diabetic and matched non-diabetic control rats following 7-w
oral trientine treatment, wherein cardiac sections were cut
following functional studies. Each image is representative of 5
independent sections per heart.times.3 hearts per treatment. a-d,
Laser confocal images of 120-.mu.M LV sections co-stained for actin
(Phalloidin-488, orange) and immunostained for
.beta..sub.1-integrin (CY5-conjugated secondary antibody, purple)
(scale-bar=33 .mu.m). a, Untreated-control; b, Untreated-diabetic;
c, Trientine treated diabetic; d, Trientine-treated non-diabetic
control. e-h, TEM images of corresponding 70-nM sections stained
with uranyl acetate/lead citrate (scale-bar=158 nm); e,
Untreated-control; f, Untreated-diabetic; g, Trientine-treated
diabetic; h, Trientine-treated non-diabetic control.
[0120] FIG. 23 shows effect of 6 months' oral trientine treatment
on LV mass in humans with T2DM, wherein trientine (600 mg
twice-daily) or matched placebo were administered to subjects with
diabetes (n=15) or matched controls (n=15) in a double-blind,
parallel-group study, and wherein differences in LV mass (g; mean
and 95% confidence interval) were determined by tagged-cardiac
MRI.
[0121] FIG. 24 shows a randomized, double blind, placebo-controlled
trial comparing effects of oral trientine and placebo on urinary Cu
excretion from male humans with uncomplicated T2DM and matched
non-diabetic controls, wherein urinary Cu excretion (.mu.mol.2
h.sup.-1 on day 1 (baseline) and day 7 following a single 2.4-g
oral dose of trientine or matched placebo to subjects described in
Table 9, placebo-treated T2DM, .smallcircle., placebo-treated
control, .cndot., trientine-treated T2DM, .quadrature.; trientine
treated control, .box-solid.. Cu excretion from T2DM following
trientine-treatment was significantly greater than that from
trientine-treated non-diabetic controls (P<0.05).
[0122] FIG. 25 shows mean arterial pressure (MAP) response in
diabetic and nondiabetic animals to 10 mg.kg.sup.-1 Trientine in 75
.mu.l+125 .mu.l saline flush (or an equivalent volume of saline).
Each point represents one minute averages of data points collected
every 2 seconds. The time of drug (or saline) administration is
indicated by the arrow. Error bars show SEM, and
[0123] FIG. 26 shows the ultraviolet-visible spectral trace of the
trientine containing formulation after being stored for 15 days and
upon the addition of copper to form the trientine-copper complex.
The traces were taken on day 0 (control formulation) and day 15.
There were three formulations containing trientine one was stored
in the dark at 4.degree. C., the second at room temperature
(21.degree. C.) in the dark and a third at room temperature in
daylight. When the spectral was taken copper was added.
DETAILED DESCRIPTION OF THE INVENTION
[0124] We have now shown in the STZ rat model for both diabetic and
non-diabetic humans a diminishment in available free copper has an
affect in ameliorating or reversing, in whole or in part, for
example, cardiac structure damage. This includes damage resulting
from, for example, atrophy, loss of myocytes, expansion of the
extra cellular space and increased deposition of extra cellular
matrix (and its consequences), and coronary artery structure injury
(and its consequences). In demonstrating reversal of damage in the
STZ rat, as further described herein, dose relativity for man has
been discovered insofar as copper scavenging into the urine is
concerned. Additionally, under physiological conditions injury to
the cardiac structure is sensed by distant stem cells, which
migrate to the site of damage then undergo alternate stem cell
differentiation, i.e., these events promote structural and
functional repair. However, it has been determined that the
accumulation of redox-active transition metals, particularly copper
in cardiac tissues and coronary arteries in subjects with diabetes,
is accompanied by a suppression of the normal tissue regeneration
effected by the migration of stem cells. In other words, elevated
tissue levels of copper suppress these normal biological behaviors
of such undifferentiated cells. Even in the non-diabetic mammal
(e.g., without type 2 diabetes mellitus) and even in a mammal
without a glucose mechanism abnormality (e.g., without IGT or
without IFG), a reduction in extra-cellular copper values will be
advantageous in providing a reduction in and/or a reversal of
copper-associated damage, for example, in whole or in part, as well
as improved tissue repair by restoration of normal tissue stem cell
responses.
[0125] A proof of principle Phase 2 study has shown positive
results. However, the dosage regimen was sub-optimal when compared
with its pharmacokinetic profile and the recently discovered
site-of-action profile. The bioavailability of the active species
of, for example, trientine dihydrochloride after oral
administration is low (<10%) due to poor absorption and marked
first-pass metabolism. Trientine dihydrochloride and its
transformed metabolite, N-acetyl-trientine hydrochloride, are both
capable of binding copper, although the chelating activity of the
analogue N-acetyl-trientine hydrochloride is reportedly
significantly lower than trientine dihydrochloride. See, Kodama H.,
et al., Life Sciences 61:899-907 (1997). Additionally, food,
mineral supplements and other drugs adversely affect absorption of
trientine dihydrochloride. The half-life of various copper
chelators, for example, trientine, indicated for the treatment and
reversal of heart failure and coronary heart disease, is relatively
short--being approximately 2 hours. Ideally trientine should be
taken in addition to current therapies, at a maximum tolerated
dose, utilizing a dose regimen that fits its pharmacokinetic and
site of action profiles. Regarding the plasma concentration of
trientine after oral administration to a patient, see Miyazaki, K.,
et al., "Determination of trientine in plasma of subjects with
high-performance liquid chromatography," Chem Pharm Bull 38:1035-38
(1998). Subjects with heart failure and/or coronary artery disease
are frequently on multiple drug regimens. Improved copper chelator
doses, dose preparation, and/or routes of administration for said
doses and dose preparations is needed for this reason as well.
[0126] The invention is related to and provides novel doses and
dose formulations, and routes of administration of various doses
and dose formulations, of copper chelators such as, for example,
trientine active agents. Trientine active agents include, for
example, trientine, salts of trientine, prodrugs of trientine and
salts of such prodrugs, analogs of trientine and salts and prodrugs
of such analogs, and/or active metabolites of trientine and salts
and prodrugs of such metabolites, including but not limited to
N-acetyl trientine and salts and prodrugs of N-acetyl trientine. It
is believed, without wishing to be bound by any particular
mechanism or theory of operation or effectiveness, that the dose
and dose formulations, and routes of administration, provide
unexpected benefits in the amelioration and reversal, in whole or
in part, of disorders, diseases, and conditions as set forth or
referenced or suggested herein, and in which copper is believed to
play a role.
[0127] Wilson's disease is due to an inherited defect in copper
excretion into the bile by the liver. The resulting copper
accumulation and copper toxicity results in liver disease, and in
some patients, brain damage. Patients present, generally between
the ages of 10 and 40 years, with liver disease, neurological
disease of a movement disorder type, or behavioral abnormalities,
and often with a combination of these. Wilson's disease is
effectively treated with orally administered copper chelators. It
has been demonstrated that chelated copper in patients with
Wilson's disease is excreted primarily through the feces, either by
the effective chelation of copper in the gut (or inhibition of
absorption), or by partial restoration of mechanisms that allow for
excretion of excess copper via urine or into the bile, or a
combination of the two. See Siegemund R, et al., "Mode of action of
triethylenetetramine dihydrochloride on copper metabolism in
Wilson's disease," Acta Neurol Scand. 83(6):364-6 (June 1991).
[0128] In contrast, experiments described herein unexpectedly
revealed that administration of the copper chelator trientine
dihydrochloride, for example, to non-Wilson's disease patients does
not result in increased excretion of copper in the feces. See
Example 9 and Table 11. Rather, excretion of excess copper in
non-Wilson's disease patients treated with copper chelators occurs
primarily, if not virtually exclusively, through the urine rather
than the feces. See Example 8 and FIG. 12. These data support the
idea that systemic (parenteral) administration of doses of copper
chelators that are lower than those given orally, or controlled
release administration of doses of copper chelators that are lower
than those given orally, or oral administration of lower dose forms
that avoid undesired first pass clearance such that more active
ingredient is available for its intended purpose outside the gut,
will be of significant benefit in the indications described herein,
for example. This includes administration of doses and dose forms
that provide for metered release directly into the circulatory
system (including intramuscular, intraperitoneal, subcutaneous and
intravenous administration) rather than indirectly through the gut.
Thus, the compounds may also be formulated for parenteral injection
(including, for example, by bolus injection or continuous infusion)
and may be presented in unit dose form in ampules, pre-filled
syringes, small bolus infusion containers, or in multi-does
containers with an added preservative.
[0129] According to the invention, doses and dose formulations of
copper chelators, including for example, trientine, that maintain
desired blood and tissue levels may be prepared that are highly
effective in causing removal of systemic copper from the body via
the urine and at lower doses than required for oral administration
given that gut copper need not be excreted, and will be more
effective in the treatment of any condition in which pathologically
increased or undesired tissue copper plays a role in disease
initiation or progression. Such diseases include any of the
indications identified herein, including but not limited to the
following: heart failure, diabetic heart disease, acute coronary
syndrome, hypertensive heart disease, ischemic heart disease,
coronary artery disease, peripheral arterial disease, and forms of
cancer amenable to treatment by copper chelation.
[0130] Trientine is a strongly basic moiety with multiple nitrogens
that can be converted into a large number of suitable associated
acid addition salts using an acid, for example, by reaction of
stoichiometrically equivalent amounts of trientine and of the acid
in an inert solvent such as ethanol or water and subsequent
evaporation if the dosage form is best formulated from a dry salt.
Possible acids for this reaction are in particular those that yield
physiologically acceptable salts. Nitrogen-containing copper
chelators, for example, trientine active agents such as, for
example, trientine, that can be delivered as a salt(s) (such as
acid addition salts, e.g., trientine dihydrochloride) act as
copper-chelating agents, which aids the elimination of copper from
the body by forming a stable soluble complex that is readily
excreted by the kidney. Thus inorganic acids can be used, e.g.,
sulfuric acid, nitric acid, hydrohalic acids such as hydrochloric
acid or hydrobromic acid, phosphoric acids such as orthophosphoric
acid, sulfamic acid. This is not an exhaustive list. Other organic
acids can be used to prepare suitable salt forms, in particular
aliphatic, alicyclic, araliphatic, aromatic or heterocyclic mono-or
polybasic carboxylic, sulfonic or sulfuric acids, (e.g., formic
acid, acetic acid, propionic acid, pivalic acid, diethylacetic
acid, malonic acid, succinic acid, pimelic acid, fumaric acid,
maleic acid, lactic acid, tartaric acid, malic acid, citric acid,
gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid,
methane-or ethanesulfonic acid, ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid,
p-toluenesulfonic acid, naphthalenemono-and-disulfonic acids, and
laurylsulfuric acid). Those in the art will be able to prepare
other suitable salt forms. Nitrogen-containing copper chelators,
for example, trientine active agents such as, for example,
trientine, can also be in the form of quaternary ammonium salts in
which the nitrogen atom carries a suitable organic group such as an
alkyl, alkenyl, alkynyl or aralkyl moiety. In one embodiment such
nitrogen-containing copper chelators are in the form of a compound
or buffered in solution and/or suspension to a near neutral pH much
lower than the pH 14 of a solution of trientine itself.
[0131] Other trientine active agents include derivative trientine
active agents, for example, trientine in combination with picolinic
acid (2-pyridinecarboxylic acid). These derivatives include, for
example, trientine picolinate and salts of trientine picolinate,
for example, trientine picolinate HCl. These also include, for
example, trientine di-picolinate and salts of trientine
di-picolinate, for example, trientine di-picolinate HCl. Picolinic
acid moieties may be attached to trientine, for example one or more
of the CH.sub.2 moieties, using chemical techniques known in the
art. Those in the art will be able to prepare other suitable
derivatives, for example, trientine-PEG derivatives, which may be
useful for particular dosage forms including oral dosage forms
having increased bioavailablity.
[0132] Other trientine active agents include trientine analogue
active agents. Such analogues include cyclic and acyclic analogues
according to the following formulae, for example: ##STR1## Acyclic
analogs of trientine are provided as follows based on the above
Formula I for tetra-heteroatom acyclic analogues, where X1, X2, X3,
and X4 are independently chosen from the atoms N, S or O such
that,
[0133] (a) for a four-nitrogen series, i.e., when X1, X2, X3, and
X4 are N then: R1, R2, R3, R4, R5, and R6 are independently chosen
from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10
cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri,
tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6
alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted
aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH.sub.2COOH,
CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH);
n1, n2, and n3 are independently chosen to be 2 or 3; and, R7, R8,
R9, R10, R11, and R12 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl. In addition, one or several of R1, R2, R3,
R4, R5, or R6 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.
Furthermore one or several of R7, R8, R9, R10, R11, or R12 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein.
[0134] (b) for a first three-nitrogen series, i.e., when X1, X2,
X3, are N and X4 is S or O then: R6 does not exist; R1, R2, R3, R4,
R5, and R6 are independently chosen from H, CH3, C2-C10 straight
chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10
cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl,
heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di,
tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and, R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R1, R2, R3, R4, or R5 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, C1-C10 alkyl-S-protein. Furthermore one or several
of R7, R8, R9, R10, R11, or R12 may be functionalized in order to
be attached to peptides, proteins, polyethylene glycols and other
such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0135] (c) for a second three-nitrogen series, i.e., when X1, X2,
and X4 are N and X3 is O or S then: R4 does not exist and R1, R2,
R3, R5, and R6 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and, R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R1, R2, R3, R5, or R6 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 allyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, C1-C10 alkyl-S-protein. Furthermore one or several
of R7, R8, R9, R10, R11, or R12 may be functionalized in order to
be attached to peptides, proteins, polyethylene glycols and other
such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
allyl-S-protein.
[0136] (d) for a first two-nitrogen series, i.e., when X2, and X3
are N and X1 and X4 are O or S then: R1 and R6 do not exist; R2,
R3, R4, and R5 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R2, R3, R4, or R5 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, C1-C10 alkyl-S-protein. Furthermore one or several
of R7, R8, R9, R10, R11, or R12 may be functionalized in order to
be attached to peptides, proteins, polyethylene glycols and other
such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0137] (e) for a second two-nitrogen series, i.e., when X1, and X3
are N and X2 and X4 are O or S then: R3 and R6 do not exist; R1,
R2, R4, and R5 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R1, R2, R4, or R5 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, R10, R11, or R12 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0138] (f) for a third three-nitrogen series, i.e., when X1, and X2
are N and X3 and X4 are O or S then: R4 and R6 do not exist; R1,
R2, R3, and R5 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R1, R2, R3, or R5 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, R10, R11, or R12 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0139] (g) for a fourth three-nitrogen series, i.e., when X1, and
X4 are N and X2 and X3 are O or S then: R3 and R4 do not exist; R1,
R2, R5 and R6 are independently chosen from H, CH3, C2-C10 straight
chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10
cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl,
heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di,
tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R1, R2, R5, or R6 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, R10, R11, or R12 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0140] Second, for a tetra-heteroatom cyclic series of analogues,
R1 and R6 are joined together by a bridging group in the form of
(CR13R14)n4, and X1, X2, X3, and X4 are independently chosen from
the atoms N, S or O such that,
[0141] (a) for a four-nitrogen series, i.e., when X1, X2, X3, and
X4 are N then: R2, R3, R4, and R5 are independently chosen from H,
CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl,
C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta
substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6
alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl
heteroaryl, C1-C6 alkyl fused aryl, CH.sub.2COOH,
CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH);
n1, n2, n3, and n4 are independently chosen to be 2 or 3; and R7,
R8, R9, R10, R11, R12, R13 and R14 are independently chosen from H,
CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl,
C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta
substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6
alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl
heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of
R2, R3, R4, or R5 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, C1-C10 alkyl-S-protein.
Furthermore one or several of R7, R8, R9, R10, R11, R12, R13 or R14
may be functionalized in order to be attached to peptides,
proteins, polyethylene glycols and other such chemical entities in
order to modify the overall pharmacokinetics, deliverability and/or
half lives of the constructs. Examples of such functionalization
include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein.
[0142] (b) for a three-nitrogen series, i.e., when X1, X2, X3, are
N and X4 is S or O then: R5 does nor exist; R2, R3, and R4 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl,
CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1, n2, n3, and n4 are independently
chosen to be 2 or 3; and R7, R8, R9, R10, R11, R12, R13 and R14 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl.
In addition, one or several of R2, R3 or R4 may be functionalized
in order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half-lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, R10,
R11, R12, R13 or R14 may be functionalized in order to be attached
to peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0143] (c) for a first two-nitrogen series, i.e., when X2, and X3
are N and X1 and X4 are O or S then: R2 and R5 do not exist; R3 and
R4 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1, n2, n3, and n4 are independently
chosen to be 2 or 3; and R7, R8, R9, R10, R11, R12, R13 and R14 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl.
In addition, one or both of R3, or R4 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half-lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, R10,
R11, R12, R13 or R14 may be functionalized in order to be attached
to peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0144] (d) for a second two-nitrogen series, i.e., when X1, and X3
are N and X2 and X4 are O or S then: R3 and R5 do not exist; R2 and
R4 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1, n2, n3, and n4 are independently
chosen to be 2 or 3; and R7, R8, R9, R10, R11, R12, R13 and R14 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl.
In addition, one or both of R2, or R4 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half-lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, R10,
R11, R12, R13 or R14 may be functionalized in order to be attached
to peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0145] (e) for a one-nitrogen series, i.e., when X1 is N and X2, X3
and X4 are O or S then: R3, R4 and R5 do not exist; R2 is
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl,
CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1, n2, n3, and n4 are independently
chosen to be 2 or 3; and R7, R8, R9, R10, R11, R12, R13 and R14 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl.
In addition, R2 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, R10,
R11, R12, R13 or R14 may be functionalized in order to be attached
to peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
allyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. ##STR2## Tri-heteroatom acyclic analogues
according to the above Formula II are provided where X1, X2, and X3
are independently chosen from the atoms N, S or O such that,
[0146] (a) for a three-nitrogen series, when X1, X2, and X3 are N
then: R1, R2, R3, R5, and R6 are independently chosen from H, CH3,
C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6
alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta
substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6
alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl
heteroaryl, C1-C6 alkyl fused aryl, CH.sub.2COOH,
CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH);
n1, and n2 are independently chosen to be 2 or 3; and R7, R8, R9,
and R10 are independently chosen from H, CH3, C2-C10 straight chain
or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10
cycloalkyl, aryl, mono, di, tri, tetra and penta substituted aryl,
heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di,
tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl. In addition, one or several of R1, R2, R3,
R5 or R6 may be functionalized in order to be attached to peptides,
proteins, polyethylene glycols and other such chemical entities in
order to modify the overall pharmacokinetics, deliverability and/or
half lives of the constructs. Examples of such functionalization
include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, or R10 may be functionalized in order to be
attached to peptides, proteins, polyethylene glycols and other such
chemical entities in order to modify the overall pharmacokinetics,
deliverability and/or half-lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0147] (b) for a first two-nitrogen series, when X1, and X3, are N
and X2 is S or O then: R3 does not exist; R1, R2, R3, R5, and R6
are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1, and n2 are independently chosen to be
2 or 3; and R7, R8, R9, and R10 are independently chosen from H,
CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl,
C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta
substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6
alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl
heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of
R1, R2, R5 or R6 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, or R10
may be functionalized in order to be attached to peptides,
proteins, polyethylene glycols and other such chemical entities in
order to modify the overall pharmacokinetics, deliverability and/or
half-lives of the constructs. Examples of such functionalization
include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein.
[0148] (c) for a second, two-nitrogen series, when X1 and X2 are N
and X3 is O or S then: R3 does not exist; R1, R2, R5, and R6 are
independently chosen from H, CH3, C2-C10 straight chain or branched
alkyl, C3-C10 cycloalkyl, C1-C6 allyl C3-C10 cycloalkyl, aryl,
mono, di, tri, tetra and penta substituted aryl, heteroaryl, fused
aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta
substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl,
CH.sub.2COOH, CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2,
CH.sub.2P(CH.sub.3)O(OH); n1 and n2 are independently chosen to be
2 or 3; and R7, R8, R9, and R10 are independently chosen from H,
CH3, C2-C10 straight chain or branched alkyl, C3-C10 cycloalkyl,
C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta
substituted aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6
alkyl mono, di, tri, tetra and penta substituted aryl, C1-C5 alkyl
heteroaryl, C1-C6 alkyl fused aryl. In addition, one or several of
R1, R2, R5, or R6 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, or R10
may be functionalized in order to be attached to peptides,
proteins, polyethylene glycols and other such chemical entities in
order to modify the overall pharmacokinetics, deliverability and/or
half-lives of the constructs. Examples of such functionalization
include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein.
[0149] A second series of tri-heteroatom acyclic analogues
according to the above Formula II are provided in which R1 and R6
are joined together by a bridging group in the form of (CR11R12)n3,
and X1, X2, and X3 are independently chosen from the atoms N, S or
O such that:
[0150] (a) for a three-nitrogen series, when X1, X2, and X3 are N
then: R2, R3, and R5 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl, CH.sub.2COOH, CH.sub.2SO.sub.3H,
CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH); n1, n2, and n3 are
independently chosen to be 2 or 3; and R7, R8, R9, R10, R11, and
R12 are independently chosen from H, CH3, C2-C10 straight chain or
branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl,
aryl, mono, di, tri, tetra and penta substituted aryl, heteroaryl,
fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and
penta substituted aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused
aryl. In addition, one or several of R2, R3, or R5 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or half
lives of the constructs. Examples of such functionalization include
but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, R10, R11, or R12 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0151] (b) for a two-nitrogen series, when X1, X2, are N and X3 is
S or O then: R5 does not exist; R2, and R3 are independently chosen
from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10
cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri,
tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6
alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted
aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH.sub.2COOH,
CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH);
n1, n2, and n3 are independently chosen to be 2 or 3; and R7, R8,
R9, R10, R11, and R12 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl. In addition, one or both of R2 or R3 may be
functionalized in order to be attached to peptides, proteins,
polyethylene glycols and other such chemical entities in order to
modify the overall pharmacokinetics, deliverability and/or
half-lives of the constructs. Examples of such functionalization
include but are not limited to C1-C10 alkyl-CO-peptide, C1-C10
alkyl-CO-protein, C1-C10 alkyl-CO-PEG, C1-C10 alkyl-NH-peptide,
C1-C10 alkyl-NH-protein, C1-C10 alkyl-NH--CO-PEG, C1-C10
alkyl-S-peptide, and C1-C10 alkyl-S-protein. Furthermore one or
several of R7, R8, R9, R10, R11, or R12 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0152] (c) for a one-nitrogen series, when X1 is N and X2, and X3
are O or S then: R3, and R5 do not exist; R2 is independently
chosen from H, CH3, C2-C10 straight chain or branched alkyl, C3-C10
cycloalkyl, C1-C6 alkyl C3-C10 cycloalkyl, aryl, mono, di, tri,
tetra and penta substituted aryl, heteroaryl, fused aryl, C1-C6
alkyl aryl, C1-C6 alkyl mono, di, tri, tetra and penta substituted
aryl, C1-C5 alkyl heteroaryl, C1-C6 alkyl fused aryl, CH.sub.2COOH,
CH.sub.2SO.sub.3H, CH.sub.2PO(OH).sub.2, CH.sub.2P(CH.sub.3)O(OH);
n1, n2, and n3 are independently chosen to be 2 or 3; and R7, R8,
R9, R10, R11, and R12 are independently chosen from H, CH3, C2-C10
straight chain or branched alkyl, C3-C10 cycloalkyl, C1-C6 alkyl
C3-C10 cycloalkyl, aryl, mono, di, tri, tetra and penta substituted
aryl, heteroaryl, fused aryl, C1-C6 alkyl aryl, C1-C6 alkyl mono,
di, tri, tetra and penta substituted aryl, C1-C5 alkyl heteroaryl,
C1-C6 alkyl fused aryl. In addition, R2 may be functionalized in
order to be attached to peptides, proteins, polyethylene glycols
and other such chemical entities in order to modify the overall
pharmacokinetics, deliverability and/or half lives of the
constructs. Examples of such functionalization include but are not
limited to C1-C10 alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10
alkyl-CO-PEG, C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein,
C1-C10 alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein. Furthermore one or several of R7, R8, R9, R10,
R11, or R12 may be functionalized in order to be attached to
peptides, proteins, polyethylene glycols and other such chemical
entities in order to modify the overall pharmacokinetics,
deliverability and/or half lives of the constructs. Examples of
such functionalization include but are not limited to C1-C10
alkyl-CO-peptide, C1-C10 alkyl-CO-protein, C1-C10 alkyl-CO-PEG,
C1-C10 alkyl-NH-peptide, C1-C10 alkyl-NH-protein, C1-C10
alkyl-NH--CO-PEG, C1-C10 alkyl-S-peptide, and C1-C10
alkyl-S-protein.
