U.S. patent application number 14/071705 was filed with the patent office on 2014-02-27 for use of relaxin to increase arterial compliance.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is Kirk P. Conrad, Sanjeev G. Shroff. Invention is credited to Kirk P. Conrad, Sanjeev G. Shroff.
Application Number | 20140057832 14/071705 |
Document ID | / |
Family ID | 35136707 |
Filed Date | 2014-02-27 |
United States Patent
Application |
20140057832 |
Kind Code |
A1 |
Conrad; Kirk P. ; et
al. |
February 27, 2014 |
USE OF RELAXIN TO INCREASE ARTERIAL COMPLIANCE
Abstract
The present invention provides methods for increasing arterial
compliance. The methods generally involve administering to an
individual in need thereof an effective amount of relaxin. The
present invention further provides methods of increasing arterial
compliance in individuals who have Type 1 or Type 2 diabetes. The
present invention further provides methods of increasing arterial
compliance in perimenopausal, menopausal, and post-menopausal
women. The present invention further provides methods of increasing
arterial compliance in individuals who have or who are at risk of
developing age-associated arterial stiffness.
Inventors: |
Conrad; Kirk P.;
(Gainesville, FL) ; Shroff; Sanjeev G.;
(Pittsburg, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Conrad; Kirk P.
Shroff; Sanjeev G. |
Gainesville
Pittsburg |
FL
PA |
US
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
35136707 |
Appl. No.: |
14/071705 |
Filed: |
November 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12985714 |
Jan 6, 2011 |
8602998 |
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14071705 |
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11084670 |
Mar 18, 2005 |
7878978 |
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12985714 |
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Current U.S.
Class: |
514/1.9 ;
514/12.7; 514/6.9; 514/7.3; 514/7.4 |
Current CPC
Class: |
A61B 5/02007 20130101;
A61K 38/2221 20130101 |
Class at
Publication: |
514/1.9 ;
514/12.7; 514/7.3; 514/6.9; 514/7.4 |
International
Class: |
A61K 38/22 20060101
A61K038/22 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The US, government may have certain rights in this
invention, pursuant to Grant No. ROI HL67937 awarded by the
National Institutes of Health.
Claims
1. A method of treating a subject having a disease selected from
one or more of atherosclerosis, Type 1 diabetes, Type 2 diabetes,
coronary artery disease, scleroderma, stroke, diastolic
dysfunction, hypercholesterolemia, familial hypercholesterolemia,
isolated systolic hypertension, primary hypertension, secondary
hypertension, left ventricular hypertrophy, arterial stiffness
associated with long-term tobacco smoking, arterial stiffness
associated with obesity, arterial stiffness associated with age and
systemic lupus erythematosis comprising measuring arterial
compliance in the subject; determining that the arterial compliance
is diminished in the subject relative to arterial compliance in a
healthy subject; and administering a pharmaceutical formulation
comprising H2 relaxin to the subject, wherein the administration of
H2 relaxin increases arterial compliance and ameliorates the
disease.
2. The method of claim 1, wherein the arterial compliance is global
arterial compliance.
3. The method of claim 1, wherein the arterial compliance is
regional arterial compliance.
4. The method of claim 1, wherein the arterial compliance is local
arterial compliance.
5. The method of claim 1, wherein the arterial compliance is
increased by at least 10% following administration of the
pharmaceutical formulation.
6. The method of claim 5, wherein the arterial compliance is
increased by 15-20% following administration of the pharmaceutical
formulation.
7. The method of claim 1, wherein the pharmaceutical formulation is
administered parenterally.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
12/985,714, filed on Jan. 6, 2011, that is a continuation
application of application Ser. No. 11/084,670, filed Mar. 18,
2005, which claims benefit of U.S. Provisional Application No.
60/554,716, filed Mar. 19, 2004, the teachings of which are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0003] The present invention is in the field of arterial
compliance, and in particular, the use of relaxin to increase
arterial compliance.