[0153] The analogues of the invention may be made using any of a
variety of chemical synthesis, isolation, and purification methods
known in the art.
[0154] Aspects of the invention include controlled or other drug
dose and drug dose delivery formulations and devices containing one
or more copper chelators, for example, trientine or salts thereof.
The present invention includes, for example, doses and dosage forms
for at least oral administration, transdermal delivery, topical
application, suppository delivery, transmucosal delivery, injection
(including subcutaneous administration, subdermal administration,
intramuscular administration, depot administration, and intravenous
administration (including delivery via bolus, slow intravenous
injection, and intravenous drip), infusion devices (including
implantable infusion devices, both active and passive),
administration by inhalation or insufflation, buccal
administration, sublingual administration, and ophthalmic
administration.
[0155] Indications in which the doses, dose formulations, and
routes of administration thereof will be useful include, for
example, diabetic cardiomyopathy, diabetic acute coronary syndrome
(e.g.; myocardial infarction--MI), diabetic hypertensive
cardiomyopathy, acute coronary syndrome associated with impaired
glucose tolerance (IGT), acute coronary syndrome associated with
impaired fasting glucose (IFG), hypertensive cardiomyopathy
associated with IGT, hypertensive cardiomyopathy associated with
IFG, ischemic cardiomyopathy associated with IGT, ischemic
cardiomyopathy associated with IFG, ischemic cardiomyopathy
associated with coronary heart disease (CHD), disorders of the
heart muscle (cardiomyopathy or myocarditis) that include, for
example, idiopathic cardiomyopathy, metabolic cardiomyopathy which
includes diabetic cardiomyopathy, alcoholic cardiomyopathy,
drug-induced cardiomyopathy, ischemic cardiomyopathy, and
hypertensive cardiomyopathy, acute coronary syndrome not associated
with any abnormality of glucose metabolism, hypertensive
cardiomyopathy not associated with any abnormality of glucose
metabolism, ischemic cardiomyopathy not associated with any
abnormality of glucose metabolism (irrespective of whether or not
such ischemic cardiomyopathy is associated with coronary heart
disease or not), and any one or more diseases of the vascular tree
including, by way of example, disease states of the aorta, carotid,
and of the arteries including cerebrovascular, coronary, renal,
retinal, iliac, femoral, popliteal, vasa nervorum, arteriolar tree
and capillary bed, atheromatous disorders of the major blood
vessels (macrovascular disease) such as the aorta, the coronary
arteries, the carotid arteries, the cerebrovascular arteries, the
renal arteries, the iliac arteries, the femoral arteries, and the
popliteal arteries, cardiac structure damage which includes, but is
not limited to, for example, atrophy, loss of myocytes, expansion
of the extracellular space and increased deposition of
extracellular matrix (and its consequences) and/or coronary artery
structure damage selected from at least media (the muscle layer)
and/or intima (the endothelial layer) damage (and its
consequences), plaque rupture of atheromatous lesions of major
blood vessels such as the aorta, the coronary arteries, the carotid
arteries, the cerebrovascular arteries, the renal arteries, the
iliac arteries, the femoral arteries and the popliteal arteries,
systolic dysfunction, diastolic dysfunction, aberrant
contractility, recoil characteristics and ejection fraction, toxic,
drug-induced, and metabolic (including hypertensive and/or diabetic
disorders of small blood vessels (microvascular disease) such as
the retinal arterioles, the glomerular arterioles, the vasa
nervorum, cardiac arterioles, and associated capillary beds of the
eye, the kidney, the heart, and the central and peripheral nervous
systems. Thus, the present invention also is directed to novel
doses and dose formulations of one or more copper chelators, for
example, trientine or salts thereof, useful for the pharmacological
therapy of diseases in humans and other mammals as disclosed
herein. The use of these doses, formulations and devices of, for
example, trientine enables effective treatment of these conditions,
through novel and improved formulations of the drug suitable for
administration to humans and other mammals.
[0156] The invention provides, for example, drug delivery
formulations containing one or more copper chelators, for example,
trientine or salts thereof. Thus, the present invention is directed
in part to novel delivery formulations of one or more copper
chelators, for example, trientine to optimize bioavailability and
to maintain plasma concentrations within the therapeutic range,
including for extended periods, and results in increases in the
time that trientine plasma concentrations of one or more copper
chelators, for example, trientine or salts thereof, remain within a
desired therapeutic range at the site or sites of action.
Controlled delivery preparations also optimize the drug
concentration at the site of action and minimize periods of under
and over medication, for example.
[0157] The invention also in part provides drug delivery
formulations and devices containing one or more copper chelators,
for example, one or more trientine active agents, including but not
limited to, trientine, trientine dihydrochloride or other
pharmaceutically acceptable salts thereof, the formulation being
suitable for periodic administration, including once daily
administration, to provide low dose controlled and/or low dose
long-lasting in vivo release of a copper chelator for chelation of
copper and excretion of chelated copper via the urine.
[0158] The invention also in part provides a drug delivery
formulations and devices containing one or more copper chelators,
for example, one or more trientine active agents, including but not
limited to, trientine, trientine dihydrochloride or other
pharmaceutically acceptable salts thereof, the formulation being
suitable for periodic administration, including once daily
administration, to provide enhanced bioavailability of a copper
chelator for chelation of copper and excretion of chelated copper
via the urine.
[0159] Examples of controlled drug formulations useful for delivery
of the compounds and formulations of the invention are found in,
for example, Sweetman, S. C. (Ed.). Martindale. The Complete Drug
Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483
pp.; Aulton, M. E. (Ed.) Pharmaceutics. The Science of Dosage Form
Design. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and,
Ansel, H. C., Allen, L. V. and Popovich, N. G. Pharmaceutical
Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999,
676 pp. Excipients employed in the manufacture of drug delivery
systems are described in various publications known to those
skilled in the art including, for example, Kibbe, E. H. Handbook of
Pharmaceutical Excipients, 3rd Ed., American Pharmaceutical
Association, Washington, 2000, 665 pp. The USP also provides
examples of modified-release oral dosage forms, including those
formulated as tablets or capsules. See, for example, The United
States Pharmacopeia 23/National Formulary 18, The United States
Pharmacopeial Convention, Inc., Rockville Md., 1995 (hereinafter
"the USP"), which also describes specific tests to determine the
drug release capabilities of extended-release and delayed-release
tablets and capsules. The USP test for drug release for
extended-release and delayed-release articles is based on drug
dissolution from the dosage unit against elapsed test time.
Descriptions of various test apparatus and procedures may be found
in the USP. The individual monographs contain specific criteria for
compliance with the test and the apparatus and test procedures to
be used. Examples have been given, for example for the release of
aspirin from Aspirin Extended-release Tablets (for example, see:
Ansel, H. C., Allen, L. V. and Popovich, N. G. Pharmaceutical
Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999,
p. 237). Modified-release tablets and capsules must meet the USP
standard for uniformity as described for conventional dosage units.
Uniformity of dosage units may be demonstrated by either of two
methods, weight variation or content uniformity, as described in
the USP. Further guidance concerning the analysis of extended
release dosage forms has been provided by the F.D.A. (see Guidance
for Industry. Extended release oral dosage forms: development,
evaluation, and application of in vitro/in vivo correlations.
Rockville, Md.: Center for Drug Evaluation and Research, Food and
Drug Administration, 1997). Compliance of a dosage regime is always
essential in order to derive the best benefit from a treatment
regime. The present invention recognizes an additional benefit from
dosage forms that can provide such levels of delivery to a subject
as are required to elicit the advantages now seen from the prospect
of lower overall dose delivery of trientine formulations when one
compares them to BID (two times a day), TID (three times a day),
QID (four times a day), and so on, multiple dosage oral regimes
hitherto used with, for example, trientine formulations for
Wilson's disease.
[0160] Aspects of the invention also include various drug delivery
systems for the delivery of one or more copper chelators, for
example, trientine or salts thereof Thus, the present invention
also is directed to novel types of drug delivery systems. These
include, for example, modified-release (MR) dosage forms of the
present invention, including delayed-release (D)R) forms;
prolonged-action (PA) forms; controlled-release (CR) forms;
extended-release (ER) forms; timed-release (TR) forms; and
long-acting (LA) forms. For the most part, these terms are used to
describe orally administered dosage forms, whereas the term
rate-controlled delivery is applied to certain types of drug
delivery systems in which the rate of drug delivery is controlled
by features of the device rather than by physiological or
environmental conditions such as gastrointestinal pH or drug
transit time through the gastrointestinal tract These formulations
effect (1) delayed total drug release form some time after drug
administration, (2) drug release in small aliquots intermittently
after administration, (3) drug release slowly at a controlled rate
governed by the delivery system, (4) drug release at a constant
rate that does not vary, and/or (5) drug release for a
significantly longer period than usual formulations. Within the
scope of the terms "modified", "delayed", "slow", "prolonged",
"timed", "long-acting", "controlled", and/or "extended" release
dosage units as used herein are any appropriate delivery form.
[0161] Advantages of these formulations for administration of one
or more copper chelators, for example, trientine or salts thereof,
include convenience to the subject; increased compliance and
achievement of steady state drug levels with twice-daily (or less)
dosing; smoothening of plasma drug profiles to a constant level for
extended time periods; prevention of drug toxicity; and elimination
of breakthrough of therapeutic failure, especially at night.
Modified-release dosage forms of the invention include dosage forms
having drug release features based on time, course, and/or location
which are designed to accomplish therapeutic or convenience
objectives not offered by conventional or immediate-release forms.
See, for example, Bogner, R. H. Bioavailability and bioequivalence
of extended-release oral dosage forms. U.S. Pharmacist 22
(Suppl.):3-12 (1997); Scale-up of oral extended-release drug
delivery systems: part I, an overview. Pharmaceutical Manufacturing
2:23-27 (1985). Extended-release dosage forms of the invention
include, for example, as defined by The United States Food and Drug
Administration (F. D. A.), a dosage form that one that allows a
reduction in dosing frequency to that presented by a conventional
dosage form, e.g., a solution or an immediate-release dosage form.
See, for example, Bogner, R. H. Bioavailability and bioequivalence
of extended-release oral dosage forms. US Pharmacist 22
(Suppl.):3-12 (1997); Guidance for industry. Extended release oral
dosage forms: development, evaluation, and application of the in
vitro/in vivo correlations. Rockville, MD: Center for Drug
Evaluation and Research, Food and Drug Administration (1997).
Repeat action dosage forms of the invention include, for example,
forms that contain two single doses of medication, one for
immediate release and the second for delayed release. Bi-layered
tablets, for example, may be prepared with one layer of drug for
immediate release with the second layer deigned to release drug
later as either a second dose or in an extended-release manner.
Targeted-release dosage forms of the invention include, for
example, formulations that facilitate drug release and which are
directed towards isolating or concentrating a drug in a body
region, tissue, or site for absorption or for drug action.
[0162] One example is oral delivery forms of tablet, capsule,
lozenge, or the like form, or any liquid form such as syrups,
aqueous solutions, emulsion and the like, capable of providing over
the period of time between dosages an ongoing release of an
effective level of the active ingredient, e.g., one or more the
compounds and formulations of the invention.
[0163] Examples of dosage units for transdermal delivery of the
compounds and formulations of the invention include transdermal
patches, transdermal bandages, and the like.
[0164] Examples of dosage units for topical delivery of the
compounds and formulations of the invention are any lotion, stick,
spray, ointment, paste, cream, gel, etc. whether applied directly
to the skin or via an intermediary such as a pad, patch or the like
but which again has a slow release action in delivery of the active
agent into the body of the subject.
[0165] Examples of dosage units for suppository delivery of the
compounds and formulations of the invention include any solid
dosage form inserted into a bodily orifice particularly those
inserted rectally, vaginally and urethrally.
[0166] Examples of dosage units for transmucosal delivery of the
compounds and formulations of the invention include depositories
solutions for enemas, pessaries, tampons, creams, gels, pastes,
foams, nebulised solutions, powders and similar formulations
containing in addition to the active: ingredients such carriers as
are known in the art to be appropriate.
[0167] Examples of dosage units for injection of the compounds and
formulations of the invention include delivery via bolus such as
single or multiple administrations by intravenous injection,
subcutaneous, subdermal, and intramuscular administration or oral
administration.
[0168] Examples of dosage units for depot administration of the
compounds and formulations of the invention include pellets or
small cylinders of active agent or solid forms wherein the active
agent is entrapped in a matrix of biodegradable polymers,
microemulsions, liposomes or is microencapsulated.
[0169] Examples of infusion devices for compounds and formulations
of the invention include infusion pumps containing one or more
copper chelators, for example, for example, trientine or salts
thereof, at a desired amount for a desired number of doses or
steady state administration, and include implantable drug pumps.
Examples of implantable infusion devices include any solid form in
which the active agent is encapsulated within or dispersed
throughout a biodegradable polymer or synthetic, polymer such as
silicone, silicone rubber, silastic or similar polymer.
[0170] Examples of dosage units for inhalation or insufflation of
the compounds and formulations of the invention include
compositions comprising solutions and/or suspensions in
pharmaceutically acceptable, aqueous, or organic solvents, or
mixture thereof and/or powders.
[0171] Examples of dosage units for buccal delivery of the
compounds and formulations of the invention include lozenges,
tablets and the like, compositions comprising solutions and/or
suspensions in pharmaceutically acceptable, aqueous, or organic
solvents, or mixture thereof and/or powders
[0172] Examples of dosage units for sublingual delivery of the
compounds and formulations of the invention include lozenges,
tablets and the like, compositions comprising solutions and/or
suspensions in pharmaceutically acceptable, aqueous, or organic
solvents, or mixture thereof and/or powders
[0173] Examples of dosage units for opthalmic delivery of the
compounds and formulations of the invention include compositions
comprising solutions and/or suspensions in pharmaceutically
acceptable, aqueous, or organic solvents, inserts,
[0174] The invention in part provides dose delivery devices and
formulations incorporating one or more copper chelators, for
example, trientine or salts thereof, complexed with one or more
suitable anions to yield complexes that are only slowly soluble in
body fluids. One such example of modified release forms of one or
more copper chelators, for example, trientine or salts thereof, is
produced by the incorporation of the active agent or agents into
certain complexes such as those formed with the anions of various
forms of tannic acid (for example, see: Merck Index 12th Ed.,
9221). Dissolution of such complexes may depend, for example, on
the pH of the environment. This slow dissolution rate provides for
the extended release of the drug. For example, trientine salts of
tannic acid, trientine tannates, provide for this quality, and are
expected to possess utility for the treatment of conditions in
which increased copper plays a role. Examples of equivalent
products are provided by those having the tradename Rynatan
(Wallace: see, for example, Madan, P. L., "Sustained release dosage
forms," U.S. Pharmacist 15:39-50 (1990); Ryna-12 S, which contains
a mixture of mepyramine tannate with phenylephrine tannate,
Martindale 33rd Ed., 2080.4).
[0175] Also included in the invention are coated beads, granules or
microspheres containing one or more copper chelators, for example,
trientine or salts thereof Thus, the invention also provides a
method to achieve modified release of one or more copper chelators,
for example, trientine or salts thereof, by incorporation of the
drug into coated beads, granules, or microspheres. Such
formulations of one or more copper chelators, for example trientine
or salts thereof, have utility for the treatment of diseases in
humans and other mammals in which a copper chelator, for example,
trientine, is indicated. In such systems, the drug is distributed
onto beads, pellets, granules or other particulate systems. Using
conventional pan-coating or air-suspension coating techniques, a
solution of the drug substance is placed onto small inert nonpareil
seeds or beads made of sugar and starch or onto microcrystalline
cellulose spheres. The nonpareil seeds are most often in the 425 to
850 micrometer range whereas the microcrystalline cellulose spheres
are available ranging from 170 to 600 micrometers (see Ansel, H.
C., Allen, L. V. and Popovich, N. G. Pharmaceutical Dosage Forms
and Drug Delivery Systems, 7th Ed., Lippincott 1999, p. 232). The
microcrystalline spheres are considered more durable during
production than sugar-based cores (see: Celphere microcrystalline
cellulose spheres. Philadelphia: FMC Corporation, 1996). Methods
for manufacture of microspheres suitable for drug delivery have
been described (see, for example, Arshady, R. Microspheres and
microcapsules: a survey of manufacturing techniques. 1: suspension
and cross-linking. Polymer Eng Sci 30:1746-1758 (1989); see also,
Arshady, R. Micro-spheres and microcapsules: a survey of
manufacturing techniques. 2: coacervation. Polymer Eng Sci
30:905-914 (1990); see also: Arshady R Microspheres and
micro-capsules: a survey of manufacturing techniques. 3: solvent
evaporation. Polymer Eng Sci 30:915-924 (1990). In instances in
which the drug dose is large, the starting granules of material may
be composed of the drug itself. Some of these granules may remain
uncoated to provide immediate drug release. Other granules (about
two-thirds to three-quarters) receive varying coats of a lipid
material such as beeswax, carnauba wax, glycerylmonostearate, cetyl
alcohol, or a cellulose material such as ethylcellulose (infra).
Subsequently, granules of different coating thickness are blended
to achieve a mixture having the desired drug-release
characteristics. The coating material may be coloured with one or
more dyes to distinguish granules or beads of different coating
thickness (by depth of colour) and to provide distinctiveness to
the product. When properly blended, the granules may be placed in
capsules or tableted. Various coating systems are commercially
available which are aqueous-based and which use ethylcellulose and
plasticizer as the coating material (e.g., Aquacoat.TM. [FMC
Corporation, Philadelphia] and Surerelease.TM. [Colorcon]; Aquacoat
aqueous polymeric dispersion. Philadelphia: FMC Corporation, 1991;
Surerelease aqueous controlled release coating system. West Point,
Pa.: Colorcon, 1990; Butler, J., Cumming, I, Brown, J. et al. A
novel multiunit controlled-release system. Pharm Tech 22:122-138
(1998); Yazici, E., Oner, L., Kas, H. S. & Hincal, A. A.
Phenytoin sodium microspheres: bench scale formulation, process
characterization and release kinetics. Pharmaceut Dev Technol
1:175-183 (1996)). Aqueous-based coating systems eliminate the
hazards and environmental concerns associated with organic
solvent-based systems. Aqueous and organic solvent-based coating
methods have been compared (see, for example, Hogan, J. E. Aqueous
versus organic solvent coating. Int J Pharm Tech Prod Manufacture
3:17-20 (1982)). The variation in the thickness of the coats and in
the type of coating materials used affects the rate at which the
body fluids are capable of penetrating the coating to dissolve the
drug. Generally, the thicker the coat, the more resistant to
penetration and the more delayed will be drug release and
dissolution. Typically, the coated beads are about 1 mm in
diameter. They are usually combined to have three or four release
groups among the more than 100 beads contained in the dosing unit
(see Madan, P. L. Sustained release dosage forms. U. S. Pharmacist
15:39-50 (1990)). This provides the different desired sustained or
extended release rates and the targeting of the coated beads to the
desired segments of the gastrointestinal tract. One example of this
type of dosage form is the Spansule.TM. (SmithKline Beecham
Corporation, U.K.). Examples of film-forming polymers which can be
used in water-insoluble release-slowing intermediate layer(s) (to
be applied to a pellet, spheroid or tablet core) include
ethylcellulose, polyvinyl acetate, Eudragit.RTM. RS, Eudragit.RTM.
RL, etc. (Each of Eudragit.RTM. RS and Eudragit.RTM. RL is an
ammonio methacrylate copolymer.) The release rate can be controlled
not only by incorporating therein suitable water-soluble pore
formers, such as lactose, mannitol, sorbitol, etc., but also by the
thickness of the coating layer applied. Multi tablets include small
spheroid-shaped compressed minitablets that may have a diameter of
between 3 to 4 mm and can be placed in gelatin capsule shell to
provide the desired pattern of drug release. Each capsule may
contain 8-10 minitablets, some uncoated for immediate release and
others coated for extended drug release.
[0176] The following methods may be employed to generate delivery
systems containing modified-release delivery forms of one or more
copper chelators, for example trientine or salts thereof or other
trientine active agents, suitable for oral administration to humans
and other mammals. Two basic mechanisms are available to achieve
modified release drug delivery. These are altered dissolution or
diffusion of drugs and excipients. Within this context, for
example, four processes may be employed, either simultaneously or
consecutively. These are as follows: (i) hydration of the device
(e.g., swelling of the matrix); (ii) diffusion of water into the
device; (iii) controlled or delayed dissolution of the drug; and
(iv) controlled or delayed diffusion of dissolved or solubilized
drug out of the device. Continuous release is ideally zero-order,
and is produced by a constant rate of diffusion or osmosis.
Modified release dosage forms commonly fit into one of three
categories of system,: monolithic or matrix; reservoir- or
membrane-controlled; or osmotic pump systems. Each comprises the
following components: active drug; release controlling agents;
matrix modifiers; drug modifiers; supplementary coatings; and
conventional formulation excipients, such as those described in
reference works known to those skilled in the art (see, for
example, Kibble A. H (ed.) Handbook of Pharmaceutical Excipients,
3rd Edition, American Pharmaceutical Association, 2000, 665
pp.).
[0177] For orally administered dosage forms of the compounds and
formulations of the invention, extended drug action may be achieved
by affecting the rate at which the drug is released from the dosage
form and/or by slowing the transit time of the dosage form through
the gastrointestinal tract (see Bogner, R. H. Bioavailability and
bioequivalence of extended-release oral dosage forms. US Pharmacist
22 (Suppl.):3-12 (1997)). The rate of drug release from solid
dosage forms may be modified by the technologies described below
which, in general, are based on the following: 1) modifying drug
dissolution by controlling access of biologic fluids to the drug
through the use of barrier coatings; 2) controlling drug diffusion
rates from dosage forms; and 3) chemically reacting or interacting
between the drug substance or its pharmaceutical barrier and
site-specific biological fluids. Systems by which these objectives
are achieved are also provided herein. In one approach, employing
digestion as the release mechanism, the active agent is either
coated or entrapped in a substance that is slow digested or
dispersed into the intestinal tract. The rate of availability of
the active agent is a function of the rate of digestion of the
dispersible material. Therefore, the release rate, and thus the
effectiveness of the agent, varies from subject to subject
depending upon the ability of the subject to digest the material.
In another approach such as disclosed in U.S. Pat. No. 3,247,066,
the active agent is dispersed in a water-soluble colloid and then
coated with a rupturable plastic, non-digestible material that is
permeable to the diffusion of water. After ingestion and upon
entering the gastrointestinal tract, water in the body fluids
diffuses through the coating and causes the colloid to swell. The
coating is ruptured by the swelling colloid and the total content
of active agent is released. Although there is substantially less
variation in the rate of release from subject to subject,
substantially the entire active agent is released at once resulting
in an initially high blood level content that decreases rapidly
with time.
[0178] U.S. Pat. No. 3,115,441 discloses another encapsulation
method useful for delivery of the compounds and formulations of the
invention wherein particles of active agent are first given a quick
thin coating of a film-forming material and a non-toxic,
hydrophobic material that is then coated with successive coatings
of an organic solvent-resistant material. The coated particles are
mixed with uncoated active agent and this mixture is then formed
into a tablet with the coated tablets being entrapped in a matrix
of the uncoated active agent. Tablets made according to this method
have the advantage of providing immediate delivery of the compounds
and formulations of the invention because the matrix material
(which comprises the initial dosage) dissolves immediately upon
ingestion.
[0179] Another approach, as in U.S. Pat. No. 4,025,613, is to
provide an improved blood level profile of the compounds and
formulations of the invention that results from simply applying a
film of a non-aqueous solution of cellulose acetate over either
individual particles of active agent before tableting or over the
outside of tablets formed from untreated active agent particles,
which upon drying forms a coating of cellulose acetate. Depending
on the role attributed to the film-coating, persons skilled in the
art will be able to choose the film-forming agent from among the
following categories: cellulose derivatives such as
hydroxypropylmethylcellulose (HPMC), ethyl cellulose, cellulose
acetophthalate, cellulose acetopropionate, cellulose trimelliate,
the polymers and copolymers of methacrylic acid and its
derivatives. The film-forming agent may be supplemented with:
plasticizers (such as polyoxyethylene glycols of high molecular
weight, esters of polyacids such as citric acid or phthalic acid)
fillers (such as talc, metal oxides such as titanium oxide)
colorants chosen from those usable and approved by the
pharmaceutical and food industries.
[0180] A further form of slow release form of the compounds and
formulations of the invention is any suitable osmotic system where
semipermeable membranes of cellulose acetate, cellulose acetate
butyrate, cellulose acetate propionate, to control the release of
active ingredients. These can be coated with aqueous dispersions of
enteric lacquers without changing release rate. An example of such
an osmotic system is an osmotic pump device, an example of which is
the Oros.TM. device developed by Alza Inc. (U.S.A.). This system
comprises a core tablet surrounded by a semi-permeable membrane
coating having a 0.4 mm diameter hole produced by a laser beam. The
core tablet has two layers, one containing the drug (the "active"
layer) and the other containing a polymeric osmotic agent (the
"push" layer). The core layer consists of active drug, filler, a
viscosity modulator, and a solubilizer. The system operates on the
principle of osmotic pressure. This system is suitable for delivery
of a wide range of drugs, including trientine or salts thereof. The
coating technology is straightforward, and release is zero-order.
When the tablet is swallowed, the semi-permeable membrane permits
aqueous fluid to enter from the stomach into the core tablet,
dissolving or suspending the drug. As pressure increases in the
osmotic layer, it forces or pumps the drug solution out of the
delivery orifice on the side of the tablet Only the drug solution
(not the undissolved drug) is capable of passing through the hole
in the tablet. The system is designed such that only a few drops of
water are drawn into the tablet each hour. The rate of inflow of
aqueous fluid and the function of the tablet depends on the
existence of an osmotic gradient between the contents of the
bi-layer and the fluid in the gastrointestinal tract. Drug delivery
is essentially constant as long as the osmotic gradient remains
unchanged. The drug release rate may be altered by changing the
surface area, the thickness or composition of the membrane, and/or
by changing the diameter of the drug release orifice. The
drug-release rate is not affected by gastrointestinal acidity,
alkalinity, fed conditions, or gut motility. The biologically inert
components of the tablet remain intact during gut transit and are
eliminated in the feces as an insoluble shell. Other examples of
the application of this technology are provided by Glucotrol XL
Extended Release Tablets (Pfizer Inc.) and Procardia XL Extended
Release Tablets (Pfizer Inc.; see, Martindale 33rd Ed., p.
2051.3).
[0181] The invention also provides delivery devices for compounds
and formulations of the invention that utilize monolithic matrices
including, for example, slowly eroding or hydrophilic polymer
matrices, in which one or more copper chelators, for example,
trientine or salts thereof, is compressed or embedded.
[0182] Monolithic matrix devices for delivery of the compounds and
formulations of the invention comprise those formed using either of
the following systems, for example: (I), drug particles are
dispersed in a soluble matrix, in which they become increasingly
available as the matrix dissolves or swells; examples include
hydrophilic colloid matrices, such as hydroxypropylcellulose (BP)
or hydroxypropyl cellulose (USP); hydroxypropyl methylcellulose
(HPMC; BP, USP); methylcellulose (MC; BP, USP); calcium
carboxymethylcellulose (Calcium CMC; BP, USP); acrylic acid polymer
or carboxy polymethylene (Carbopol) or Carbomer (BP, USP); or
linear glycuronan polymers such as alginic acid (BP, USP), for
example those formulated into microparticles from alginic acid
(alginate)-gelatin hydrocolloid coacervate systems, or those in
which liposomes have been encapsulated by coatings of alginic acid
with poly-L-lysine membranes. Drug release occurs as the polymer
swells, forming a matrix layer that controls the diffusion of
aqueous fluid into the core and thus the rate of diffusion of drug
from the system. In such systems, the rate of drug release depends
upon the tortuous nature of the channels within the gel, and the
viscosity of the entrapped fluid, such that different release
kinetics can be achieved, for example, zero-order, or first-order
combined with pulsatile release. Where such gels are not
cross-linked, there is a weaker, non-permanent association between
the polymer chains, which relies on secondary bonding. With such
devices, high loading of the active drug is achievable, and
effective blending is frequent. Devices contain 20-80% of drug
(w/w), along with gel modifiers that can enhance drug diffusion;
examples of such modifiers include sugars that can enhance the rate
of hydration, ions that can influence the content of cross-links,
and pH buffers that affect the level of polymer ionization.
Hydrophilic matrix devices typically contain pH buffers,
surfactants, counter-ions, lubricants such as magnesium stearate
(BP, USP) and a glidant such as colloidal silicon dioxide (USP;
colloidal anhydrous silica, BP) in addition to drug substance and
hydrophilic matrix; (II) drug particles are dissolved in an
insoluble matrix, from which drug becomes available as solvent
enters the matrix, often through channels, and dissolves the drug
particles. Examples include systems formed with a lipid matrix, or
insoluble polymer matrix, including preparations formed from
Carnauba wax (BP; USP); medium-chain triglyceride such as
fractionated cocoanut oil (BP) or triglycerida saturata media
(PhEur); or cellulose ethyl ether or ethylcellulose (BP, USP).
Lipid matrices are simple and easy to manufacture, and incorporate
the following blend of powdered components: lipids (20-40%
hydrophobic solids w/w) which remain intact during the release
process; drug substance; channeling agent, such as sodium chloride
or sugars, which leaches from the formulation, forming aqueous
micro-channels (capillaries) through which solvent enters, and
through which drug is released. In the alternative system, which
employs an insoluble polymer matrix, the drug is embedded in an
inert insoluble polymer and is released by leaching of aqueous
fluid, which diffuses into the core of the device through
capillaries formed between particles, and from which drug diffuses
out of the device. The rate of release is controlled by the degree
of compression, particle size, and the nature and relative content
(w/w) of excipients. An example of such a device is that of Ferrous
Gradumet (Martindale 33rd Ed., 1360.3). A further example of a
suitable insoluble matrix is an inert plastic matrix. By this
method, trientine active agent is granulated with an inert plastic
material such as polyethylene, polyvinyl acetate, or
polymethacrylate, and the granulated mixture is then compressed
into tablets. Once ingested, the drug is slowly released from the
inert plastic matrix by diffusion (see, for example, Bodmeier, R.
& Paeratakul, O., "Drug release from laminated polymeric films
prepared from aqueous latexes," J Pharm Sci 79:32-26 (1990);
Laghoueg, N., et al., "Oral polymer-drug devices with a core and an
erodable shell for constant drug delivery," Int J Pharm 50:133-139
(1989); Buckton, G., et al., "The influence of surfactants on drug
release from acrylic matrices. Int J Pharm 74:153-158 (1991)). The
compression of the tablet creates the matrix or plastic form that
retains its shape during the leaching of the drug and through its
passage through the gastrointestinal tract. An immediate-release
portion of drug may be compressed onto the surface of the tablet.
The inert tablet matrix, expended of drug, is excreted with the
feces. An example of a successful dosage form of this type is
Gradumet (Abbott; see, for example, Ferro-Gradumet, Martindale 33rd
Ed., p. 1860.4).
[0183] Further useful approaches have compounds and formulations of
the invention incorporated in pendent attachments to a polymer
matrix (see, for example, Scholsky, K. M. & Fitch, R. M.
Controlled release of pendant bioactive materials from acrylic
polymer colloids. J Controlled Release 3:87-108 (1986)). In these
devices, drugs are attached by means of an ester linkage to
poly(acrylate) ester latex particles prepared by aqueous emulsion
polymerization.
[0184] Further embodiments incorporate dosage forms of the
compounds and formulations of the invention in which the drug is
bound to a biocompatible polymer by a labile chemical bond, e.g.,
polyanhydrides prepared from a substituted anhydride (itself
prepared by reacting an acid chloride with the drug: methacryloyl
chloride and the sodium salt of methoxy benzoic acid) have been
used to form a matrix with a second polymer (Eudragit RL) which
releases drug on hydrolysis in gastric fluid (see: Chafi, N.,
Montheard, J. P. & Vergnaud, J. M. Release of 2-aminothiazole
from polymeric carriers. Int J Pharm 67:265-274 (1992)).
[0185] In formulating a successful hydrophilic matrix system for
the compounds and formulations of the invention, the polymer
selected for use must form a gelatinous layer rapidly enough to
protect the inner core of the tablet from disintegrating too
rapidly after ingestion. As the proportion of polymer is increased
in a formulation so is the viscosity of the gel formed with a
resulting decrease in the rate of drug diffusion and release (see
Formulating for controlled release with Methocel Premium cellulose
ethers. Midland, Mich.: Dow Chemical Company, 1995). In general,
20% (w/w) of HPMC results in satisfactory rates of drug release for
an extended-release tablet formulation. However, as with all
formulations, consideration must be given to the possible effects
of other formulation ingredients such as fillers, tablet binders,
and disintegrants. An example of a proprietary product formulated
using a hydrophilic matrix base of HPMC for extended drug release
is that of Oramorph SR Tablets (Roxane; see Martindale 33rd Ed., p.
2014.4).
[0186] Two-layered tablets can be manufactured containing one or
more of the compounds and formulations of the invention, with one
layer containing the uncombined drug for immediate release and the
other layer having the drug imbedded in a hydrophilic matrix for
extended-release. Three-layered tablets may also be similarly
prepared, with both outer layers containing the drug for immediate
release. Some commercial tablets are prepared with an inner core
containing the extended-release portion of drug and an outer shell
enclosing the core and containing drug for immediate release.
[0187] The invention also provides forming a complex between the
active agent, e.g., one or more compounds and formulations of the
invention and an ion exchange resin, whereupon the complex may be
tableted, encapsulated or suspended in an aqueous vehicle. Release
of the active agent is dependent on the local pH and electrolyte
concentration such that the choice of ion exchange resin may be
made so as to preferentially release the active agent in a given
region of the alimentary canal. Delivery devices incorporating such
a complex are also provided. For example, a modified release dosage
form of trientine can be produced by the incorporation of trientine
into complexes with an anion-exchange resin. Solutions of trientine
may be passed through columns containing an ion-exchange resin to
form a complex by the replacement of H.sub.3O.sup.+ ions. The
resin-trientine complex is then washed and may be tableted,
encapsulated, or suspended in an aqueous vehicle. The release of
the trientine is dependent on the pH and the electrolyte
concentration in the gastrointestinal fluid. Release is greater in
the acidity of the stomach than in the less acidic environment of
the small intestine. Alternative examples of this type of extended
release preparation are provided by hydrocodone polistirex and
chorpheniramine polistirex suspension (Medeva; Tussionex
Pennkinetic Extended Release Suspension, see: Martindale 33rd Ed.,
p. 2145.2) and by phentermine resin capsules (Pharmanex; Ionamin
Capsules see: Martindale 33rd Ed., p. 1916.1). Such resin-trientine
active agent systems can additionally incorporate polymer barrier
coating and bead technologies in addition to the ion-exchange
mechanism. The initial dose comes from an uncoated portion, and the
remainder from the coated beads. The coating does not dissolve, and
release may be extended over a 12-hour period by ion exchange. The
drug containing particles are minute, and may be also suspended to
produce a liquid with extended-release characteristics, as well as
solid dosage forms. Such preparations may also be suitable for
administration, for example in depot preparations suitable for
intramuscular injection.
[0188] The invention also provides a method to produce modified
release preparations of one or more copper chelators, for example,
trientine or salts thereof, by microencapsulation. Such
microencapsulated preparations are useful for the treatment of
humans and other mammals, in which copper chelation therapy is
indicated. Microencapsulation is a process by which solids,
liquids, or even gasses may be encapsulated into microscopic size
particles through the formation of thin coatings of "wall" material
around the substance being encapsulated such as disclosed in U.S.
Pat. Nos. 3,488,418; 3,391,416 and 3,155,590. Gelatin (BP, USP) is
commonly employed as a wall-forming material in microencapsulated
preparations, but synthetic polymers such as polyvinyl alcohol
(USP), ethylcellulose (BP, USP), polyvinyl chloride, and other
materials may also be used (see, for example, Zentner, G. M., Rork,
G. S. & Himmelstein, K. J. Osmotic flow through controlled
porosity films: an approach to delivery of water soluble compounds.
J Controlled Release 2:217-229 (1985); Fites, A. L., Banker, G. S.
& Smolen, V. F. Controlled drug release through polymeric
films. J Pharm Sci 59:610-613 (1970); Samuelov, Y., Donbrow, M.
& Friedman, M. Sustained release of drugs from
ethylcellulose-polyethylene glycol films and kinetics of drug
release. J Pharm Sci 68:325-329 (1979)).
[0189] Encapsulation begins with the dissolving of the prospective
wall material, say gelatin, in water. One or more copper chelators,
for example, trientine or one or more salts thereof, is then added
and the two-phase mixture is thoroughly stirred. With the material
to be encapsulated broken up to the desired particle size, a
solution of a second material is added, that can be acacia (BP,
USP). This additive material is chosen to have the ability to
concentrate the gelatin (polymer) into tiny liquid droplets. These
droplets (the coacervate) then form a film or coat around the
particles of the solid trientine as a consequence of the extremely
low interfacial tension of the residual water or solvent in the
wall material so that a continuous, tight, film-coating remains on
the particle (see Ansel, H. C., Allen, L. V. and Popovich, N. G.
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.,
Lippincott 1999, p. 233). The final dry microcapsules are free
flowing, discrete particles of coated material. Of the total
particle weight, the wall material usually represents between 2 and
20% (w/w). The coated particles are then admixed with tableting
excipients and formed into dosage-sized tablets. Different rates of
trientine release may be obtained by changing the core-to-wall
ratio, the polymer used for the coating, or the method of
microencapsulation (for example, see: Yazici, E., Oner, L., Kas, H.
S. & Hincal, A. A. Phenytoin sodium microspheres: bench scale
formulation, process characterization and release kinetics.
Pharmaceut Dev Technol 1996;1:175-183).
[0190] One of the advantages of microencapsulation is that the
administered dose of one or more copper chelators, for example,
trientine or salts thereof, is subdivided into small units that are
spread over a large area of the gastrointestinal tract, which may
enhance absorption by diminishing localized drug concentrations
(see Yazici et al., supra). An example of a drug that is
commercially available in a microencapsulated extended-release
dosage form is potassium chloride (Micro-K Exten-caps,
Wyeth-Ayerst, Martindale 33rd Ed., p 1968.1). Other useful
approaches include those in which the drug is incorporated into
polymeric colloidal particles or microencapsulates (microparticles,
microspheres or nanoparticles) in the form or reservoir and matrix
devices (see: Douglas, S. J., et al., "Nanoparticles in drug
delivery," C. R. C. Crit Rev Therap Drug Carrier Syst 3:233-261
(1987); Oppenheim, R. C., "Solid colloidal drug delivery systems:
nanoparticles." Int J Pharm 8:217-234 (1981); Higuchi, T.
"Mechanism of sustained action medication: theoretical analysis of
rate of release of solid drugs dispersed in solid matrices." J
Pharm Sci 52:1145-1149 (1963)).
[0191] The invention also includes repeat action tablets containing
one or more copper chelators, for example, trientine or salts
thereof. Further examples of a method by which modified release
forms of one or more copper chelators, for example, trientine or
salts thereof, suitable for treatment of humans or other mammals,
can be produced are provided by the incorporation of trientine into
repeat action tablets. These are prepared so that an initial dose
of the drug is released immediately followed later by a second
dose. The tablets may be prepared with the immediate-release dose
in the tablet's outer shell or coating with the second dose in the
tablet's inner core, separated by a slowly permeable barrier
coating. In general, the drug from the inner core is exposed to
body fluids and released 4 to 6 hours after administration. An
example of this type of product is proved by Repetabs (Schering
Inc.). Repeat action dosage forms are suitable for the
administration of one or more copper chelators, for example,
trientine or salts thereof, for the indications noted herein,
including but not limited to chronic conditions such as heart
failure, diabetic heart disease, acute coronary syndrome,
hypertensive heart disease, ischemic heart disease, coronary artery
disease, peripheral arterial disease, or any form of cancer. This
form of delivery is particularly suitable for delivery of
trientine, since it has a rapid rate of absorption and
excretion.
[0192] The invention also includes delayed-release oral dosage
forms containing one or more copper chelators, for example,
trientine or salts thereof. The release of one or more copper
chelators, for example, trientine or salts thereof from an oral
dosage form can be intentionally delayed until it reaches the
intestine by way of, for example, enteric coating. Enteric coatings
by themselves are not an efficient method for the delivery of
copper chelators such as, for example, trientine or salts thereof
including trientine dihydrochloride, because of the inability of
such coating systems to provide or achieve a sustained therapeutic
effect after release onset. Enteric coats are designed to dissolve
or breakdown in an alkaline environment. The presence of food may
increase the pH of the stomach. Therefore, the concurrent
administration of enteric-coated trientine dihydrochloride with
food or the presence of food in the stomach may lead to dose
dumping and unwanted secondary effects. Furthermore, given the fact
that, for example, trientine dihydrochloride can give rise to
gastrointestinal side-effects, it would be desirable to have a drug
delivery system that is capable of providing the controlled
delivery of trientine dihydrochloride or other pharmaceutically
acceptable salts of trientine in a predictable manner over a long
period of time.
[0193] Enteric coatings also have application in the present
invention when combined or incorporated with one or more of the
other dose delivery formulations or devices described herein. This
form of delivery conveys the advantage of minimizing the gastric
irritation that may be caused in some subjects by trientine. The
enteric coating may be time-dependent, pH-dependent where it breaks
down in the less acidic environment of the intestine and erodes by
moisture over time during gastrointestinal transit, or
enzyme-dependent where it deteriorates due to the
hydrolysis-catalyzing action of intestinal enzymes (see, for
example, Muhammad, N. A., et al. "Modifying the release properties
of Eudragit L30D," Drug Dev Ind Pharm. 17:2497-2509 (1991)). Among
the many agents used to enteric coat tablets and capsules known to
those skilled in the art are fats including triglycerides, fatty
acids, waxes, shellac, and cellulose acetate phthalate although
further examples of enteric coated preparations can be found in the
USP.
[0194] The invention also provides drug delivery devices
incorporating one or more copper chelators, for example, trientine
or salts thereof, in a membrane-control system. Such devices
comprise a rate-controlling membrane surrounding a drug reservoir.
Following oral administration the membrane gradually becomes
permeable to aqueous fluids, but does not erode or swell. The drug
reservoir may be composed of a conventional tablet, or a
microparticle pellet containing multiple units that do not swell
following contact with aqueous fluids. The cores dissolve without
modifying their internal osmotic pressure, thereby avoiding the
risk of membrane rupture, and typically comprise 60:40 mixtures of
lactulose: microcrystalline cellulose (w/w). Drug is released
through a two-phase process, comprising diffusion of aqueous fluids
into the matrix, followed by diffusion of the drug out of the
matrix. Multiple-unit membrane-controlled systems typically
comprise more than one discrete unit. They can contain discrete
spherical beads individually coated with rate-controlling membrane
and may be encapsulated in a hard gelatin shell (examples of such
preparations include Contac 400; martindale 33rd Ed., 1790.1 and
Feospan; Martindale 33rd Ed., p. 1859.4). Alternatively,
multiple-unit membrane-controlled systems may be compressed into a
tablet (for example, Suscard; Martindale 33rd Ed., p. 2115.1).
Alternative implementations of this technology include devices in
which the drug substance is coated around inert sugar spheres, and
devices prepared by extrusion spheronization employing a
conventional matrix system. Advantages of such systems include the
more consistent gastrointestinal transit rate achieved by
multiple-unit systems, and the fact that such systems infrequently
suffer from catastrophic dose dumping. They are also ideal for the
delivery of more than one drug at a time.
[0195] Preferred for oral delivery is a sustained release form of
one or more compounds and formulations of the invention which is a
matrix formation, such a matrix formation taking the form of film
coated spheroids containing as active ingredient one or more copper
chelators, for example, trientine or salts thereof such as
trientine dihydrochloride, and a non water soluble spheronising
agent. The term "spheroid" is known in the pharmaceutical art and
means spherical granules having a diameter usually of between 0.01
mm and 4 mm. The spheronising agent may be any pharmaceutically
acceptable material that, together with the active ingredient, can
be spheronised to form spheroids. Microcrystalline cellulose is
preferred. Suitable microcrystalline cellulose includes, for
example, the material sold as Avicel PH 101 (Trade Mark, FMC
Corporation). According to a preferred aspect of the present
invention, the film-coated spheroids contain between 70% and 99%
(by wt), especially between 80% and 95% (by wt), of the
spheronising agent, especially microcrystalline cellulose. In
addition to the active ingredient and spheronising agent, the
spheroids may also contain a binder. Suitable binders, such as low
viscosity, water soluable polymers, will be well known to those
skilled in the pharmaceutical art. A suitable binder is, in
particular polyvinylpyrrolidone in various degrees of
polymerization. However, water-soluble hydroxy lower alkyl
celluloses, such as hydroxy propyl cellulose, are preferred.