BACKGROUND OF THE INVENTION
[0004] Arterial compliance declines with age even in healthy
individuals with no overt cardiovascular disease. With age, a
decrease is seen in the ability of the large and small arteries to
distend in response to an increase in pressure. The age-associated
reduction in arterial compliance is an independent risk factor for
the development of cardiovascular disease, and is associated with a
number of other pathological conditions. For example, reduced
arterial compliance is also associated with both Type 1 diabetes
mellitus and Type 2 diabetes melitus. It has been reported that
diabetic arteries appear to age at an accelerated rate compared to
arteries of nondiabetic individuals. See, e.g., Arnett et al.
(1994) Am J Epidemiol. 140:669-682; Rowe (1987) Am J Cardid. 60:68
G-71 G; Cameron et al. (2003) Diabetes Car-26(7):2133-8; Kass et
al. (2001) Circulation 104; 1464-1470; Avolio of al. (1983)
Circulation 68; 50-58; U.S. Pat. No. 6,251,863; U.S. Pat. No.
6,211,147.
[0005] There is a need in the art for methods of increasing
arterial compliance, and for treating disorders associated with or
resulting from reduced arterial compliance. The present invention
addresses these needs.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods of treating
individuals with diminished arterial compliance an effective amount
of a formulation comprising a relaxin receptor agonist. In a
preferred embodiment the relaxin receptor agonist is a recombinant
human relaxin, e.g., human H2 relaxin.
[0007] In one embodiment of the invention, the invention provides a
method of increasing arterial compliance in a subject, wherein said
method comprises measuring global arterial compliance in said
subject; determining that said global arterial compliance is
diminished in said subject relative to global arterial compliance
in a healthy subject; and administering to said subject a
pharmaceutical formulation comprising relaxin to increase arterial
compliance in said subject. Global arterial compliance may be
measured, in one embodiment, from the diastolic decay of the aortic
pressure waveform using the area method. In another embodiment,
global arterial compliance may be calculated as the stroke
volume-to-pulse pressure ratio, where the stroke volume is defined
as the ratio of cardiac output to heart rate.
[0008] In related embodiments, the local arterial compliance or the
regional arterial compliance of a subject may be measured in
addition to or as an alternative to the global arterial compliance
measurement and, if the local or regional arterial compliance is
diminished relative to the local or regional arterial compliance
expected for a similarly situated healthy individual, relaxin may
be administered to increase arterial compliance in that
individual.
[0009] In further embodiments, the subject to whom relaxin is
administered suffers from one or more of the following disorders:
atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary artery
disease, scleroderma, stroke, diastolic dysfunction, familial
hypercholesterolemia, isolated systolic hypertension, primary
hypertension, secondary hypertension, left ventricular hypertrophy,
arterial stiffness associated with long-term tobacco smoking,
arterial stiffness associated with obesity, arterial stiffness
associated with age, systemic lupus erythematosus, preeclampsia,
and hypercholesterolemia. In related embodiments, the invention
provides methods of increasing arterial compliance in
perimenopausal, menopausal, and post-menopausal women and in
individuals who are at risk of one of the aforementioned
disorders.
[0010] In an additional embodiment of the invention, administration
of relaxin increases arterial compliance by at least 10%, 15%, 20%
or more, relative to the measured arterial compliance before
administration. In still further embodiments, the invention provide
for the administration of relaxin to individuals with diminished
arterial compliance at a predetermined rate so as to maintain a
serum concentration of relaxin from 0.5 to 80 ng/ml. In one
embodiment, the relaxin is recombinant human relaxin. In yet
another embodiment, the relaxin is recombinant H2 relaxin. In
related embodiments, the relaxin may be administered daily, in an
injectable formulation, as a sustained release formulation, or as a
continuous infusion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-C depict the percent change from baseline for
cardiac output (FIG. 1A), heart rate (FIG. 1B), and stroke volume
(FIG. 1C) in female rats administered low dose recombinant human
relaxin (rhRLX; 4 .mu.g/h), high dose rhRLX (25 .mu.g/h), or
vehicle.
[0012] FIGS. 2A-D depict percent change from baseline for systemic
vascular resistance (FIG. 2A), mean arterial pressure (FIG. 2B),
global arterial compliance (FIG. 2C), and ratio of stroke
volume-to-pulse pressure in female rats administered low dose
rhRLX, high dose rhRLX, or vehicle.