Additionally (or alternatively) the spheroids may contain a water
insoluble polymer, especially an acrylic polymer, an acrylic
copolymer, such as a methacrylic acid-ethyl acrylate copolymer, or
ethyl cellulose. Other thickening agents or binders include:the
lipid type, among which are vegetable oils (cotton seed, sesame and
groundnut oils) and derivatives of these oils (hydrogenated oils
such as hydrogenated castor oil, glycerol behenate,the waxy type
such as natural carnauba wax or natural beeswax, synthetic waxes
such as cetyl ester waxes, the amphiphilic type such as polymers of
ethylene oxide (polyoxyethylene glycol of high molecular weight
between 4000 and 100000) or propylene and ethylene oxide copolymers
(poloxamers), the cellulosic type (semisynthetic derivatives of
cellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose,
hydroxymethylcellulose, of high molecular weight and high
viscosity, gum) or any other polysaccharide such as alginic acid,
the polymeric type such as acrylic acid polymers (such as
carbomers), and the mineral type such as colloidal silica,
bentonite.
[0196] Suitable diluents for the active ingredient in the pellets,
spheroids or core are, e.g., microcrystalline cellulose, lactose,
dicalcium phosphate, calcium carbonate, calcium sulphate, sucrose,
dextrates, dextrin, dextrose, dicalcium phosphate dihydrate,
kaolin, magnesium carbonate, magnesium oxide, maltodextrin,
cellulose, microcrystalline cellulose, sorbitol, starches,
pregelatinized starch, talc, tricalcium phosphate and lactose.
Suitable lubricants are e.g., magnesium stearate and sodium stearyl
fumarate. Suitable binding agents are e.g., hydroxypropyl methyl
cellulose, polyvidone and methyl cellulose.
[0197] Suitable binders that may be included are: gum arabic, gum
tragacanth, guar gum, alginic acid, sodium alginate, sodium
carboxymethylcellulose, dextrin, gelatin, hydroxyethylcellulose,
hydroxypropylcellulose, liquid glucose, magnesium and aluminium.
Suitable disintegrating agents are starch, sodium starch glycolate,
crospovidone and croscarmalose sodium. Suitable surface active are
Poloxamer 188.RTM., polysorbate 80 and sodium lauryl sulfate.
Suitable flow aids are talc colloidal anhydrous silica. Suitable
lubricants that may be used are glidants (such as anhydrous
silicate, magnesium trisilicate, magnesium silicate, cellulose,
starch, talc or tricalcium phosphate) or alternatively antifriction
agents (such as calcium stearate, hydrogenated vegetable oils,
paraffin, magnesium stearate, polyethylene glycol, sodium benzoate,
sodium lauryl sulphate, fumaric acid, stearic acid or zinc stearate
and talc). Suitable water-soluble polymers are PEG with molecular
weights in the range 1000 to 6000.
[0198] Delayed release through the use of a tablet, pellet,
spheroid or core itself, which besides having a filler and binder,
other ancillary substances, in particular lubricants and nonstick
agents, and disintegrants. Examples of lubricants and nonstick
agents, which may be mentioned, are higher fatty acids and their
alkali metal and alkaline-earth-metal salts, such as calcium
stearate. Suitable disintegrants are, in particular, chemically
inert agents. Disintegrants that may be mentioned as preferred are
cross-linked polyvinylpyrrolidone, cross-linked sodium
carboxymethylcelluloses, and sodium starch glycolate.
[0199] The dosage unit if oral preferably delivers more than about
than 50% of a copper chelator, for example, trientine
dihydrochloride, in 12 hrs at a pH of about <6.5 in a controlled
manner during in vivo and in vitro dissolution. Other formulations
and dose forms are set forth below.
[0200] Yet further embodiments of the invention include forms of
one or more copper chelators, for example, trientine or salts
thereof, incorporated into transdermal drug delivery systems, such
as those described in: Transdermal Drug Delivery Systems, Chapter
10. In: Ansel, H. C., Allen, L. V. and Popovich, N. G.
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.,
Lippincott 1999, pp. 263-278). Transdermal drug delivery systems
facilitate the passage of therapeutic quantities of drug substances
through the skin and into the systemic circulation to exert
systemic effects, as originally described (see Stoughton, R. D.
Percutaneous absorption. Toxicol Appl Pharmacol 7:1-8 (1965)).
Evidence of percutaneous drug absorption may be found through
measurable blood levels of the drug, detectable excretion of the
drug and/or its metabolites in the urine, and through the clinical
response of the subject to its administration. For transdermal drug
delivery, it is considered ideal if the drug penetrates through the
skin to the underlying blood supply without drug build up in the
dermal layers (Black, C. D., "Transdermal drug delivery systems,"
U.S. Pharm 1:49 (1982)). Formulations of drugs suitable for
trans-dermal delivery are known to those skilled in the art, and
are described in references such as Ansel et al. (supra). Methods
known to enhance the delivery of drugs by the percutaneous route
include chemical skin penetration enhancers, which increase skin
permeability by reversibly damaging or otherwise altering the
physicochemical nature of the stratum corneum to decrease its
resistance to drug diffusion (see Shah, V. P., Peck, C. C. &
Williams, R. L. Skin penetration enhancement: clinical
pharmacological and regulatory considerations. In: Walters, K. A.
& Hadgraft, J. (Eds.) Pharmaceutical skin penetration
enhancement. New York: Dekker, 1993). Among effective alterations
are increased hydration of the stratum corneum and/or a change in
the structure of the lipids and lipoproteins in the intercellular
channels brought about through solvent action or denaturation (see
Walters K. A., "Percutaneous absorption and transdermal therapy,"
Pharm Tech 10:30-42 (1986)). Skin penetration enhances suitable for
formulation with trientine in Transdermal Drug Delivery Systems may
be chosen from the following list: acetone, laurocapram,
dimethylacetamide, dimethylformamide, dimethylsulphoxide, ethanol,
oleic acid, polyethylene glycol, propylene glycol and sodium lauryl
sulphate. Further skin penetration enhancers may be found in
publications known to those skilled in the art (see, for example,
Osborne, D. W., & Henke, J. J., "Skin penetration enhancers
cited in the technical literature," Pharm Tech 21:50-66 (1997);
Rolf, D., "Chemical and physical methods of enhancing transdermal
drug delivery," Pharm Tech 12:130-139 (1988)).
[0201] In addition to chemical means, there are physical methods
that enhance transdermal drug delivery and penetration of the
compounds and formulations of the invention. These include
iontophoresis and sonophoresis. Iontophoresis involves the delivery
of charged chemical compounds across the skin membrane using an
applied electrical field. Such methods have proven suitable for
delivery of a number of drugs. Accordingly, another embodiment of
the invention comprises one or more copper chelators, for example,
trientine or salts thereof, formulated in such a manner suitable
for administration by iontophoresisor sonophoresis. Formulations of
one or more copper chelators, for example, trientine, suitable for
administration by iontophoresis or sonophoresis may be in the form
of gels, creams, or lotions. Transdermal delivery may utilize,
among others, monolithic delivery systems, drug-impregnated
adhesive delivery systems (e.g., the Latitude.TM. drug-in-adhesive
system from 3M), active transport devices and membrane-controlled
systems. Monolithic systems incorporate an active agent matrix,
comprising a polymeric material in which the active agent is
dispersed between backing and frontal layers. Drug impregnated
adhesive delivery systems comprise an adhesive polymer in which one
or more compounds and formulations of the invention and any
excipients are incorporated into the adhesive polymer. Active
transport devices incorporate an active agent reservoir, often in
liquid or gel form, a membrane that may be rate controlling, and a
driving force to propel the active agent across the membrane.
Membrane-controlled transdermal systems commonly comprise an active
agent reservoir, often in liquid or gel form, a membrane that may
be rate controlling and backing, adhesive and/or protecting layers.
Transdermal delivery dosage forms include those which substitute
the trientine active ingredient, preferably trientine
dihydrochloride for the diclofenic or other pharmaceutically
acceptable salt thereof referred to in the transdermal delivery
systems disclosed in, by way of example, U.S. Pat. Nos. 6,193,996,
6,262,121.
[0202] Topical administration of one or more compounds and
formulations of the invention ingredient can be prepared as an
admixture or other pharmaceutical formulation to be applied in a
wide variety of ways including, but are not limited to, lotions,
creams gels, sticks, sprays, ointments and pastes. These product
types may comprise several types of formulations including, but not
limited to solutions, emulsions, gels, solids, and liposomes. If
the topical composition is formulated as an aerosol and applied to
the skin as a spray-on, a propellant may be added to a solution
composition. Suitable propellants as used in the art can be
utilized. By way of example of topical administration of an active
agent, reference is made to U.S. Pat. Nos. 5,602,125, 6,426,362 and
6,420,411.
[0203] Also included in the sustained dosage forms in accordance
with the present invention are any variants of the oral forms that
are adapted for suppository or other parenteral use. When rectally
administered in the form of suppositories, for example, these
compositions may be prepared by mixing one or more compounds and
formulations of the invention with a suitable non-irritating
excipient, such as cocoa butter, synthetic glyceride esters or
polyethylene glycols, which are solid at ordinary temperatures, but
liquidity and/or dissolve in the rectal cavity to release the drug.
Suppositories are generally solid dosage forms intended for
insertion into body orifices including rectal, vaginal and
occasionally urethrally and can be long acting or slow release.
Suppositories include a base that can include, but is not limited
to, materials such as alginic acid, which will prolong the release
of the pharmaceutically acceptable active ingredient over several
hours (5-7). Such bases can be characterized into two main
categories and a third miscellaneous group: 1) fatty or oleaginous
bases, 2) water-soluble or water-miscible bases and 3)
miscellaneous bases, generally combinations of lipophilic and
hydrophilic substances. Fatty or oleaginous bases include
hydrogenated fatty acids of vegetable oils such as palm kernel oil
and cottonseed oil, fat-based compound containing compounds of
glycerin with the higher molecular weight fatty acids such as
palmitic and stearic acids, cocoa butter is also used where phenol
and chloral hydrate lower the melting point of cocoa butter when
incorporated, solidifying agents like cetyl esters wax (about 20%)
or beeswax (about 4%) may be added to maintain a solid suppository.
Other bases include other commercial products such as Fattibase
(triglycerides from palm, palm kernel and coconut oils with
self-emulsifying glycerol monostearate and poloxyl stearate),
Wecobee and Witepsol bases. Water-soluble bases are generally
glycerinated gelatin and Water-miscible bases are generally
polyethylene glycols. The miscellaneous bases include mixtures of
the oleaginous and water-soluble or water-miscible materials. An
example of such a base in this group is polyoxyl 40 stearate and
polyoxyethylene diols and the free glycols.
[0204] Transmucosal delivery of the compounds and formulations of
the invention may utilize any mucosal membrane but commonly
utilizes the nasal, buccal, vaginal and rectal tissues.
[0205] Formulations suitable for nasal administration of the
compounds and formulations of the invention may be administered in
a liquid form, for example, nasal spray, nasal drops, or by aerosol
administration by nebulizer, including aqueous or oily solutions of
the active ingredient. Formulations for nasal administration,
wherein the carrier is a solid, include a coarse powder having a
particle size, for example, of less than about 100 microns,
preferably less than about 50 microns, which is administered in the
manner in which snuff is taken, i.e., by rapid inhalation through
the nasal passage from a container of the powder held close up to
the nose. Compositions in solution may be neubulised by the use of
inner gases and such nebulised solutions may be breathed directly
from the neulising device or the nebulising device may be attached
to a facemask, tent or intermittent positive pressure-breathing
machine. Solutions, suspensions or powder compositions may be
administered orally or nasally from devices that deliver the
formulation in an appropriate manner. Formulations may be prepared
as aqueous solutions for example in saline, solutions employing
benzyl alcohol or other suitable preservatives, absorption
promoters to enhance bio-availability, fluorocarbons, and/or other
solubilising or dispersing agents known in the art.
[0206] The invention provides extended-release formulations
containing one or more copper chelators, for example, trientine or
salts thereof suitable for parenteral administration. Extended
rates of drug action following injection may be achieved in a
number of ways, including the following: crystal or amorphous drug
forms having prolonged dissolution characteristics; slowly
dissolving chemical complexes of the drug entity; solutions or
suspensions of drug in slowly absorbed carriers or vehicles (as
oleaginous); increased particle size of drug in suspension; or, by
injection of slowly eroding microspheres of drug (for example, see:
Friess, W., Lee, G. and Groves, M. J. Insoluble collagen matrices
for prolonged delivery of proteins. Pharmaceut Dev Technol
1:185-193 (1996)). The duration of action of the various forms of
insulin for example is based in part on its physical form
(amorphous or crystalline), complex formation with added agents,
and its dosage form (solution of suspension).
[0207] The copper chelator must be formulated into a stable, safe
pharmaceutical composition for administration to a patient. The
copper chelator is a trientine active agent. The composition can be
prepared according to conventional methods by dissolving or
suspending an amount of the trientine active agent ingredient in a
diluent. The amount is from between 0.1 mg to 1000 mg per ml of
diluent of the trientine active agent. An acetate, phosphate,
citrate or glutamate buffer may be added allowing a pH of the final
composition to be from 5.0 to 9.5; optionally a carbohydrate or
polyhydric alcohol tonicifier and, a preservative selected from the
group consisting of m-cresol, benzyl alcohol, methyl, ethyl, propyl
and butyl parabens and phenol may also be added. A sufficient
amount of water for injection is used to obtain the desired
concentration of solution. Additional tonicifying agents such as
sodium chloride, as well as other excipients, may also be present,
if desired. Such excipients, however, must maintain the overall
tonicity of the trientine active agent.
[0208] The terms buffer, buffer solution and buffered solution,
when used with reference to hydrogen-ion concentration or pH, refer
to the ability of a system, particularly an aqueous solution, to
resist a change of pH on adding acid or alkali, or on dilution with
a solvent Characteristic of buffered solutions, which undergo small
changes of pH on addition of acid or base, is the presence either
of a weak acid and a salt of the weak acid, or a weak base and a
salt of the weak base. An example of the former system is acetic
acid and sodium acetate. The change of pH is slight as long as the
amount of hydroxyl ion added does not exceed the capacity of the
buffer system to neutralize it.
[0209] The stability of the parenteral formulation of the present
invention is enhanced by maintaining the pH of the formulation in
the range of approximately 5.0 to 9.5. Other pH ranges, for
example, include, 5.5 to 9.0, or 6.0 to 8.5, or 6.5 to 8.0, or 7.0
to 7.5.
[0210] The buffer used in the practice of the present invention is
selected from any of the following, for example, an acetate buffer,
a phosphate buffer or glutamate buffer, the most preferred buffer
being a phosphate buffer.
[0211] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, polyethylene glycols and physiologically
compatible solvents.
[0212] A stabilizer may be included in the present formulation but,
and importantly, is not needed. If included, however, a stabilizer
useful in the practice of the present invention is a carbohydrate
or a polyhydric alcohol. The polyhydric alcohols include such
compounds as sorbitol, mannitol, glycerol, and polyethylene glycols
(PEGs). The carbohydrates include, for example, mannose, ribose,
trehalose, maltose, inositol, lactose, galactose, arabinose, or
lactose.
[0213] Suitable stabilizers include, for example, polyhydric
alcohols such as sorbitol, mannitol, inositol, glycerol, xylitol,
and polypropylene/ethylene glycol copolymer, as well as various
polyethylene glycols (PEG) of molecular weight 200, 400, 1450,
3350, 4000, 6000, and 8000).
[0214] The United States Pharmacopeia (USP) states that
anti-microbial agents in bacteriostatic or fungistatic
concentrations must be added to preparations contained in multiple
dose containers. They must be present in adequate concentration at
the time of use to prevent the multiplication of microorganisms
inadvertently introduced into the preparation while withdrawing a
portion of the contents with a hypodermic needle and syringe, or
using other invasive means for delivery, such as pen injectors.
Antimicrobial agents should be evaluated to ensure compatibility
with all other components of the formula, and their activity should
be evaluated in the total formula to ensure that a particular agent
that is effective in one formulation is not ineffective in another.
It is not uncommon to find that a particular agent will be
effective in one formulation but not effective in another
formulation.
[0215] A preservative is, in the common pharmaceutical sense, a
substance that prevents or inhibits microbial growth and may be
added to a pharmaceutical formulation for this purpose to avoid
consequent spoilage of the formulation by microorganisms. While the
amount of the preservative is not great, it may nevertheless effect
the overall stability of the trientine active agent. Thus, even
selection of a preservative can be difficult.
[0216] While the preservative for use in the practice of the
present invention can range from 0.005 to 1.0% (w/v), the preferred
range for each preservative, alone or in combination with others,
is: benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol
(0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl or
propyl or butyl (0.005%-0.03%) parabens. The parabens are lower
alkyl esters of para-hydroxybenzoic acid.
[0217] detailed description of each preservative is set forth in
"Remington's Pharmaceutical Sciences" as well as Pharmaceutical
Dosage Forms: Parenteral Medications, Vol. 1, 1992, Avis et al. For
these purposes, the crystalline trientine dihydrochloride salt may
be administered parenterally (including subcutaneous injections,
intravenous, intramuscular, intradermal injection or infusion
techniques) or by inhalation spray in dosage unit formulations
containing conventional non-toxic pharmaceutically-acceptable
carriers, adjuvants and vehicles.
[0218] It may also be desirable to add sodium chloride or other
salt to adjust the tonicity of the pharmaceutical formulation,
depending on the tonicifier selected. However, this is optional and
depends on the particular formulation selected. Parenteral
formulations must be isotonic or substantially isotonic otherwise
significant irritation and pain would occur at the site of
administration.
[0219] The desired isotonicity may be accomplished using sodium
chloride or other pharmaceutically acceptable agents such as
dextrose, boric acid, sodium tartrate, propylene glycol, polyols
(such as mannitol and sorbitol), or other inorganic or organic
solutes. Generally, the composition is isotonic with the blood of
the subject.
[0220] If desired, the parenteral formulation may be thickened with
a thickening agent such as methyl cellulose. The formulation may be
prepared in an emulsified form, either water in oil or oil in
water. Any of a wide variety of pharmaceutically acceptable
emulsifying agents may be employed including, for example, acacia
powder, a non-ionic surfactant or an ionic surfactant.
[0221] It may also be desirable to add suitable dispersing or
suspending agents to the pharmaceutical formulation these may
include, for example, aqueous suspensions such as synthetic and
natural gums i.e. tragacanth, acacia, alginate, dextran, sodium
carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or
gelatin.
[0222] The vehicle of greatest importance for parenteral products
is water. Water of suitable quality for parenteral administration
must be prepared either by distillation or by reverse osmosis. Only
by these means is it possible to separate adequately various
liquid, gas and solid contaminating substances from water. Water
for injection is the preferred aqueous vehicle for use in the
pharmaceutical formulation of the present invention. The water may
be purged with nitrogen gas to remove any oxygen or free radicals
of oxygen from the water.
[0223] It is possible that other ingredients may be present in the
parenteral pharmaceutical formulation of the present invention.
Such additional ingredients may include wetting agents, oils (e.g.,
a vegetable oil such as sesame, peanut or olive), analgesic agents,
emulsifiers, antioxidants, bulking agents, tonicity modifiers,
metal ions, oleaginous vehicles, proteins (e.g., human serum
albumin, gelatin or proteins) and a zwitterion (e.g., an amino acid
such as betaine, taurine, arginine, glycine, lysine and histidine).
Such additional ingredients, of course, should not adversely affect
the overall stability of the pharmaceutical formulation of the
present invention.
[0224] Containers are also an integral part of the formulation of
an injection and may be considered a component, for there is no
container that is totally insoluble or does not in some way affect
the liquid it contains, particularly if the liquid is aqueous.
Therefore, the selection of a container for a particular injection
must be based on a consideration of the composition of the
container, as well as of the solution, and the treatment to which
it will be subjected.
[0225] In order to permit introduction of a needle from a
hypodermic syringe into a multiple-dose vial and provide for
resealing as soon as the needle is withdrawn, each vial is sealed
with a rubber closure held in place by an aluminum band.
[0226] Stoppers for glass vials, such as, West 4416/50, 4416/50
(Teflon faced) and 4406/40, Abbott 5139 or any equivalent stopper
can be used as the closure for the dose vial. These stoppers pass
the stopper integrity test when tested using patient use patterns,
e.g., the stopper can withstand at least about 100 injections.
[0227] Each of the components of the pharmaceutical formulation
described above is known in the art and is described in
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2nd
ed., Avis et al. Ed., Mercel Dekker, New York, N.Y. 1992, which is
incorporated by reference in its entirety herein.
[0228] The manufacturing process for the above formulation involves
compounding, sterile filtration and filling steps. The compounding
procedure, may for example, involve the dissolution of ingredients
in a specific order, such as the preservative first followed by the
stabilizer/tonicity agents, buffers and then the trientine active
agent or dissolving all of the ingredients forming the parenteral
formulation at the same time. An example of one method of preparing
a parenteral formulation for administration is the dissolution of
the trientine active form, for example, trientine hydrochloride, in
water and diluting the resultant mixture to 154 mM in a phosphate
buffered saline.
[0229] Alternatively, parenteral formulations of the present
invention are prepared by mixing the ingredients following
generally accepted procedures. For example, the selected components
may be mixed in a blender or other standard device to produce a
concentrated mixture which may then be adjusted to the final
concentration and viscosity by the addition of water, a thickening
agent, a buffer, 5% human serum albumin or an additional solute to
control tonicity.
[0230] Alternatively, the trientine active agent can be packaged as
a dry solid and/or powder to be reconstituted with a solvent to
yield a parenteral formulation in accordance with the present
invention for use at the time of reconstitution.
[0231] In addition the manufacturing process may include any
suitable sterilization process when developing the parenteral
formulation of the present invention. Typical sterilization
processes include filtration, steam (moist heat), dry heat, gases
(e.g., ethylene oxide, formaldehyde, chlorine dioxide, propylene
oxide, beta-propiolacctone, ozone, chloropicrin, peracetic acid
methyl bromide and the like), radiant exposure and aseptic
handling.
[0232] Suitable routes of parenteraladministration include
intramuscular, intravenous, subcutaneous, intradermal,
intraarticular, intrathecal and the like. The subcutaneous route of
administration is preferred. Mucosal delivery is also permissible.
The dose and dosage regimen will depend upon the weight and health
of the subject.
[0233] Routes for parenteral administration therefore include
intravenous, intramuscular, intraperitoneal, sub dermal, and
subcutaneous administration.
[0234] In addition to the above means of achieving extended drug
action, the rate and duration of drug delivery may be controlled
by, for example by using mechanically controlled drug infusion
pumps.