[0013] FIGS. 3A and 3B depict representative arterial pressure
tracings from one rat (FIG. 3A); and ensemble average arterial
pressure waveforms for the three groups (vehicle, low dose rhRLX,
and high dose rhRLX) at day 10 after implantation of the osmotic
minipump (FIG. 3B).
[0014] FIGS. 4A and 4B depict circumferential stress
(.theta.)-midwall radius (R.sub.m) (FIG. 4A); and incremental
elastic modulus (E.sub.inc)-R.sub.m (FIG. 4B) relationships for
small renal arteries isolated from rats treated with rhRLX or
vehicle for 5 days.
[0015] FIG. 5 shows temporal changes in systemic hemodynamics in
response to low dose (4 .mu.g/h) recombinant human relaxin
administration in male and female rats. Heart rate (A), stroke
volume (B), cardiac output (C), and mean arterial pressure (D) data
are presented as percentages of baseline. * P<0.05 vs. baseline
(post-hoc Fisher's LSD). Significant increments in SV are shown
only for days 6, 8 and 10.
[0016] FIG. 6 depicts temporal changes in systemic arterial
properties in response to low dose (4 .mu.g/h) recombinant human
relaxin administration in male and female rats. Systemic vascular
resistance (A) and two measures of global arterial compliance,
AC.sub.area (B) and SV/PP (C), data are presented as percentages of
baseline. * P<0.05 vs. baseline (post-hoc Fisher's LSD).
Significant increments in AC.sub.area and SV/PP are shown only for
days 8 and 10.
[0017] FIG. 7 depicts temporal changes in systemic hemodynamics in
response to three doses of recombinant human relaxin administration
in female rats: low (4 .mu.g/h), medium (25 .mu.g/h), and high (50
.mu.g/h). Heart rate (A), stroke volume (B), cardiac output (C),
and mean arterial pressure (D) data are presented as percentages of
baseline. * P<0.05 vs. baseline (post-hoc Fisher's LSD).
[0018] FIG. 8 depicts temporal changes in systemic hemodynamics in
response to three doses of recombinant human relaxin administration
in female rats: low (4 .mu.g/h), medium (25 .mu.g/h), and high (50
.mu.g/h). Systemic vascular resistance (A) and two measures of
global arterial compliance, AC.sub.area (B) and SV/PP (C), data are
presented as percentages of baseline. * P<0.05 vs. baseline
(post-hoc Fisher's LSD).
[0019] FIG. 9 depicts temporal changes in systemic hemodynamics in
response to short-term high dose recombinant human relaxin
administration in female rats. Heart rate (A), stroke volume (B),
cardiac output (C), and mean arterial pressure (D) data are
presented as percentages of baseline. * P<0.05 vs. baseline
(post-hoc Fisher's LSD).
[0020] FIG. 10 depicts temporal changes in systemic arterial
properties in response to short-term high dose recombinant human
relaxin administration in female rats. Systemic vascular resistance
(A) and two measures of global arterial compliance, AC.sub.area (B)
and SV/PP (C), data are presented as percentages of baseline.
[0021] FIG. 11 depicts relationships between composite percentage
changes from baseline and baseline values for systemic vascular
resistance (A) and two measures of global arterial compliance,
AC.sub.area (B) and SV/PP (C), in male and female rats administered
low dose recombinant human relaxin (4 .mu.g/h). These relationships
were gender-independent. The solid line in each panel corresponds
to the plot of the relationship obtained by linear regression (male
and female rats combined).
DEFINITIONS
[0022] The terms "subject," "host," "individual," and "patient,"
used interchangeably herein, refer to any subject, particularly a
mammalian subject, for whom diagnosis or therapy is desired,
particularly humans. Other subjects may include cattle, dogs, cats,
guinea pigs, rabbits, rats, mice, horses, and so on. In many
embodiments, a subject is a human in need of treatment for a
disease or condition related to or resulting from reduced arterial
compliance.
[0023] The terms "treatment," "treating," "therapy," and the like
are used herein to generally refer to obtaining a desired
therapeutic, pharmacologic or physiologic effect. The effect may be
prophylactic in terms of completely or partially preventing a
disease or symptom thereof and/or may be therapeutic in terms of a
partial or complete cure for a disease and/or adverse effect
attributable to the disease. "Treatment" as used herein covers any
treatment of a disease in a mammal, e.g. a human, and includes: (a)
preventing the disease from occurring in a subject which may be
predisposed to the disease but has not yet been diagnosed as having
it; (b) inhibiting the disease, i.e., arresting its development;
and (c) relieving the disease, i.e., causing regression of the
disease.