[0235] The pharmaceutically acceptable active agent, for example,
one or more copper chelators, such as, for example, trientine or
salts thereof such as trientine dihydrochloride, can be
administered in the form of a depot injection that may be
formulated in such a manner as to permit a sustained release of the
active ingredient. The active ingredient can be compressed into
pellets or small cylinders and implanted subcutaneously or
intramuscularly. The pellets, or cylinders may additionally be
coated with a suitable biodegradable polymer chosen so as to
provide a desired release profile. The active ingredient may
alternatively be micropelleted. Active agent micropellets using
bioacceptable polymers can be designed to allow release rates to be
manipulated to provide a desired release profile. Alternatively,
injectable depot forms can be made by forming microencapsulated
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations can also be
prepared by entrapping the drug in liposomes, examples of which
include unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearyl aamine or
phosphatidylcholines. Depot injectable formulations can also be
prepared by entrapping the drug in microemulsions which are
compatible with body tissue. By way of example reference is made to
U.S. patent application Ser. Nos. 6,410,041 and 6,362,190.
[0236] The invention in part provides infusion dose delivery
formulations and devices, including but not limited to implantable
infusion devices. Implantable infusion devices may employ inert
material such as biodegradable polymers listed above or synthetic
silicones for example cylastic, silicone rubber or other polymers
manufactured by the Dow-Corning Corporation. The polymer may be
loaded with active agent and any excipients. Implantable infusion
devices may also comprise a coating of, or a portion of, a medical
device wherein the coating comprises the polymer loaded with active
agent and any excipient. Such an implantable infusion device may be
prepared as disclosed in U.S. Pat. No. 6,309,380 by coating the
device with an in vivo biocompatible and biodegradable or
bioabsorbable or bioerodable liquid or gel solution containing a
polymer with the solution comprising a desired dosage amount of
active ingredient and any excipients. The solution is converted to
a film adhering to the medical device thereby forming the
implantable drug-deliverable medical device.
[0237] An implantable infusion device may also be prepared by the
in situ formation of an active agent containing solid matrix as
disclosed in U.S. Pat. No. 6,120,789, herein incorporated in its
entirety. Implantable infusion devices may be passive or active. An
active implantable infusion device may comprise an active agent
reservoir, a means of allowing the active agent to exit the
reservoir, for example a permeable membrane, and a driving force to
propel the active agent from the reservoir. Such an active
implantable infusion device may additionally be activated by an
extrinsic signal, such as that disclosed in WO 02/45779, wherein
the implantable infusion device comprises a system configured to
deliver the active agent comprising an external activation unit
operable by a user to request activation of the implantable
infusion device, including a controller to reject such a request
prior to the expiration of a lockout interval. Examples of an
active implantable infusion device include implantable drug pumps.
Implantable drug pumps include, for example, miniature,
computerized, programmable, refillable drug delivery systems with
an attached catheter that inserts into a target organ system,
usually the spinal cord or a vessel. See Medtronic Inc.
Publications: UC9603124EN NP-2687, 1997; UC199503941b EN NP-2347
182577-101,2000; UC199801017a EN NP3273a 182600-101, 2000;
UC200002512 EN NP4050, 2000; UC199900546bEN NP-3678EN, 2000.
Minneapolis, Minn.: Medtronic Inc; 1997-2000. many pumps have 2
ports: one into which drugs can be injected and the other that is
connected directly to the catheter for bolus administration or
analysis of fluid from the catheter. Implantable drug infusion
pumps (SynchroMed EL and Synchromed programmable pumps; Medtronic)
are indicated for long-term intrathecal infusion of morphine
sulfate for the treatment of chronic intractable pain;
intravascular infusion of floxuridine for treatment of primary or
metastatic cancer; intrathecal injection (baclofen injection) for
severe spasticity; long-term epidural infusion of morphine sulfate
for treatment of chronic intractable pain; long-term intravascular
infusion of doxorubicin, cisplatin, or methotrexate for the
treatment or metastatic cancer; and long-term intravenous infusion
of clindamycin for the treatment of osteomyelitis. Such pumps may
also be used for the long-term infusion of one or more copper
chelators, for example, for example, trientine or salts thereof, at
a desired amount for a desired number of doses or steady state
administration. One form of a typical implantable drug infusion
pump (Synchromed EL programmable pump; Medtronic) is titanium
covered and roughly disk shaped, measures 85.2 mm in diameter and
22.86 mm in thickness, weighs 185 g, has a drug reservoir of 10 mL,
and runs on a lithium thionyl-chloride battery with a 6- to 7-year
life, depending on use. The downloadable memory contains programmed
drug delivery parameters and calculated amount of drug remaining,
which can be compared with actual amount of drug remaining to
access accuracy of pump function, but actual pump function over
time is not recorded. The pump is usually implanted in the right or
left abdominal wall. Other pumps useful in the invention include,
for example, portable disposable infuser pumps (PDIPs).
Alternatively, implantable infusion devices may employ liposome
delivery systems such as a small unilamellar vesicles, large
unilamellar vesicles, and multilamellar vesicles can be formed from
a variety of phospholipids, such as cholesterol, stearyl amine or
phosphatidylcholines.
[0238] The invention also includes delayed-release ocular
preparations containing one or more copper chelators, for example,
trientine or salts thereof. Disease of the retinal arteries,
leading to leading to leakage of plasma and ultimately to diabetic
retinopathy, is a leading cause of impaired vision and blindness
consequent upon diabetes. Trientine therapy is effective in
treating diabetic arterial disease. This aspect of the invention
provides ocular preparations of trientine suitable for
administration to humans for the treatment of the disease of the
retinal arteries in diabetes. Such administration is expected to
yield high, localized concentrations of drug, suitable for
treatment of diabetic arterial disease in the retina, and diabetic
retinopathy.
[0239] One of the problems associated with the use of ophthalmic
solutions is the rapid loss of administered drug due to blinking of
the eye and the flushing effect of lacrimal fluids. Up to 80% of an
administered dose may be lost through tears and the action of
nasolacrimal drainage within 5 minutes of installation. Extended
periods of therapy may be achieved by formulations that increase
the contact time between the medication and the corneal surface.
This may be accomplished through use of agents that increase the
viscosity of solutions; by ophthalmic suspensions in which the drug
particles slowly dissolve; by slowly dissipating ophthalmic
ointments; or by use of ophthalmic inserts. Preparations of one or
more copper chelators, for example, trientine or its salts suitable
for ocular administration to humans may be formulated using
synthetic high molecular weight cross-linked polymers such as those
of acrylic acid (e.g., Carbopol 940) or gellan gum (Gelrite; see,
Merck Index 12th Ed., 4389), a compound that forms a gel upon
contact with the precorneal tear film (e.g. as employed in
Timoptic-XE by Merck, Inc.).
[0240] Further embodiments include delayed-release ocular
preparations containing trientine in ophthalmic inserts, such as
the OCUSERT system (Alza Inc.). Typically, such inserts are
elliptical with dimensions of about 13.4 mm by 5.4 mm by 0.3 mm
(thickness). The insert is flexible and has a drug-containing core
surrounded on each side by a layer of hydrophobic ethylene/vinyl
acetate copolymer membranes through which the drug diffuses at a
constant rate. The white margin around such devices contains white
titanium dioxide, an inert compound that confers visibility. The
rate of drug diffusion is controlled by the polymer composition,
the membrane thickness, and the drug solubility. During the first
few hours after insertion, the drug release rate is greater than
that which occurs thereafter in order to achieve initially
therapeutic drug levels. The drug-containing inserts may be placed
in the conjunctival sac from which they release their medication
over a typical 7-d period in the treatment of diabetic retinal
disease. Another form of an ophthalmic insert is a rod shaped,
water soluble structure composed of hydroxypropyl cellulose in
which trientine is embedded. The insert is placed into the inferior
cul-de-sac of the eye once or twice daily in the treatment of
diabetic retinal disease. The inserts soften and slowly dissolve,
releasing the drug that is then taken up by the ocular fluids. A
further example of such a device is furnished by Lacrisert (Merck
Inc.).
[0241] Also targeted release delivery systems where the active
agent is isolated or concentrated in a body region, tissue or site
for absorption or action.
[0242] The invention also provides in part dose delivery
formulations and devices formulated to enhance bioavailability of
trientine active agent. This may be in addition to or in
combination with any of the dose delivery formulations or devices
described above.
[0243] Despite good hydrosolubility, trientine is poorly absorbed
in the digestive tract and consequently its bioavailability is
incomplete, and may be irregular or vary from one person to
another. A therapeutically effective amount of trientine active
agent is an amount capable of providing an appropriate level of
trientine active agent in the bloodstream. By increasing the
bioavailability of trientine active agent, a therapeutically
effective level of trientine active agent may be achieved by
administering lower dosages than would otherwise be necessary.
[0244] An increase in bioavailability of trientine active agent may
be achieved by complexation of trientine active agent with one or
more bioavailability or absorption enhancing agents or in
bioavailability or absorption enhancing formulations.
[0245] The invention in part provides for the formulation of
trientine active agent with other agents useful to enhance
bioavailability or absorption. Such bioavailability or absorption
enhancing agents include, but are not limited to, various
surfactants such as various triglycerides, such as from butter oil,
monoglycerides, such as of stearic acid and vegetable oils, esters
thereof, esters of fatty acids, propylene glycol esters, the
polysorbates, sodium lauryl sulfate, sorbitan esters, sodium
sulfosuccinate, among other compounds. By altering the surfactant
properties of the delivery vehicle it is possible to, for example,
allow an active agent to have greater intestinal contact over a
longer period of time which increases uptake and reduces side
effects. Further examples of such agents include carrier molecules
such as cyclodextrin and derivatives thereof, well known in the art
for their potential as complexation agents capable of altering the
physicochemical attributes of drug molecules. For example,
cyclodextrin may stabilize (both thermally and oxidatively), reduce
the volatility of, and alter the solubility of, active agents with
which they are complexed. Cyclodextrins are cyclic molecules
composed of glucopyranose ring units which form toroidal
structures. The interior of the cyclodextrin molecule is
hydrophobic and the exterior is hydrophilic, making the
cyclodextrin molecule water soluble. The degree of solubility can
be altered through substitution of the hydroxyl groups on the
exterior of the cyclodextrin. Similarly, the hydrophobicity of the
interior can be altered through substitution, though generally the
hydrophobic nature of the interior allows accommodation of
relatively hydrophobic guests within the cavity. Accommodation of
one molecule within another is known as complexation and the
resulting product is referred to as an inclusion complex. Examples
of cyclodextrin derivatives include sulfobutylcyclodextrin,
maltosylcyclodextrin, hydroxypropylcyclodextrin, and salts thereof.
Complexation of trientine with a carrier molecule such as
cyclodextrin to form an inclusion complex may thereby reduce the
size of the trientine dose needed for therapeutic efficacy by
enhancing the bioavailability of the administered trientine.
[0246] The invention in part provides for the formulation of
trientine active agent in a microemulsions to enhance
bioavailability. A microemulsion is a fluid and stable homogeneous
solution composed of four major constituents, respectively, a
hydrophilic phase, a lipophilic phase, at least one surfactant (SA)
and at least one cosurfactant (CoSA). A surfactant is a chemical
compound possessing two groups, the first polar or ionic, which has
a great affinity for water, the second which contains a longer or
shorter aliphatic chain and is hydrophobic. These chemical
compounds having marked hydrophilic character are intended to cause
the formation of micelles in aqueous or oily solution. Examples of
suitable surfactants include mono-, di- and triglycerides and
polyethylene glycol (PEG) mono- and diesters. A cosurfactant, also
sometimes known as "co-surface-active agent", is a chemical
compound having hydrophobic character, intended to cause the mutual
solubilization of the aqueous and oily phases in a microemulsion.
Examples of suitable co-surfactants include ethyl diglycol, lauric
esters of propylene glycol, oleic esters of polyglycerol, and
related compounds.
[0247] The invention in part provides for the formulation of
trientine active agent with various polymers to enhance
bioavailability by increasing adhesion to mucosal surfaces, by
decreasing the rate of degradation by hydrolysis or enzymatic
degradation of the active agent, and by increasing the surface area
of the active agent relative to the size of the particle. Suitable
polymers can be natural or synthetic, and can be biodegradable or
non-biodegradable. Delivery of low molecular weight active agents
such as trientine active agent may occur by either diffusion or
degredation of the polymeric system. Representative natural
polymers include proteins such as zein, modified zein, casein,
gelatin, gluten, serum albumin, and collagen, polysaccharides such
as cellulose, dextrans, and polyhyaluronic acid. Synthetic polymers
are generally preferred due to the better characterization of
degradation and release profiles. Representative synthetic polymers
include polyphosphazenes, poly(vinyl alcohols), polyamides,
polycarbonates, polyacrylates, polyalkylenes, polyacrylamides,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl
halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,
polyurethanes and copolymers thereof. Examples of suitable
polyacrylates include poly(methyl methacrylate), poly(ethyl
methacrylate), poly(butyl methacrylate), poly(isobutyl
methacrylate), poly(hexyl methacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate) and poly(octadecyl acrylate). Synthetically
modified natural polymers include cellulose derivatives such as
alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers,
cellulose esters, and nitrocelluloses. Examples of suitable
cellulose derivatives include methyl cellulose, ethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
hydroxybutyl methyl cellulose, cellulose acetate, cellulose
propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxymethyl cellulose, cellulose triacetate and
cellulose sulfate sodium salt. Each of the polymers described above
can be obtained from commercial sources such as Sigma Chemical Co.,
St. Louis, Mo., Polysciences, Warrenton, Pa., Aldrich Chemical Co.,
Milwaukee, Wis., Fluka, Ronkonkoma, N.Y., and BioRad, Richmond,
Calif. or can be synthesized from monomers obtained from these
suppliers using standard techniques. The polymers described above
can be separately characterized as biodegradable,
non-biodegradable, and bioadhesive polymers, as discussed in more
detail below. Representative synthetic degradable polymers include
polyhydroxy acids such as polylactides, polyglycolides and
copolymers thereof, poly(ethylene terephthalate), poly(butic acid),
poly(valeric acid), poly(lactide-co-caprolactone), polyanhydrides,
polyorthoesters and blends and copolymers thereof Representative
natural biodegradable polymers include polysaccharides such as
alginate, dextran, cellulose, collagen, and chemical derivatives
thereof (substitutions, additions of chemical groups, for example,
alkyl, alkylene, hydroxylations, oxidations, and other
modifications routinely made by those skilled in the art), and
proteins such as albumin, zein and copolymers and blends thereof,
alone or in combination with synthetic polymers. In general, these
materials degrade either by enzymatic hydrolysis or exposure to
water in vivo, by surface or bulk erosion. Examples of
non-biodegradable polymers include ethylene vinyl acetate,
poly(meth)acrylic acid, polyamides, polyethylene, polypropylene,
polystyrene, polyvinyl chloride, polyvinylphenol, and copolymers
and mixtures thereof. Hydrophilic polymers and hydrogels tend to
have bioadhesive properties. Hydrophilic polymers that contain
carboxylic groups (e.g., poly[acrylic acid]) tend to exhibit the
best bioadhesive properties. Polymers with the highest
concentrations of carboxylic groups are preferred when
bioadhesiveness on soft tissues is desired. Various cellulose
derivatives, such as sodium alginate, carboxymethylcellulose,
hydroxymethylcellulose and methylcellulose also have bioadhesive
properties. Some of these bioadhesive materials are water-soluble,
while others are hydrogels. Polymers such as
hydroxypropylmethylcellulose acetate succinate (HPMCAS), cellulose
acetate trimellitate (CAT), cellulose acetate phthalate (CAP),
hydroxypropylcellulose acetate phthalate (HPCAP),
hydroxypropylmethylcellulose acetate phthalate (HPMCAP), and
methylcellulose acetate phthalate (MCAP) may be utilized to enhance
the bioavailibity of drugs with which they are complexed. Rapidly
bioerodible polymers such as poly(lactide-co-glycolide),
polyanhydrides, and polyorthoesters, whose carboxylic groups are
exposed on the external surface as their smooth surface erodes, can
also be used for bioadhesive drug delivery systems. In addition,
polymers containing labile bonds, such as polyanhydrides and
polyesters, are well known for their hydrolytic reactivity. Their
hydrolytic degradation rates can generally be altered by simple
changes in the polymer backbone. Upon degradation, these materials
also expose carboxylic groups on their external surface, and
accordingly, these can also be used for bioadhesive drug delivery
systems.
[0248] Other agents that may enhance bioavailability or absorption
can act by facilitating or inhibiting transport across the
intestinal mucosa For example, it has long been suggested that
blood flow in the stomach and intestine is a factor in determining
intestinal drug absorption and drug bioavailability, so that agents
that increase blood flow, such as vasodilators, may increase the
rate of absorption of orally administered drugs by increasing the
blood flow to the gastrointestinal tract. Vasodilators have been
used in combination with other drugs. For example, in EPO
Publication 106335, the use of a coronary vasodilator, diltiazem,
is reported to increase oral bioavailability of drugs which have an
absolute bioavailability of not more than 20%, such as adrenergic
beta-blocking agents (e.g., propranolol), catecholamines (e.g.,
dopamine), benzodiazepine derivatives (e.g., diazepam),
vasodilators (e.g., isosorbide dinitrate, nitroglycerin or amyl
nitrite), cardiotonics or antidiabetic agents, bronchodilators
(e.g., tetrahydroisoquinoline), hemostatics (e.g., carbazochrome
sulfonic acid), antispasmodics (e.g., timepidium halide) and
antitussives (e.g., tipepidine). Vasodilators therefore constitute
another class of agents which may enhance the bioavailability of
trientine.
[0249] Other mechanisms of enhancing bioavailability of the
compounds and formulations of the invention include the inhibition
of reverse active transport mechanisms. For example, it is now
thought that one of the active transport mechanisms present in the
intestinal epithelial cells is p-glycoprotein transport mechanism
which facilitates the reverse transport of substances, which have
diffused or have been transported inside the epithelial cell, back
into the lumen of the intestine. It has been speculated that the
p-glycoprotein present in the intestinal epithelial cells may
function as a protective reverse pump which prevents toxic
substances which have been ingested and diffused or transported
into the epithelial cell from being absorbed into the circulatory
system and becoming bioavailable. One of the unfortunate aspects of
the function of the p-glycoprotein in the intestinal cell however
is that it can also function to prevent bioavailability of
substances which are beneficial, such as certain drugs which happen
to be substrates for the p-glycoprotein reverse transport system.
Inhibition of this p-glycoprotein mediated active transport system
will cause less drug to be transported back into the lumen and will
thus increase the net drug transport across the gut epithelium and
will increase the amount of drug ultimately available in the blood.
Various p-glycoprotein inhibitors are well known and appreciated in
the art. These include, water soluble vitamin E; polyethylene
glycol; poloxamers including Pluronic F-68; Polyethylene oxide;
polyoxyethylene castor oil derivatives including Cremophor EL and
Cremophor RH 40; Chrysin, (+)-Taxifolin; Naringenin; Diosmin;
Quercetin; and the like.
[0250] By analogy, inhibition of a reverse active transport system
of which, for example, a trientine active agent is a substrate may
thereby enhance the bioavailability of said trientine active
agent.
[0251] Surprisingly, as shown in Example 2, and in FIGS. 3 and 4 in
particular, trientine dihydrochloride is effective at removing Cu
from diabetic rats at doses far lower than have been previously
shown to be effective. As can be seen in FIG. 3 and particularly in
FIG. 4 which presents Cu excretion normalised to body weight, Cu
excretion in the urine of diabetic rats administered trientine at a
dose of 0.1 mg.kg.sup.-1 (the lowest dose administered in the
studies presented herein) is significantly increased over that of
diabetic rats administered saline.
[0252] These data show that trientine active agents, including but
not limited to trientine, trientine salts, trientine analogues of
formulae I and II, and so on, will be effective at doses lower
than, for example, the 1.2 g.d.sup.-1 herein shown to be effective
in treating human heart disease. It may be effective at doses in
the order of 1/10, 1/100 and even 1/1000 of those we have already
employed (e.g. in the order of 120 mg.d.sup.-1, 12 mg.d.sup.-1 or
even 1.2 mg.d.sup.-1).
[0253] The invention accordingly in part provides low-dose dose
delivery formulations and devices comprising one or more trientine
active agents, including but not limited to trientine, trientine
salts, trientine analogues of formulae I and II, and so on, in an
amount sufficient to provide, for example, dosage rates from 0.01
mg.kg.sup.-1 to 5 mg.kg.sup.-1, 0.01 mg.kg.sup.-1 to 4.5
mg.kg.sup.-1, 0.02 mg.kg.sup.-1 to 4 mg.kg.sup.-1, 0.02 to 3.5
mg.kg.sup.-1, 0.02 mg.kg.sup.-1 to 3 mg.kg.sup.-1, 0.05
mg.kg.sup.-1 to 2.5 mg.kg.sup.-1, 0.05 mg.kg.sup.-1 to 2
mg.kg.sup.-1, 0.05-0.1 mg.kg.sup.-1 to 5 mg.kg.sup.-1, 0.05-0.1
mg.kg.sup.-1 to 4 mg.kg.sup.-1, 0.05-0.1 mg.kg.sup.-1 to 3
mg.kg.sup.-1, 0.05-0.1 mg.kg.sup.-1 to 2 mg.kg.sup.-1, 0.05-0.1
mg.kg.sup.-1 to 1 mg.kg.sup.-1, and/or any other rate within the
ranges as set forth.
[0254] Any such dose may be administered by any of the routes or in
any of the forms herein described. It will be appreciated that any
of the dose delivery formulations or devices described herein
particularly for oral administration may be utilized, where
applicable or desirable, in a dose delivery formulation or device
for administration by any of the other routes herein contemplated
or commonly employed. For example, it could be given parenterally
using a dose form suitable for parenteral administration, or be
delivered in an oral dosage form such as a modified release,
extended release, delayed release, slow release or repeat action
oral dosage form.
[0255] Another aspect of the invention, base on results of studies
described herein that equate human copper values depletion against
those of the STZ rat, a dosage form each with less than 250 mg of
trientine dihydrochloride (or trientine active agent when expressed
as the dihydrochloride). Envisaged are capsule forms having less
than 250 mg trientine dihydrochloride or equivalent thereof of
trientine active agent per capsule or tablets or capsules of any
suitable form.
[0256] As used herein "at risk" refers to mammals subjected to a
risk assessment of a kind exemplified in the Journal of American
Medical Association, May 16, 2001, Volume 285 No. 19, 2486-2497
where Framingham risk scoring which takes account of age, total
cholesterol, HDL cholesterol, systolic blood pressure, treatment
for hypertension and cigarette smoking is mentioned and to which
can be added glucose abnormalities of any of the kinds herein
described.
[0257] Reference herein to "elevated" in relation to the presence
of copper values in a mammal, for example, a human, will include
undesired copper levels, copper to be removed for therapeutic
benefit, and/or copper levels of at least about 10 mcg free
copper/dL of serum when measured as discussed by Merck & Co
Inc.
[0258] Histological evidence from experiments showed that six
months of treatment with trientine appears to protect the hearts of
diabetic Wistar rats from development of diabetic damage
(cardiomyopathy), as judged by histology. The doses of trientine
required for copper and iron to be excreted in the urine have also
been investigated, for example, as well as possible differences
between the excretion of these metals in diabetic and nondiabetic
animals. For example, the excretion profiles of copper and iron in
the urine of normal and diabetic rats were compared after acute
intravenous administration of increasing doses of trientine.
Additionally, it was ascertained whether acute intravenous
administration of trientine has acute adverse cardiovascular side
effects.
[0259] A better understanding of the invention will be gained by
reference to the following experimental section. The following
experiments are illustrative of the present invention and are not
intended to limit the invention in any way.
EXAMPLE 1
[0260] This Example was carried out to determine for the sake of
subsequent comparison baseline physiological data relating to the
effects of streptozotocin (STZ) treatment in rats, in addition to
baseline physiological data from diabetic and nondiabetic rats.
[0261] All methods used in this study were approved by the
University of Auckland Animal Ethics Committee and were in
accordance with The Animals Protection Act and Regulations of New
Zealand.
[0262] In order to induce diabetes, male Wistar rats (n=28,
303.+-.2.9 g) were divided randomly into diabetic and nondiabetic
groups. Following induction of anesthesia (5% halothane and
21.min.sup.-1 O2), animals in the diabetic group received a single
intravenous dose of streptozotocin (STZ, 55 mg.kg.sup.-1 body
weight, Sigma; St. Louis, Mo.) in 0.5 ml saline administered via
the tail vein. Nondiabetic animals received an equivalent volume of
saline. Following injection, both diabetic and nondiabetic rats
were housed in like-pairs and provided with access to normal rat
chow (Diet 86 pellets; New Zealand Stock Feeds, Auckland, NZ) and
deionized water ad libitum. Blood glucose and body weight were
measure at day 3 following STZ/saline injection and then weekly
throughout the study. Diabetes was identified by polydipsia,
polyuria and hyperglycemia (>11 mmol.1.sup.-1, Advantage II,
Roche Diagnostics, NZ Ltd).
[0263] Results were as follows. With regard to Effects of STZ on
blood glucose and body weight, blood glucose increased to 25.+-.2
mmol.1.sup.-1 three days following STZ injection (Table 1). Despite
a greater daily food intake, diabetic animals lost weight whilst
nondiabetic animals continued to gain weight during the 44 days
following STZ/saline injection. On the day of the experiment blood
glucose levels were 24.+-.1 and 5.+-.0 mmol.1.sup.-1 and body
weight 264.+-.7 g and 434.+-.9 g for diabetic and nondiabetic
animals respectively. TABLE-US-00002 TABLE 1 Blood glucose, body
weight and food consumption in diabetic versus nondiabetic animals
Diabetic Nondiabetic Body weight prior to 303 .+-. 3 g 303 .+-. 3 g
STZ/saline Blood glucose 3 days *25 .+-. 2 mmol l.sup.-1 5 .+-. 0.2
mmol l.sup.-1 following STZ/saline Daily food consumption *58 .+-.
1 g 28 .+-. 1 g Blood glucose on *24 .+-. 1 mmol l.sup.-1 5 .+-.
0.2 mmol l.sup.-1 experimental day Body weight on *264 .+-. 7 g 434
.+-. 9 g experimental day
Diabetic animals n=14, nondiabetic animals n=14. Values shown as
mean.+-.SEM. Asterisk indicates a significant difference
(P<0.05).
[0264] Thus, results showed that STZ treatment resulted in elevated
blood glucose, increased food intake, and decreased body weight
consistent with induction of diabetes.
EXAMPLE 2
[0265] This Example assessed the effect of acute intravenous
administration of increasing doses of trientine on the excretion
profiles of copper and iron in the urine of diabetic and
nondiabetic rats.
[0266] Six to seven weeks (mean=44.+-.1 days) after administration
of STZ, animals underwent either a control or drug experimental
protocol. All animals were fasted overnight prior to surgery but
continued to have ad libitum access to deionized water. Induction
and maintenance of surgical anesthesia was by 3-5% halothane and
21.min.sup.-1 O2. The femoral artery and vein were cannulated with
a solid-state blood pressure transducer (Mikrotip.TM. 1.4F, Millar
Instruments, Texas, USA) and a saline filled PE 50 catheter
respectively. The ureters were exposed via a midline abdominal
incision, cannulated using polyethylene catheters (external
diameter 0.9 mm, internal diameter 0.5 mm) and the wound sutured
closed. The trachea was cannulated and the animal ventilated at
70-80 breaths.min.sup.-1 with air supplemented with O2 (Pressure
Controlled Ventilator, Kent Scientific, Connecticut, USA). The
respiratory rate and end-tidal pressure (10-15 cmH2O) were adjusted
to maintain end-tidal CO2 at 35-40 mmHg (SC-300 CO2 monitor, Pryon
Corporation, Wisconsin, USA). Body temperature was maintained at
37.degree. C. throughout surgery and the experiment by a heating
pad. Estimated fluid loss was replaced with intravenous
administration of 154 mmol.1.sup.-1 NaCl solution at a rate of 5
ml.kg.sup.-1.h.sup.-1.
[0267] Following surgery and a 20 min stabilization period, the
experimental protocol was started. Trientine was administered
intravenously over 60 s in hourly doses of increasing concentration
(0.1, 1.0, 10 and 100 mg.kg-1 in 75 .mu.l saline followed by 125
.mu.l saline flush). Control animals received an equivalent volume
of saline. Urine was collected in 15 min aliquots throughout the
experiment in pre-weighed polyethylene epindorf tubes. At the end
of the experiment a terminal blood sample was taken by cardiac
puncture and the separated serum stored at -80.degree. C. until
future analysis. Hearts were removed through a rapid mid-sternal
thoracotomy and processed as described below.
[0268] Mean arterial pressure (MAP), heart rate (HR, derived from
the MAP waveform) oxygen saturation (Nonin 8600V Pulse Oximeter,
Nonin Medical Inc., Minnesota, USA) and core body temperature, were
all continuously monitored throughout the experiment using a
PowerLab/16s data acquisition module (AD Instruments, Australia).
Calibrated signals were displayed on screen and saved to disc as 2
s averages of each variable.
[0269] Urine and tissue analysis was carried out as follows.
Instrumentation: A Perkin Elmer (PE) Model 3100 Atomic Absorption
Spectrophotometer equipped with a PE HGA-600 Graphite Furnace and
PE AS-60 Furnace Autosampler was used for Cu and Fe determinations
in urine. Deuterium background correction was employed. A Cu or Fe
hollow-cathode lamp (Perkin Elmer Corporation) was used and
operated at either 10 W (Cu) or 15 W (Fe). The 324.8 nm atomic line
was used for Cu and the 248.3 nm atomic line for Fe. The slit width
for both Cu and Fe was 0.7 nm. Pyrolytically coated graphite tubes
were used for all analyses. The injection volume was 20 .mu.L. A
typical graphite furnace temperature program is shown below:
TABLE-US-00003 GF-AAS temperature program Procedure Temp/.degree.
C. Ramp/s Hold/s Int. Flow/mL min.sup.-1 Drying 90 1 5 300 120 60 5
300 Pre-treatment 1250* 20 10 300 20 1 10 300 Atomization -
2300/2500 1 8 0 Cu/Fe Post-treatment 2600 1 5 300 *A pre-treatment
temperature of 1050.degree. C. was used for tissue digest analyses
(see Example 3)
[0270] Reagents: All reagents used were of the highest purity
available and at least of analytical grade. GF-AAS standard working
solutions of Cu and Fe were prepared by stepwise dilution of 1000
mg.1.sup.-1 (Spectrosol standard solutions; BDH). Water was
purified by a Millipore Milli-Q ultra-pure water system to a
resistivity of 18 M.OMEGA..
[0271] Sample pretreatment was carried out as follows. Urine: Urine
was collected in pre-weighed 1.5 ml micro test tubes (eppendorf).
After reweighing, the urine specimens were centrifuged and the
supernatant diluted 25:1 with 0.02 M 69% Aristar grade HNO.sub.3.
The sample was stored at 4.degree. C. prior to GF-AAS analysis. If
it was necessary to store a sample for a period in excess of 2
weeks, it was frozen and kept at -20.degree. C. Serum: Terminal
blood samples were centrifuged and serum treated and stored as per
urine until analysis. From the trace metal content of serum from
the terminal blood sample and urine collected over the final hour
of the experiment, renal clearance was calculated using the
following equation: renal .times. .times. .times. clearance .times.
.times. of .times. .times. .times. trace .times. .times. .times.
metal .times. .times. ( .mu. .times. .times. 1. .times. .times. min
- 1 ) = concentration .times. .times. of .times. .times. .times.
metal .times. .times. in .times. .times. urine .times. .times. (
.mu.g . .times. .mu. .times. .times. 1 - 1 ) * rate .times. .times.
of .times. .times. urine .times. .times. flow .times. .times. (
.mu.1 . min - 1 ) .times. ( concentration .times. .times. of
.times. .times. metal .times. .times. in .times. .times. serum
.times. .times. ( .mu.g . .times. .mu. .times. .times. 1 - 1 )
##EQU1##
[0272] Statistical analyses were carried out as follows. All values
are expressed as mean.+-.SEM and P values<0.05 were considered
statistically significant. Student's unpaired t-test was initially
used to test for weight and glucose differences between the
diabetic and control groups. For comparison of responses during
drug exposure, statistical analyses were performed using analysis
of variance (Statistics for Windows v.6.1, SAS Institute Inc.,
Calfornia, USA). Subsequent statistical analysis was performed
using a mixed model repeated measures ANOVA design (see Example
4).
[0273] The results were as follows. With regard to cardiovascular
variables during infusion, baseline levels of MAP during the
control period prior to infusion were not significantly different
between nondiabetic and diabetic animals (99.+-.4 mmHg). HR was
significantly lower in diabetic than nondiabetic animals (287.+-.11
and 364.+-.9 bpm respectively, P<0.001). Infusion of trientine
or saline had no effect on these variables except at the highest
dose where MAP decreased by a maximum of 19.+-.4 mmHg for the 2 min
following administration and returned to pre-dose levels within 10
min. Body temperature and oxygen saturation remained stable in all
animals throughout the experiment.
[0274] With regard to urine excretion, diabetic animals
consistently excreted significantly more urine than nondiabetic
animals except in response to the highest dose of drug (100
mg.kg.sup.-1) or equivalent volume of saline (FIG. 16).
Administration of the 100 mg.kg.sup.-1 dose of trientine also
increased urine excretion in nondiabetic animals to greater than
that of nondiabetic animals receiving the equivalent volume of
saline (FIG. 17). This effect was not seen in diabetic animals.
[0275] With regard to urinary excretion of Cu and Fe analysis of
the dose response curves showed that, at all doses, diabetic and
nondiabetic animals receiving drug excreted more Cu than animals
receiving an equivalent volume of saline (FIG. 18). To provide some
correction for the effects of lesser total body growth of the
diabetic animals, and thus to allow more appropriate comparison
between diabetic and nondiabetic animals, excretion rates of trace
elements were also calculated per gram of body weight. FIG. 19
shows that diabetic animals had significantly greater copper
excretion per gram of body weight in response to each dose of drug
than did nondiabetic animals. The same pattern was seen in response
to saline, however the effect was not always significant.
[0276] Total copper excreted over the entire duration of the
experiment was significantly increased in both nondiabetic and
diabetic animals administered trientine compared with their
respective saline controls (FIG. 20). Diabetic animals receiving
drug also excreted more total copper per gram of body weight than
nondiabetic animals receiving drug. The same significant trend was
seen in response to saline administration (FIG. 21).
[0277] In comparison, iron excretion in both diabetic and
nondiabetic animals receiving trientine was not greater than
animals receiving an equivalent volume of saline (FIG. 22).
Analysis per gram of body weight shows diabetic animals receiving
saline excrete significantly more iron than nondiabetic animals,
however this trend was not evident between diabetic and nondiabetic
animals receiving trientine (FIG. 23). Total iron excretion in both
diabetic and nondiabetic animals receiving drug was not different
from animals receiving saline (FIG. 24). In agreement with analysis
of dose response curves, total iron excretion per gram of body
weight was significantly greater in diabetic animals receiving
saline than nondiabetic animals but this difference was not seen in
response to trientine (FIG. 25).
[0278] Electron paramagnetic resonance spectroscopy showed that the
urinary Cu from drug-treated animals was mainly complexed as
trientine-Cu.sup.II (FIG. 28), indicating that the increased tissue
Cu in diabetic rats is mainly divalent. These data indicate that
rats with severe hyperglycaemia develop increased systemic
Cu.sup.II that can be extracted by selective chelation.
[0279] With regard to Serum content and renal clearance of Cu and
Fe, while there was no significant difference in serum copper
content, there was a significant increase in renal clearance of
copper in diabetic animals receiving drug compared with diabetic
animals receiving saline (Table 2). The same pattern was seen in
nondiabetic animals, although the trend was not statistically
significant (P=0.056). There was no effect of drug or state
(diabetic versus nondiabetic) on serum content or renal clearance
of iron. TABLE-US-00004 TABLE 2 Serum content and renal clearance
of Cu and Fe in diabetic and nondiabetic animals receiving drug or
saline. 1.1.a.a.1 1.1.a.a.1 1.1.a.a.2 1.1.a.a.2 diabetic diabetic
nondiabetic nondiabetic trientine Saline trientine Saline n = 6 n =
7 n = 4 n = 7 Serum Cu 7.56 .+-. 0.06 9.07 .+-. 1.74 7.11 .+-. 0.41
7.56 .+-. 0.62 (.mu.g .mu.l.sup.-1 .times. 10.sup.-4) Serum Fe 35.7
.+-. 7.98 63.2 .+-. 16.4 33.6 .+-. 1.62 31.4 .+-. 8.17 (.mu.g
.mu.l.sup.-1 .times. 10.sup.-4) Renal *28.5 .+-. 4.8 1.66 .+-. 0.82
19.9 .+-. 6.4 0.58 .+-. 0.28 clearance Cu (.mu.l min.sup.-1) Renal
0.25 .+-. 0.07 0.38 .+-. 0.15 0.46 .+-. 0.22 0.11 .+-. 0.03
clearance Fe (.mu.l min.sup.-1) Values shown as mean .+-. SEM.
Asterisk indicates a significant difference (P < 0.05) between
diabetic animals receiving trientine and diabetic animals receiving
an equivalent volume of saline.
[0280] In summary, acute intravenous administration of trientine
significantly increased total copper excretion in both nondiabetic
and diabetic animals compared with their respective saline
controls. Furthermore, following acute intravenous administration
of increasing doses of trientine, diabetic animals had
significantly greater copper excretion per gram of body weight than
did nondiabetic animals. In contrast, total iron excretion in both
diabetic and nondiabetic animals receiving drug was not different
from animals receiving saline.
EXAMPLE 3
[0281] This example was carried out to determine the effect of
acute intravenous administration of increasing doses of trientine
on the copper and iron content of cardiac tissue in normal and
diabetic rates.
[0282] Methods were carried out as follows. Spectrophotometric
analysis was conducted as described in Example 2. Cu, Fe and Zn in
tissue digests were determined at Hill Laboratories (Hamilton, New
Zealand) using either a PE Sciex Elan-6000 or PE Sciex Elan-6100
DRC ICP-MS. The operating parameters are summarized in the Table
below. TABLE-US-00005 Instrumental operating parameters for ICP-MS
Parameter Value Inductively coupled plasma Radiofrequency power
1500 W Argon plasma gas flow rate 15 l min.sup.-1 Argon auxiliary
gas flow rate 1.2 l min.sup.-1 Argon nebuliser gas flow rate 0.89 l
min.sup.-1 Interface Sampler cone and orifice diameter Ni/1.1 mm
Skimmer cone and orifice diameter Ni/0.9 mm Data acquisition
parameters Scanning mode Peak hopping Dwell time 30 ms (Cu, Zn)/100
ms (Fe) Sweeps/replicate 20 Replicates 3 Sample uptake rate 1 ml
min.sup.-1
[0283] Reagents were as follows. Standard Reference Material 1577b
Bovine Liver was obtained from the National Institute of Standards
and Technology and used to evaluate the efficiency of tissue
digestion. The results obtained are reported below: TABLE-US-00006
GF-AAS and ICP-MS results for NIST SRM 1577b bovine liver* Element
Certified value GF-AAS ICP-MS Cu 160 .+-. 8 142 .+-. 12 164 .+-. 12
Fe 184 .+-. 15 182 .+-. 21 166 .+-. 14 Zn 127 .+-. 16 -- 155 .+-.
42 *Measured in .mu.g g.sup.-1 of dry matter.
[0284] Sample pre-treatment was carried out as follows. Heart:
Following removal from the animal, the heart was cleaned of excess
tissue, rinsed in buffer to remove excess blood, blotted dry and a
wet ventricular weight recorded. Using titanium instruments a
segment of left ventricular muscle was dissected and placed in a
pre-weighed 5.0 ml polystyrene tube. The sample was freeze-dried
overnight to constant weight before 0.45 ml of 69% Aristar grade
HNO.sub.3 was added. The sample tube was heated in a water bath at
65.degree. C. for 60 minutes. The sample was brought to 4.5 ml with
Milli-Q H.sub.2O. The resulting solution was diluted 2:1 in order
to reduce the HNO.sub.3 concentration below the maximum permitted
for ICP-MS analysis.
[0285] The results were as follows. With regard to the metal
content of cardiac tissue, wet heart weights in diabetic animals
were significantly less than those in nondiabetic animals while
heart/body weight ratios were increased (see Table 3). Cardiac
tissue from some animals was also analysed for Cu and Fe content.
There was no significant difference in content of copper between
diabetic and nondiabetic animals receiving saline or trientine.
Iron content of the non-diabetic animals administered saline was
significantly greater than that of the diabetic animals
administered saline (see Table 3). TABLE-US-00007 TABLE 3 Heart
weight, heart weight/body weight ratios and trace metal content of
heart tissue in diabetic versus nondiabetic animals Diabetic
Nondiabetic Wet heart weight *0.78 .+-. 0.02 g 1.00 .+-. 0.02 g
Heart weight/ *2.93 .+-. 0.05 mg g.sup.-1 2.30 .+-. 0.03 mg
g.sup.-1 body weight Cu content .mu.g g.sup.-1 dry tissue Trientine
treated 24.7 .+-. 1.5 27.1 .+-. 1.0 Saline treated 21.3 .+-. 0.9
27.2 .+-. 0.7 Fe content .mu.g g.sup.-1 dry tissue Trientine
treated 186 .+-. 46 235 .+-. 39 Saline treated .sup..dagger.180
.+-. 35.sup. 274 .+-. 30 Diabetic animals: n = 14; nondiabetic
animals: n = 14. Values shown as mean .+-. SEM. Asterisk indicates
a significant difference (P < 0.05) between diabetic and
non-diabetic animals. .sup..dagger.indicates a significant
difference (P < 0.05) between diabetic and non-diabetic animals
receiving saline.
[0286] In summary, it was demonstrated that acute intravenous
administration of increasing doses of trientine had no significant
effect on the copper content of cardiac tissue in normal and
diabetic rates.
EXAMPLE 4
[0287] In this Example, a mixed linear model was applied to the
data generated above in Examples 1-3.
[0288] Methods were as follows. With regard to statistical analysis
using a mixed linear model, data for each dose level were analyzed
using a mixed linear model (PROC MIXED; SAS, Version 8). The model
included diabetes, drug and their interaction as fixed effects,
time as a repeated measure, and rats as the subjects in the
dataset. Complete independence was assumed across subjects. The
full model was fitted to each dataset using a maximum likelihood
estimation method (REML) fits mixed linear models (i.e., fixed and
random effects models). A mixed model is a generalization of the
standard linear model, the generalization being that one can
analyze data generated from several sources of variation instead of
just one. A level of significance of 0.05 was used for all tests.
Results were as follows.
[0289] With regard to copper, diabetic rats excreted significantly
higher levels of copper across all dose levels (see FIG. 27).
Baseline copper excretion was also significantly higher in diabetic
rats compared to nondiabetic rats. There was no difference at
baseline levels between the drug and saline groups. The interaction
effect for the model was significant at dose levels of 1.0
mg.kg.sup.-1 and above. The presence of a significant interaction
term means that the influence of one effect varies with the level
of the other effect. Therefore, the outcome of a significant
interaction between the diabetes and drug factors is increased
copper excretion above the predicted additive effects of these two
factors.
[0290] With regard to iron, diabetic rats in the saline only group
excreted significantly higher levels of iron at all dose levels.
This resulted in all factors in the model being significant across
all dose levels.
[0291] In sum, the acute effect of intravenous trientine
administration on the cardiovascular system and urinary excretion
of copper and iron was studied in anesthetized, diabetic (6 weeks
of diabetes, Streptozotocin induced) and nondiabetic rats. Animals
were assigned to one of four groups: diabetic+trientine,
diabetic+saline, nondiabetic+trientine, nondiabetic+saline. Drug,
or an equivalent volume of saline, was administered hourly in doses
of increasing strength (0.1, 1.0, 10, 100 mg.kg.sup.-1) and urine
was collected throughout the experiment in 15 min aliquots. A
terminal blood sample was taken and cardiac tissue harvested.
Analysis of urine samples revealed: (1) At all drug doses, diabetic
and nondiabetic animals receiving drug excreted more Cu (.mu.mol)
than animals receiving an equivalent volume of saline; (2) When
analyzed per gram of bodyweight, diabetic animals excreted
significantly more copper (.mu.mol.gBW.sup.-1) at each dose of
trientine than did nondiabetic animals. The same pattern was seen
in response to saline but the effect was not significant at every
dose; (3) At most doses, in diabetic animals iron excretion
(.mu.mol) was greater in animals administered saline than in those
administered drug. In nondiabetic animals there was no difference
between iron excretion in response to saline or trientine
administration; (4) Analysis per gram of body weight shows no
difference between iron excretion in nondiabetic and diabetic
animals receiving trientine. Diabetic animals receiving saline
excrete more iron per gram of bodyweight than nondiabetic animals
receiving saline; (5) Analysis of heart tissue showed no
significant difference in total copper content between diabetic and
nondiabetic animals, nor any effect of drug on cardiac content of
iron and copper; and (6) Renal clearance calculations showed a
significant increase in clearance of copper in diabetic animals
receiving trientine compared with diabetic animals receiving
saline. The same trend was seen in nondiabetic animals but the
affect was not significant There was no effect of trientine on
renal clearance of iron.
[0292] There were no adverse cardiovascular effects were observed
after acute administration of trientine. Trientine treatment
effectively increases copper excretion in both diabetic and
nondiabetic animals. The excretion of copper in urine following
trientine administration is greater per gram of bodyweight in
diabetic than in nondiabetic animals. Iron excretion was not
increased by trientine treatment in either diabetic or nondiabetic
animals.
EXAMPLE 5
[0293] Experiments relating to the efficacy of trientine to restore
cardiac function in STZ diabetic rats were carried out. As noted
above, histological evidence showed that treatment with trientine
appears to protect the hearts of diabetic Wistar rats from
development of cardiac damage (diabetic cardiomyopathy), as judged
by histology. However, it was unknown whether this histological
improvement may lead to improved cardiac function.
[0294] This experiment was carried out to compare cardiac function
in trientine-treated and non-treated, STZ diabetic and normal rats
using an isolated-working-rodent heart model.
[0295] Methods were as follows. The animals used in these
experiments received care that complied with the "Principles of
Laboratory Animal Care" (National Society for Medical Research),
and the University of Auckland Animal Ethics Committee approved the
study.
[0296] Male albino Wistar rats weighing 330-430 g were assigned to
four experimental groups as shown in Table 4. TABLE-US-00008 TABLE
4 Experimental groups Group Code N Treatment Group A STZ 8 Diabetes
for 13 weeks Group B STZ/D7 8 Diabetes for 13 weeks (Drug therapy
week 7-13) Group C Sham 9 Non-diabetic controls Group D Sham/D7 11
Non-diabetic controls (Drug therapy week 7-13) STZ =
Streptozotocin; D7 = trientine treatment for 7 consecutive weeks
commencing 6 weeks after the start of the experiment.
[0297] Diabetes was induced by intravenous streptozotocin (STZ;
Sigma; St. Louis, Mo.). All rats were given a short inhalational
anesthetic (Induction: 5% halothane and 2 L/min oxygen, maintained
on 2% halothane and 2 L/min oxygen). Those in the two diabetic
groups then received a single intravenous bolus dose of STZ (57
mg/kg body weight) in 0.5 ml of 0.9% saline administered via a tail
vein. Non-diabetic sham-treated animals received an equivalent
volume of 0.9% saline. Diabetic and non-diabetic rats were housed
in like-pairs and provided with free access to normal rat chow
(Diet 86 pellets; New Zealand Stock Feeds, Auckland, NZ) and
deionized water ad libitum. Each cage had two water bottles on it
to ensure equal access to water or drug for each animal. Animals
were housed at 21 degrees and 60% humidity in standard rat cages
with a sawdust floor that was changed daily.
[0298] Blood glucose was measured in tail-tip capillary blood
samples (Advantage II, Roche Diagnostics, NZ Ltd). Sampling was
performed on all groups at the same time of the day. Blood glucose
and body weight were measured on day 3 following STZ/saline
injection and then weekly throughout the study. Diabetes was
confirmed by presence of polydipsia, polyuria and hyperglycemia
(>11 mmol.L.sup.-1).
[0299] In the drug treated diabetic group, trientine was prepared
in the drinking water for each cage at a concentration of 50 mg/L.
The trientine-containing drinking water was administered
continuously from the start of week 7 until the animal was
sacrificed at the end of week 13. In the case of the Sham/D7
non-diabetic group that drank less water per day than diabetic
animals, the drug concentration in their drinking water was
adjusted so that they consumed approximately the same dose as the
corresponding STZ/D7 group. Trientine treated animals ingested mean
drug doses of between 8 to 11 mg per day.
[0300] At the time the drug started in the diabetic group the
diabetic animals were expected to have to have established
cardiomyopathy, as shown by preliminary studies (data not shown)
and confirmed in the literature. See Rodrigues B, et al., Diabetes
37(10):1358-64 (1988).
[0301] On the last day of the experiment, animals were anesthetized
(5% halothane and 2 L.min.sup.-1 O.sub.2), and heparin (500
IU.kg.sup.-1) (Weddel Pharmaceutical Ltd., London) administered
intravenously via tail vein. A 2 ml blood sample was then taken
from the inferior vena cava and the heart was then rapidly excised
and immersed in ice-cold Krebs-Henseleit bicarbonate buffer to
arrest contractile activity. Hearts were then placed in the
isolated perfused working heart apparatus.
[0302] The aortic root of the heart was immediately ligated to the
aortic cannula of the perfusion apparatus. Retrograde (Langendorff)
perfusion at a hydrostatic pressure of 100 cm H.sub.2O and at
37.degree. C. was established and continued for 5 min while
cannulation of the left atrium via the pulmonary vein was
completed. The non-working (Langendorff) preparation was then
converted to the working heart model by switching the supply of
perfusate buffer from the aorta to the left atrium at a filling
pressure of 10 cm H.sub.2O. The left ventricle spontaneously
ejected into the aortic cannula against a hydrostatic pressure
(after-load) of 76 cmH.sub.2O (55.9 mmHg). The perfusion solution
was Krebs-Henseleit bicarbonate buffer (mM: KCl 4.7, CaCl.sub.2
2.3, KH.sub.2PO.sub.4 1.2, MgSO.sub.4 1.2, NaCl 118, and
NaHCO.sub.3 25), pH 7.4 containing 11 mM glucose and it was
continuously gassed with 95% O.sub.2:5% CO.sub.2. The buffer was
also continuously filtered in-line (initial 8 .mu.m, following 0.4
.mu.m cellulose acetate filters; Sartorius, Germany). The
temperature of the entire perfusion apparatus was maintained by
water jackets and buffer temperature was continuously monitored and
adjusted to maintain hearts at 37.degree. C. throughout
perfusion.
[0303] A modified 24 g plastic intravenous cannula (Becton Dickson,
Utah, USA) was inserted into the left ventricle via the apex of the
heart using the normal introducer-needle. This cannula was
subsequently attached to a SP844 piezo-electric pressure transducer
(AD Instruments) to continuously monitor left ventricular pressure.
Aortic pressure was continuously monitored through a side arm of
the aortic cannula with a pressure transducer (Statham Model P23XL,
Gould Inc., Calif., USA). The heart was paced (Digitimer Ltd,
Heredfordshire, England) at a rate of 300 bpm by means of
electrodes attached to the aortic and pulmonary vein cannulae using
supra-threshold voltages with pulses of 5-ms duration from the
square wave generator.
[0304] Aortic flow was recorded by an in-line flow meter (Transonic
T206, Ithaca, N.Y., USA) and coronary flow was measured by timed 30
sec collection of the coronary vein effluent at each time point
step of the protocol.
[0305] The working heart apparatus used was a variant of that
originally described by Neely, J R, et al., Am J Physiol 212:804-14
(1967). The modified apparatus allowed measurements of cardiac
function at different pre-load pressures (FIG. 14 and FIG. 15).
This was achieved by constructing the apparatus so that the inflow
height of the buffer coming to the heart could be altered through a
series of graduated steps in a reproducible manner. As in the case
of the pre-load, the outflow tubing from the aorta could also be
increased in height to provide a series of defined after-load
pressures. The after-load heights have been converted to mm Hg for
presentation in the results which is in keeping with published
convention.
[0306] All data from the pressure transducers and flow probe were
collected (Powerlab 16s data acquisition machine; AD Instruments,
Australia). The data processing functions of this device were used
to calculate the first derivative of the two pressure waves
(ventricular and aortic). The final cardiac function data available
comprised:
[0307] Cardiac output*; aortic flow, coronary flow; peak left
ventricular/aortic pressure developed; maximum rate of ventricular
pressure development (+dP/dt)**; maximum rate of ventricular
pressure relaxation (-dP/dt)**; maximum rate of aortic pressure
development (aortic +dP/dt); maximum rate of aortic relaxation
(aortic -dP/dt). [*Cardiac output (CO) is the amount of buffer
pumped per unit time by the heart and is comprised of buffer that
is pumped out the aorta as well as the buffer pumped into the
coronary vessels. This is an overall indicator of cardiac function.
** +dP/dt is the rate of change of ventricular (or aortic pressure)
and correlates well with the strength of the contraction of the
ventricle (contractility). It can be used to compare contractility
abilities of different hearts when at the same pre-load (Textbook
of Medical Physiology, Ed. A. Guyton. Saunders company 1986).
-dP/dt is an accepted measurement of the rate of relaxation of the
ventricle].
[0308] The experiment was divided into two parts, the first with
fixed after-load and variable pre-load the second, which
immediately followed on from the first, with fixed pre-load and
variable after-load.
[0309] Fixed After-load and changing Pre-load: After the initial
cannulation was completed, the heart was initially allowed to
equilibrate for 6 min at 10 cm H.sub.2O atrial filling pressure and
76 cm H.sub.2O after-load. During this period the left ventricular
pressure transducer cannula was inserted and the pacing unit
started. Once the heart was stable, the atrial filling pressure was
then reduced to 5 cm H.sub.2O of water and then progressively
increased in steps of 2.5 cmH.sub.2O over a series of 7 steps to a
maximum of 20 cmH.sub.2O. The pre-load was kept at each filling
pressure for 2 min, during which time the pressure trace could be
observed to stabilize and the coronary flow was measured. On
completion of the variable pre-load experiment, the variable
after-load portion of the experiment was immediately commenced.
[0310] Fixed Pre-load and changing After-load: During this part of
the experiment the filling pressure (pre-load) was set at 10 cm
H.sub.2O and the after-load was then increased from 76 cm H.sub.2O
(55.9 mm Hg) in 9 steps; of 2 min duration. The maximum height
(after-load) to which each individual heart was ultimately exposed,
was determined either by attainment of the maximal available
after-load height of 145 cm H.sub.2O (106.66 mm Hg), or the height
at which measured aortic flow became 0 ml/min. In the later
situation, the heart was considered to have "functionally failed."
To ensure that this failure was indeed functional and not due to
other causes (e.g., permanent ischemic or valvular damage) all
hearts were then returned to the initial perfusion conditions
(pre-load 10 cm H2O; after-load 75 cm H.sub.2O) for 4 minutes to
confirm that pump function could be restored. At the end of this
period the hearts were arrested with a retrograde infusion of 4 ml
of cold KCL (24 mM). The atria and vascular remnants were then
excised, the heart blotted dry and weighed. The ventricles were
incised midway between the apex and atrioventricular sulcus.
Measurements of the ventricular wall thickness were then made using
a micro-caliper (Absolute Digimatic, Mitutoyo Corp, Japan).
[0311] Data from the Powerlab was extracted by averaging 1 min
intervals from the stable part of the electronic trace generated
from each step in the protocol. The results from each group were
then combined and analyzed for differences between the groups for
the various cardiac function parameters (aortic flow, cardiac flow,
MLVDP, LV or aortic .+-.dP/dt). Differences between repeated
observations at different pre-load conditions were explored and
contrasted between study group using a mixed models approach to
repeated measures (SAS v8.1, SAS Institute Inc, Cary N.C.). Missing
random data were imputed using a maximum likelihood approach.
Significant mean and interaction effects were further examined
using the method of Tukey to maintain a pairwise 5% error rate for
post hoc tests. All tests were two-tailed. Survival analysis was
done using Proc Liftest (SAS V8.2); A one-way analysis of variance
was used to test for difference between groups in various weight
parameters. Tukey's tests were used to compare each group with each
other. In each graph unless otherwise stated.* indicates
p<0.05=STZ v STZ/D7, #.p<0.05=STZ/D7 v Sham/D7.
[0312] Results showing the weights of the animals at the end of the
experimental period are found in Table 5. Diabetic animals were
about 50% smaller than their corresponding age matched normals. A
graph of the percentage change in weight for each experimental
group is found in FIG. 5, wherein the arrow indicates the start of
trientine treatment. TABLE-US-00009 TABLE 5 Initial and final
animal body weights (mean .+-. SD) Number Treat- Initial weight (n)
ment weight (g) Final (g) Group A 8 STZ 361 .+-. 12 221 .+-. 27
{close oversize bracket} * {close oversize bracket} * Group B 8
STZ/D7 401 .+-. 33 290 .+-. 56 {close oversize bracket} * Group C 9
Sham 361 .+-. 16 574 .+-. 50 Group D 11 Sham/ 357 .+-. 7 563 .+-.
17 D7 *P < 0.05
[0313] Blood glucose values for the three groups of rats are
presented in FIG. 6. Generally, the presence of diabetes was
established and confirmed within 3-5 days following the STZ
injection. The Sham and Sham/D7 control group remained
normoglycemic throughout the experiment. Treatment with the drug
made no difference to the blood glucose profile (p=ns) in either
treated group compared to their respective appropriate untreated
comparison group.
[0314] Final heart weight and ventricular wall thickness
measurements are presented in Table 6. There was a small but
significant improvement in the "heart: body weight" ratio with
treatment in the diabetic animals. There was a trend toward
improved "ventricular wall thickness:bodyweight" ratio in treated
diabetics compared to non-treated but this did not reach
significance.
[0315] Fixed After-Load and Changing Pre-Load
[0316] The following graphs of FIGS. 7 to 12 represent cardiac
performance parameters of the animals (STZ diabetic; STZ
diabetic+drug; and sham-treated controls) while undergoing
increasing atrial filling pressure (5-20 cmH.sub.2O, pre-load) with
a constant after-load of 75 cm H.sub.2O. All results are
mean.+-.sem. In each graph for clarity unless otherwise stated,
only significant differences related to the STZ/D7 the other groups
are shown:* indicates p<0.05 for STZ v STZ/D7, # p<0.05 for
STZ/D7 v Sham/D7. Unless stated, STZ/D7 v Sham or Sham/D7 was not
significant.
[0317] Cardiac output (FIG. 7) is the sum to the aortic flow (FIG.
10) and the coronary flow as displayed in FIG. 8. Since the control
hearts and experimental groups have significantly different final
weights, the coronary flow is also presented (FIG. 9) as the flow
normalized to heart weight (note that coronary flow is generally
proportional to cardiac muscle mass, and therefore to cardiac
weight). TABLE-US-00010 TABLE 6 Final heart weights (g) and per g
of animal body Weight (BW) (mean .+-. Left Ventricular Left
Ventricular wall wall thickness Heart Heart weight (g)/ thickness
per BW Group weight (g) BW (g) (mm) (mm)/(g) Sham 1.58 .+-.
0.13.sup..sctn. 0.0028 .+-. 0.0002.sup..sctn. 3.89 .+-.
0.38.sup..sctn. 0.0068 .+-. 0.0009.sup..sctn. STZ/D7 1.18 .+-. 0.24
0.0041 .+-. 0.0005 3.79 .+-. 0.52 0.0127 .+-. 0.0027 {close
oversize bracket} ns {close oversize bracket} * {close oversize
bracket} ns {close oversize bracket} ns STZ 1.03 .+-. 0.17 0.0047
.+-. 0.0004 3.31 .+-. 0.39 0.0152 .+-. 0.0026 Sham/D7 1.58 .+-.
0.05.sup..sctn. 0.0028 .+-. 0.0001.sup..sctn. 4.03 .+-.
0.1.sup..sctn. 0.0072 .+-. 0.0003.sup..sctn. *P < 0.05
.sup..sctn.= significant with the STZ and STZ/D7 groups p <
0.05
[0318] The first derivative of the pressure curve gives the rate of
change in pressure development in the ventricle with each cardiac
cycle and the maximum positive rate of change (+dP/dt) value is
plotted in FIG. 11. The corresponding maximum rate of relaxation
(-dP/dt) is in FIG. 12. Similar results showing improvement in
cardiac function were found from the data derived from the aortic
pressure cannula (results not shown).
Fixed Pre-Load and Changing After-Load
[0319] Under conditions for constant pre-load and increasing
after-load the ability of the hearts to cope with additional
after-load work was assessed. The plot of functional survival, that
is, the remaining number of hearts at each after-load that still
had an aortic output of greater than 0 ml/min, is found in FIG.
13.
[0320] Administration of trientine improved cardiac function in STZ
diabetic rats compared to untreated diabetic controls. For example,
cardiac output, ventricular contraction and relaxation, and
coronary flow were all improved in trientine treated diabetic rats
compared to non-treated diabetic controls.
EXAMPLE 6
[0321] This Example was carried out to further evaluate the effect
of acute trientine administration on cardiac tissue by assessing
left ventricular (LV) histology.
[0322] Methods were as follows. Following functional analysis, LV
histology was studied by laser confocal (LCM; FIG. 29a-d) and
transmission electron microscopy (TEM; FIG. 29e-h). For LCM, LV
sections were co-stained with phalloidin to visualize actin
filaments, and .beta..sub.1-integrin as a marker for the
extracellular space. Ding B, et al., "Left ventricular hypertrophy
in ascending aortic stenosis in mice: anoikis and the progression
to early failure," Circulation 101:2854-2862 (2000).
[0323] For each treatment, 5 sections from each of 3 hearts were
examined by both LCM and TEM. For LCM, LV sections were fixed (4%
paraformaldehyde, 24 h); embedded (6% agar); vibratomed (120 pm,
Campden); stained for f-actin (Phafloidin-488, molecular Probes)
and .beta..sub.1-integrin antibody with a secondary antibody of
goat anti-rabbit conjugated to CY5 (1:200; Ding B, et al., "Left
ventricular hypertrophy in ascending aortic stenosis in mice:
anoikis and the progression to early failure," Circulation
101:2854-2862 (2000)); and visualised (TCS-SP2, Leica). For TEM,
specimens were post-fixed (1:1 v/v 1% w/v 0s0 M 0s0 M PBS); stained
(aqueous uranyl acetate (2% w/v, 20 mm) then lead citrate (3 mm));
sectioned (70 nm); and visualized (CM-12, Phillips).
[0324] The results were as follows. Copper chelation normalized LV
structure in diabetic rats. Compared with controls (FIG. 29a),
diabetes caused obvious alterations in myocardial structure, with
marked loss of myocytes; thinning and disorganization of remaining
myofibrils; decreased density of actin filaments; and marked
expansion of the interstitial space (FIG. 29b). These findings are
consistent with previous reports. Jackson C V, et al., "A
functional and ultrastructural analysis of experimental diabetic
rat myocardium: manifestation of acardiomyopathy," Diabetes
34:876-883 (1985). By marked contrast, myocardial histology
following trientine treatment was improved (FIG. 29c). Importantly,
the orientation and volume of cardiomyocytes and their actin
filaments was largely normalized, consistent with the normalization
of -dP.sub.LV/dt observed in the functional studies. Trientine
treatment reversed the expanded cardiac ECM. Myocardium from
trientine-treated non-diabetics appeared normal by LCM (FIG. 29d)
suggesting that it has no detectable adverse effects on LV
structure. Thus, Cu chelation essentially restored the normal
histological appearance of the myocardium without suppressing
hyperglycaemia These data provide important structural correlates
for the functional recovery of these hearts, shown above.
[0325] TEM was largely consistent with LCM. Compared with controls
(FIG. 29e), diabetes caused unmistakable myocardial damage
characterized by loss of myocytes with evident myocytolysis;
disorganization of remaining cardiomyocytes in which swollen
mitochondria were prominent; and marked expansion of the
extracellular space (FIG. 29f). These findings are consistent with
previous reports. Jackson C V, et al., "A functional and
ultrastructural analysis of experimental diabetic rat myocardium:
manifestation of acardiomyopathy," Diabetes 34:876-883 (1985). Oral
trientine caused substantive recovery of LV structure in diabetics,
with increased numbers and normalized orientation of myocytes;
return to normal of mitochondrial structure; and marked narrowing
of the extracellular space (FIG. 29g). These data suggest that
hyperglycaemia-induced systemic Cu.sup.II accumulation might
contribute to the development of mitochondrial dysfunction.
Brownlee M, "Biochemistry and molecular cell biology of diabetic
complications," Nature 414:813-820 (2001). Myocardium from
trientine-treated non-diabetics appeared normal by TEM (FIG. 29h).
Thus, trientine treatment normalized both cellular and interstitial
aspects of hyperglycaemia-induced myocardial damage. Taken
together, these microscopic studies provide remarkable evidence
that selective Cu-chelation can substantially improve LV structure,
even in the presence of severe chronic hyperglycaemia.
[0326] In sum, it was demonstrated that (1) Treatment with
trientine had no obvious effect on blood glucose concentrations in
the two diabetic groups (as expected); (2) There was a small but
significant improvement in the (heart weight)/(body weight) ratio
in the trientine-treated diabetic group compared to that of the
untreated diabetic group; (3) When the Pre-load was increased with
the After-load held constant, cardiac output was restored to Sham
values. Both the aortic and absolute coronary flows improved in the
drug treated group; (4) Indicators for ventricular contraction and
relaxation were both significantly improved in the drug treated
group compared to equivalent values in the untreated diabetic
group. The improvement restored function to such an extent that
there was no significant difference between the drug treated and
the sham-treated control groups; (5) The aortic transducer measures
of pressure change also showed improved function in the drug
treated diabetic group compared to the untreated diabetics (data
not shown); (6) When after-load was increased in the presence of
constant pre-load, it was observed that the heart's ability to
function at higher after-loads was greatly improved in the drug
treated diabetic group compared to the untreated diabetic group.
When 50% of the untreated diabetic hearts had failed, about 90% of
the trientine treated diabetic hearts were still fimctioning; (7)
Compared to the untreated diabetic hearts, the response of the drug
treated diabetic hearts showed significant improvements in several
variables: cardiac output, aortic flow, coronary flow, as well as
improved ventricular contraction and relaxation indices; (8) Drug
treatment of normal animals had no adverse effects on cardiac
performance; and, (9) Histological observations (TEM and LCM) also
showed improvement in cardiac architecture in rats following
treatment with trientine.
[0327] Treatment of STZ diabetic rats with trientine dramatically
improves several measures of cardiac function. It is also concluded
that administration of oral trientine for 7 weeks in Wistar rats
with previously established diabetes of 6 weeks duration resulted
in a global improvement in cardiac function. This improvement was
demonstrated by improved contractile function (+dP/dT) and a
reduction in ventricular stiffness (-dP/dT). The overall ability of
the trientine treated diabetic heart to tolerate increasing
after-load was also substantially improved.
EXAMPLE 7
[0328] This Example was carried out to assess the effect of chronic
trientine administration on cardiac structure and function in
diabetic and non-diabetic humans.
[0329] Methods were as follows. Human studies were approved by
institutional ethics and regulatory committees. The absorption and
excretion of trientine, and representative plasma
concentration--time profiles of trientine after oral administration
have been reported (see Miyazaki K, et al., "Determination of
trientine in plasma of patients with high-performance liquid
chromatography," Chem. Pharm. Bull. 38:1035-1038 (1990)).
[0330] Subjects (30-70 y) who provided written informed consent
were eligible for inclusion if they had:T2DM with HbA.sub.1c>7%;
cardiac ejection fraction (echocardiography).gtoreq.45% with
evidence of diastolic dysfunction but no regional wall-motion
anomalies; no new medications for more than 6 months with no change
of .beta.-blocker dose; normal electrocardiogram (sinus rhythm,
normal PR Interval, normal T wave and QRS configuration, and
isoelectric ST segment); and greater than 90% compliance with
single-blinded placebo therapy during a 2-w run-in period. Women
were required to be post-menopausal, surgically sterile, or
non-lactating and non-pregnant and using adequate contraception.
Patients were ineligible if they failed to meet the inclusion
criteria or had: morbid obesity (B.M.I..gtoreq.45 kg.m.sup.-2)T1
DM; a history of significant cardiac valvular disease; evidence of
autonomic neuropathy; ventricular wall motion abnormality; history
of multiple drug allergies; use or misuse of substances of abuse;
abnormal laboratory tests at randomisation; or standard
contraindications to MRI.
[0331] Before randomization, potentially eligible subjects entered
a 4-w single blind run-in phase of two placebo-capsules twice-daily
and underwent screening echocardiography, being excluded if
regional wall motion abnormalities or impaired LV systolic function
(ejection fraction <50%) were detected. In addition, LV
diastolic filling was assessed using mitral inflow Doppler (with
pre-load reduction) to ensure patients had abnormalities of
diastolic filling; no patient with normal mitral filling proceeded
to randomisation. Subjects meeting inclusion criteria and with no
grounds for exclusion were then randomised to receive trientine
(600 mg twice-daily) before meals (total dose 1.2 g.d.sup.-1) or 2
identical placebo capsules twice-daily before meals, in a
double-blind, parallel-group design. Treatment assignment was
performed centrally using variable block sizes to ensure balance
throughout trial recruitment and numbered drug packs were prepared
and dispensed sequentially to randomised patients. The double-blind
treatment was continued for 6 months in each subject.
[0332] At baseline and following 6 months' treatment, LV mass was
determined using cardiac MRI, performed in the supine position with
the same 1.5 T scanner (Siemens Vision) using a phased array
surface coil. Prospectively gated cardiac cine images were acquired
in 6 short axis and 3 long axis slices with the use of a segmented
k-space pulse sequence (TR 8 ms; TE 5 ms; flip angle 10.degree.;
field of view 280-350 mm) with view sharing (11-19
frames.slice.sup.-1). Each slice was obtained during a breath-hold
of 15-19 heartbeats. The short axis slices spanned the left
ventricle from apex to base with a slice thickness of 8 mm and
inter-slice gap of 2-6 mm. The long axis slices were positioned at
equal 60.degree. intervals about the long axis of the LV. Cardiac
MRI provides accurate and reproducible estimates of LV mass and
volume. LV-mass and volume were calculated using guide point
modeling, which produces precise and accurate estimations of mass
and volume. Briefly, a three dimensional mathematical model of the
LV was interactively fitted to the epicardial and endocardial
boundaries of the LV wall in each slice of the study,
simultaneously. Volume and mass were then calculated from the model
by numerical integration (mass=wall volume.times.1.05 g.ml.sup.-1).
All measurements were performed by 1 measurer at the end of six
months' data collection. Outcome analyses were conducted by
intention-to-treat, using a maximum likelihood approach to impute
missing at random data within a mixed model, and marginal
least-squares adjusted-means were determined. Changes from baseline
were compared between treatment-groups in the mixed model with
baseline values entered as covariate. Since there were only 2
groups in the main effect and no interaction effect, no post hoc
procedures were employed. In additional analysis the influence of
clinically important differences between the treatment groups at
baseline was considered by adjusting for them as covariates in an
additional model. All P values were calculated from 2-tailed tests
of statistical significance and a 5% significance level was
maintained throughout. The effect of treatment on categorical
variables was tested using the procedures of Mantel and Haenzel
(SAS v8.01, SAS Institute).
[0333] Table 7 shows baseline information on 30 patients with
long-standing type 2 diabetes, no clinical evidence of coronary
artery disease and abnormal diastolic function who participated in
a 6-month randomized, double blind, placebo controlled study of
chronic oral therapy with trientine dihydrochloride. TABLE-US-00011
TABLE 7 Characteristics of Study Participants Trientine Placebo
dihydrochloride N 15 15 Median age (years) 54 (range 43-64) 52
(range 33-69) % female 44% 56% Median duration of diabetes (years)
10 (1-24) 8 (1-21) Mean body mass index (kg/m.sup.2) 32 (5) 34 (5)
(SD) % hypertensive 64% 80% Mean % HbA.sub.1c (SD) 9.3 (1.3) 9.3
(2.0) Initial left ventricular mass (g) 202.2 (53.1) 207.5 (48.7)
(SD)
[0334] Trientine (600 mg twice-daily, a dose at the lower end of
those employed in adult Wilson's disease, see Dahlman T, et al.,
"Long-term treatment of Wilson's disease with triethylene tetramine
dihydrochloride (trientine)," Quart. J Med 88: 609-616 (1995)) or
placebo was administered orally for 6 months to equivalent groups
of diabetic adults (n=15.group.sup.-1; Table 7), also matched for
pharmacotherapy including: .beta.-blockers, calcium antagonists,
ACE-inhibitors, cholesterol-lowering drugs, antiplatelet agents and
antidiabetic drugs. LV masses were determined by tagged-molecular
resonance imaging (MRI; see Bottini P B, et al., "Magnetic
resonance imaging compared to echocardiography to assess left
ventricular mass in the hypertensive patient," Am. J. Hypertens 8:
221-228 (1995)) at baseline and following 6 months' trientine
treatment. As expected, diabetics initially had significant LVH,
consistent with previous reports. Struthers A D & Morris A D,
"Screening for and treating left-ventricular abnormalities in
diabetes mellitus: a new way of reducing cardiac deaths," Lancet
359: 1430-1432 (2002).
[0335] Results showed that Trientine treatment reverses LVH in
type-2 diabetic humans. MRI scans of the heart at baseline and
6-months showed a significant reduction in LV mass. Mean LV mass in
diabetics significantly decreased, by 5%, following 6 months'
trientine treatment, whereas that in placebo-treated subjects
increased by 3% (FIG. 33); this highly significant effect remained
after LV mass was indexed to body surface area, and occurred
without change in systolic or diastolic blood pressure (Table 8).
Thus, trientine caused powerful regression in LV mass without
altering blood pressure or urinary volume (FIG. 32). No significant
drug-related adverse events occurred during the 6 months' trientine
therapy.
[0336] Chronic trientine treatment improves cardiac structure and
function in humans TABLE-US-00012 TABLE 8 Results of Trientine
treatment Placebo Trientine-treated .DELTA. urinary copper 0.67
-0.83 (.mu.mol L.sup.-1) (-1.16 to 2.49) (-2.4 to 0.74) .DELTA.
systolic blood pressure -1.9 -3.5 (mmHg) (-10.6 to 6.8) (-9.5 to
1.8) .DELTA. diastolic blood pressure -4.5 -3.9 (mmHg) (-9.0 to
0.01) (-13.4 to 6.5) .DELTA. left ventricular mass/body +3.49
-5.56** surface area (0.63 to 7.61) (-9.64 to -1.48) (g m.sup.-2)
Differences in key treatment-variables (6 months - baseline, mean
(95% confidence interval. *, P < 0.05 vs. placebo **P < 0.01
vs. placebo).
[0337] MRI scans of the heart at baseline and 6-months showed a
significant reduction in LV mass.
[0338] In sum, trientine administration for 6 months yielded
improvements in the structure and function of the human heart.
EXAMPLE 8
[0339] This Example was carried out to assess the effect of chronic
trientine administration on urinary metal excretion in diabetic and
non-diabetic humans.
[0340] Methods were as follows. Human studies were approved by
institutional ethics and regulatory committees. We measured urinary
metal excretion in human males with T2DM or matched non-diabetic
controls, baseline information on which is shown in Table 9, in a
randomized, double blind, placebo-controlled trial. Males with
uncomplicated T2DM (Table 9) underwent 12-d elemental balance
studies in a fully residential metabolic unit. All foods and
beverages were provided. Total daily intake (method of double
diets) and excretion (urinary and fecal) of trace elements (Ca, Mg,
Zn, Fe, Cu, Mn, Mo, Cr and Se) were determined (ICP MS). Baseline
measurements were taken during the first 6 d, after which oral
trientine (2.4 g once-daily) or matched placebo was administered in
a 2.times.2 randomized double-blind protocol and metal losses
measured for a further 6 d. TABLE-US-00013 TABLE 9 Characteristics
of Study Participants Trientine Trientine Placebo treated Placebo
treated control control diabetic diabetic Median age 42 52 51 50
(years) (range (range (range (range 32-53) 30-68) 32-66) 30-64) n
10 10 10 10 Median -- -- 5.9 7.5 duration of (range 1-13) (range
1-34) diabetes (years) Fasting plasma 4.7 .+-. 0.3 5.0 .+-. 0.4
11.5 .+-. 3.8 10.8 .+-. 4.3 glucose (mmol L.sup.-1) Mean HbA.sub.10
5.4 .+-. 0.2 5.0 .+-. 0.3 9.9 .+-. 2.7 9.1 .+-. 1.6 (%) Body 24.6
.+-. 3.5 27.9 .+-. 5.2 32.9 .+-. 4.5 30.4 .+-. 3.1 mass index (kg
m.sup.-2)
(mean.+-.S.E.M. unless otherwise stated); f.b.g., HbA.sub.1c and
B.M.I. were significantly greater in diabetics and groups were
otherwise well-matched).
[0341] Results showed that urinary Cu losses are increased
following oral trientine treatment in humans with type-2 diabetes.
Urine volumes were equivalent in drug- and placebo-treated groups.
Basal 2-h Cu-losses were measured for 10 h in diabetic (n=20) and
matched control (n=20) subjects during part of day I (FIG. 32); and
daily losses were determined throughout days 1-6.
[0342] Baseline urinary Cu-excretion was significantly greater in
diabetics than controls (mean diabetic, 0.257 .mu.mol.d.sup.-1
control, 0.196; P<0.001).
[0343] Trientine- and placebo-evoked 2-h urinary Cu-excretion was
measured again in the same subjects on day 7 following oral drug
(2.4 g once-daily) or matched placebo (n=10.group-.sup.-1.
Trientine increased urinary Cu in both groups, but the excretion
rate in diabetes was greater (FIG. 30; P<0.05). There was no
corresponding increase in trientine-evoked urinary Fe excretion,
although basal concentrations in diabetes were increased relative
to control (P<0.001; results not shown). Thus, trientine
elicited similar urinary Cu responses in rats with T1DM and in
humans with T2DM. Mean trientine-evoked urinary Cu-excretion was
5.8 .mu.mol.d.sup.-1 in T2DM compared to 4.1 .mu.mol.d.sup.-1 in
non-diabetic controls, a 40% increase. This correspondence between
the two major forms of diabetes in two species suggests that
increased systemic Cu.sup.II is likely to be widely present in
diabetes.
[0344] In sun, chronic trientine administration increased urinary
copper in both diabetic and nondiabetic groups, but the excretion
rate in diabetes was greater. No corresponding increase in urinary
Fe excretion was observed with trientine. Thus, trientine elicited
similar urinary copper responses in rats with type 1 diabetes
mellitus and in humans with type 2 diabetes mellitus.
EXAMPLE 9
[0345] This Example was carried out to determine the effect of oral
trientine (triethylene tetramine dihydrochloride) administration on
fecal output of metals in diabetic and non-diabetic humans. Methods
were as follows.
[0346] Oral trientine (2.4 g once daily) or matched placebo were
administered to matched groups (n=10/group) of humans with type-2
diabetes mellitus (T2DM) or matched controls. Total metal balance
studies were performed in a residential metabolic unit Total fecal
outputs were collected daily for 12 days, freeze dried, and
analyzed by ICP-MS for content of Cu, Fe, Zn, Ca, Mg, Mn, Cr, Mb
and Se. Baseline measurements were taken during the first 6 d after
which oral trientine or matched placebo were administered in a
2.times.2 randomized double-blind protocol and metal losses
measured for a further 6 d.
[0347] Results were as follows. Mean daily fecal losses of Cu were
not significantly different between subjects before and after
administration of trientine or placebo, nor were Cu outputs
different between diabetic and control subjects. The lack of effect
of trientine on fecal Cu output was unexpected (see Table 11), and
contrasts sharply with reports from Wilson's disease, in which
trientine reportedly increased fecal Cu excretion. TABLE-US-00014
TABLE 11 Fecal copper excretion Mean Cu Losses (mg/day) Pre-Tment
Post-Tment Diab-Plac (n = 10) 1.914503965 1.937921277 Ctrl-Plac (n
= 10) 1.670142101 2.078654892 Diab-Drug (n = 10) 1.869867293
1.965342334 Ctrl-Drug (n = 10) 2.19850868 2.045467014 SEM:
Diabetic-PrePlac 0.122570307 0.178995736 SEM: Control-PrePlac
0.1765707 0.209400786 SEM: Diabetic-PreDrug 0.228263465 0.144463066
SEM: Control-PreDrug 0.209289978 0.124516832 Reference values
Ishikawa et al (2001): control -1.00 mg/d Kenzie Pam all et al
(1988): control -1.30 mg/d Kosaka H et al (2001) control 53.5
ug/d
[0348] Results of fecal output studies of other metals were
similar. Neither diabetes nor drug had measurable effects on
outputs of Zn, Fe, Ca, Mg, Mn, Cr, Mb or Se. In sum, in normal
humans and those with T2DM, trientine did not increase fecal output
of Cu or other metals. Therefore, trientine does not act in T2DM by
increasing fecal Cu output. On the other hand, our previous results
showed that trientine administration increased urinary Cu output.
Taken in aggregate, these results indicate that trientine acts to
remove Cu from the systemic compartment by increasing its loss in
the urine. Therefore, systemically active forms of trientine are
the preferred embodiment of this invention.
[0349] The human data, taken together with those in rats above,
indicate that chronic Cu chelation can cause significant
regeneration of the heart in those with diabetes-evoked damage.
Trientine largely reversed heart failure and LV damage in severely
diabetic rats. Furthermore, six months' oral trientine
administration significantly ameliorated left ventricular
hypertrophy in humans with type-2 diabetes. Rat rats and humans
with diabetes acquire increased systemic Cu.sup.II, which can be
removed by treatment with the Cu-selective chelator, trientine.
EXAMPLE 10
[0350] This Example assessed the effect of the copper chelation
efficacy of various concentrations of parenteral administration of
trientine on anaesthetized diabetic and nondiabetic male Wistar
rats through the measurement of copper in the urine.
[0351] Stock solutions of various intravenous formulations having
concentrations of trientine hydrochloride were made up in 0.9%
saline and was stored for four months at 4.degree. C. without
appreciable deterioriation in efficacy. The concentrations of the
stock formulations were: 0.67 mg/ml, 6.7 mg/ml, 67 mg/ml, and 670
mg/ml. The formulation was then administered to the rats in doses
of 0.1 mg/kg, 1 mg/kg, 10 mg/kg, and 100 mg/kg to the animals
respectively.
[0352] Six to seven weeks (mean=44.+-.1 days) after administration
of STZ, animals underwent either a control or drug experimental
protocol. All animals were fasted overnight prior to surgery but
continued to have ad libitum access to deionized water. Induction
and maintenance of surgical anesthesia was by 3-5% halothane and
21.min.sup.-1 O2. The femoral artery and vein were cannulated with
a solid-state blood pressure transducer (Mikrotip.TM. 1.4F, Millar
Instruments, Texas, USA) and a saline filled PE 50 catheter
respectively. The ureters were exposed via a midline abdominal
incision, cannulated using polyethylene catheters (external
diameter 0.9 mm, internal diameter 0.5 mm) and the wound sutured
closed. The trachea was cannulated and the animal ventilated at
70-80 breaths.min.sup.-1 with air supplemented with O2 (Pressure
Controlled Ventilator, Kent Scientific, Connecticut, USA). The
respiratory rate and end-tidal pressure (10-15 cmH2O) were adjusted
to maintain end-tidal CO2 at 35-40 mmHg (SC-300 CO2 monitor, Pryon
Corporation, Wisconsin, USA). Body temperature was maintained at
37.degree. C. throughout surgery and the experiment by a heating
pad. Estimated fluid loss was replaced with intravenous
administration of 154 mmol.1.sup.-1 NaCl solution at a rate of 5
ml.kg.sup.-1.h.sup.-1.
[0353] Mean arterial pressure (MAP), heart rate (HR, derived from
the MAP waveform) oxygen saturation (Nonin 8600V Pulse Oximeter,
Nonin Medical Inc., Minnesota, USA) and core body temperature, were
all continuously monitored throughout the experiment using a
PowerLab/16s data acquisition module (AD Instruments, Australia).
Calibrated signals were displayed on screen and saved to disc as 2
s averages of each variable.
[0354] Following surgery and a 20 min stabilization period, the
experimental protocol was started. The trientine formulation or an
equivalent volume of saline was intravenously administered hourly
in doses of increasing strength from 0.1 mg/kg, 1.0 mg/kg, 10
mg/kg, and 100 mg/kg. Urine was collected throughout the experiment
in 15 min aliquots.
[0355] Sample pretreatment was carried out as follows. Urine: Urine
was collected in pre-weighed 1.5 ml micro test tubes (eppendorf).
After reweighing, the urine specimens were centrifuged and the
supernatant diluted 25:1 with 0.02 m 69% Aristar grade HNO.sub.3.
The sample was stored at 4.degree. C. prior to GF-AAS analysis. If
it was necessary to store a sample for a period in excess of 2
weeks, it was frozen and kept at -20.degree. C. Serum: Terminal
blood samples were centrifuged and serum treated and stored as per
urine until analysis. From the trace metal content of serum from
the terminal blood sample and urine collected over the final hour
of the experiment, renal clearance was calculated using the
following equation: renal .times. .times. .times. clearance .times.
.times. of .times. .times. .times. trace .times. .times. .times.
metal .times. .times. ( .mu. .times. .times. 1. .times. .times. min
- 1 ) = concentration .times. .times. of .times. .times. .times.
metal .times. .times. in .times. .times. urine .times. .times. (
.mu.g . .times. .mu. .times. .times. 1 - 1 ) * rate .times. .times.
of .times. .times. urine .times. .times. flow .times. .times. (
.mu.1 . min - 1 ) .times. .times. concentration .times. .times. of
.times. .times. metal .times. .times. in .times. .times. serum
.times. .times. ( .mu.g . .times. .mu. .times. .times. 1 - 1 )
##EQU2##
[0356] Statistical analyses were carried out as follows. All values
are expressed as mean.+-.SEM and P values<0.05 were considered
statistically significant. Student's unpaired t-test was initially
used to test for weight and glucose differences between the
diabetic and control groups. For comparison of responses during
drug exposure, statistical analyses were performed using analysis
of variance (Statistics for Windows v.6.1, SAS Institute Inc.,
Calfornia, USA). Subsequent statistical analysis was performed
using a mixed model repeated measures ANOVA design (see Example
4).
[0357] The results were as follows. With regard to the
cardiovascular effects there were no adverse effects from the acute
injection of trientine. See FIG. 25 that shows no adverse
cardiovascular effects after the injection, although at 100 mg/kg
this gave a transient drop in blood pressure. This change was a
maximum blood pressure fall of 19.+-.4 mmHg, however the rat
recovered in 10 minutes (not shown).
[0358] In summary, acute intravenous administration of trientine in
the concentration ranges from between 0.1 mg/kg, 1 mg/kg, 10 mg/kg,
and 100 mg/kg has no significant effect on blood pressure.
Furthermore, a trientine formulation is efficacious as a copper
chelator when given intravenously and that trientine in saline
remains active as a copper chelator after storage at 4.degree. C.
for 4 months.
EXAMPLE 11
[0359] This Example assessed the stability of a trientine
formulation after being stored by its ability to chelate
copper.
[0360] A standard 100 mM solution of Trientine HCl was made up in
deionized (MilliQ) water. One sample of the solution was stored in
the dark at 4.degree. C. and 21.degree. C. in the dark and a third
sample was stored at 21.degree. C. in daylight.
[0361] The Ultraviolet-visible spectrum of the formulation was
initially measured at day 0 and then at day 15. 20 .mu.l aliquots
of sample solutions were taken at day 15. For each aliquot 960
.mu.l of 50 mM TRIS buffer and 20 .mu.l aliquot of Copper Nitrate
standard (100 mM--Orion Research Inc) were added. This was then
measured over wavelengths 700-210 nm to determine the binding
stability of the trientine formulations. See FIG. 26 that shows
that there was no detectable change in the ability of the trientine
formulation to chelate copper over this 15 day time period
irrespective of storage conditions. Furthermore room light had no
detectable detrimental effect on copper chelation and that
trientine is stable as a chelator while in solution.
[0362] All patents, publications, scientific articles, web sites,
and other documents and materials referenced or mentioned herein
are indicative of the levels of skill of those skilled in the art
to which the invention pertains, and each such referenced document
and material is hereby incorporated by reference to the same extent
as if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such patents,
publications, scientific articles, web sites, electronically
available information, and other referenced materials or
documents.
[0363] The written description portion of this patent includes all
claims. Furthermore, all claims, including all original claims as
well as all claims from any and all priority documents, are hereby
incorporated by reference in their entirety into the written
description portion of the specification, and Applicants reserve
the right to physically incorporate into the written description or
any other portion of the application, any and all such claims.
Thus, for example, under no circumstances may the patent be
interpreted as allegedly not providing a written description for a
claim on the assertion that the precise wording of the claim is not
set forth in haec verba in written description portion of the
patent.
[0364] The claims will be interpreted according to law. However,
and notwithstanding the alleged or perceived ease or difficulty of
interpreting any claim or portion thereof, under no circumstances
may any adjustment or amendment of a claim or any portion thereof
during prosecution of the application or applications leading to
this patent be interpreted as having forfeited any right to any and
all equivalents thereof that do not form a part of the prior
art.
[0365] All of the features disclosed in this specification may be
combined in any combination. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0366] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Thus, from the foregoing, it will be appreciated
that, although specific embodiments of the invention have been
described herein for the purpose of illustration, various
modifications may be made without deviating from the spirit and
scope of the invention. Other aspects, advantages, and
modifications are within the scope of the following claims and the
present invention is not limited except as by the appended
claims.
[0367] The specific methods and compositions described herein are
representative of preferred embodiments and are exemplary and not
intended as limitations on the scope of the invention. Other
objects, aspects, and embodiments will occur to those skilled in
the art upon consideration of this specification, and are
encompassed within the spirit of the invention as defined by the
scope of the claims. It will be readily apparent to one skilled in
the art that varying substitutions and modifications may be made to
the invention disclosed herein without departing from the scope and
spirit of the invention. The invention illustratively described
herein suitably may be practiced in the absence of any element or
elements, or limitation or limitations, which is not specifically
disclosed herein as essential. Thus, for example, in each instance
herein, in embodiments or examples of the present invention, the
terms "comprising", "including", "containing", etc. are to be read
expansively and without limitation. The methods and processes
illustratively described herein suitably may be practiced in
differing orders of steps, and that they are not necessarily
restricted to the orders of steps indicated herein or in the
claims.
[0368] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by various embodiments and/or preferred embodiments and optional
features, any and all modifications and variations of the concepts
herein disclosed that may be resorted to by those skilled in the
art are considered to be within the scope of this invention as
defined by the appended claims.
[0369] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0370] It is also to be understood that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise, the
term "X and/or Y" means "X" or "Y" or both "X" and "Y", and the
letter "s" following a noun designates both the plural and singular
forms of that noun. In addition, where features or aspects of the
invention are described in terms of Markush groups, those skilled
in the art will recognize that the invention is also thereby
described in terms of any individual member or subgroup of members
of the Markush group.
[0371] Other embodiments are within the following claims. Under no
circumstances may the patent be interpreted to be limited to the
specific examples or embodiments or methods specifically and/or
expressly disclosed herein. Under no circumstances may the patent
be interpreted to be limited by any statement made by any Examiner
or any other official or employee of the Patent and Trademark
Office unless such statement is specifically and without
qualification or reservation expressly adopted in a responsive
writing by Applicants.
* * * * *