[0024] As used herein the terms "isolated" and "substantially
purified," used interchangeably herein, when used in the context of
"isolated relaxin," refer to a relaxin polypeptide that is in an
environment different from that in which the relaxin polypeptide
naturally occurs. As used herein, the term "substantially purified"
refers to a relaxin polypeptide that is removed from its natural
environment and is at least 60% free, preferably 75% free, and most
preferably 90% free from other components with which it is
naturally associated.
[0025] Before the present invention is further described, it is to
be understood that this invention is not limited to the particular
embodiments described. The scope of the present invention will be
limited only by the appended claims.
[0026] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0028] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a relaxin formulation" includes a plurality
of such formulations and reference to "the active agent" includes
reference to one or more active agents and equivalents thereof
known to those skilled in the art, and so forth.
[0029] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The present invention provides methods of treating disorders
associated with arterial stiffness; methods of increasing arterial
compliance; methods of reducing arterial stiffness in an
individual; and methods of reducing the risk that an individual
will develop one or more complications or disorders associated with
reduced arterial compliance. The methods generally involve
administering to an individual in need thereof an effective amount
of a relaxin receptor agonist. In some embodiments, the individual
has, or is at risk of developing, age-related arterial stiffness.
In other embodiments, the individual has Type 1 or Type 2 diabetes,
and thus has developed, or is at risk of developing, arterial
stiffness. In other embodiments, the individual is a perimenopausal
woman, a menopausal woman, or a post-menopausal woman, and thus has
developed, or is at risk of developing, arterial stiffness. In
still other embodiments, the individual is a women who has or is at
risk of developing preeclampsia.
[0031] One of the major cardiovascular adaptations in human
pregnancy is the increase in global arterial compliance,
accompanied by increases in relaxin levels, which reaches a peak by
the end of the first trimester just as systemic vascular resistance
(SVR) reaches a nadir. At least in theory, the rise in global
arterial compliance is critical to the maintenance of
cardiovascular homeostasis during pregnancy for several reasons:
(1) the rise in global AC prevents excessive decline in diastolic
pressure which otherwise would fall to precariously low levels due
to the significant decline in SVR; (2) the rise minimizes the
pulsatile or oscillatory work wasted by the heart which otherwise
would increase in disproportion to the rise in total work required
of and expended by the heart during pregnancy; and (3) the rise in
global AC preserves steady shear-type (or prevents oscillatory
shear-type) stress at the blood-endothelial interface despite the
hyperdynamic circulation of pregnancy, thereby favoring production
of nitric oxide rather than superoxide and other damaging reactive
oxygen species by the endothelium. The increase in global AC, along
with the reduction in SVR, can result in circulatory underfilling,
and thus, contribute to renal sodium and water retention and plasma
volume expansion during early pregnancy.
Treatment Methods
[0032] The present invention provides methods for increasing
arterial compliance which utilize the step of administering to an
individual in need thereof an effective amount of a relaxin
receptor agonist. In some embodiments, the individual has
atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary artery
disease, scleroderma, stroke, diastolic dysfunction, familial
hypercholesterolemia, isolated systolic hypertension, primary
hypertension, secondary hypertension, left ventricular hypertrophy,
arterial stiffness associated with long-term tobacco smoking,
arterial stiffness associated with obesity, arterial stiffness
associated with age, systemic lupus erythematosus, preeclampsia,
and hypercholesterolemia, or is at risk of developing age-related
arterial stiffness. In other embodiments, the individual is a
perimenopausal woman, a menopausal woman, or a post-menopausal
woman, or a woman who has ceased menstruation for non-age-related
reasons, e.g., due to excessive exercise or as a result of surgery
(e.g., hysterectomy, oophorectomy), and has developed, or is at
risk of developing, arterial stiffness.
[0033] The methods generally involve administering to an individual
an effective amount of relaxin. In some embodiments, an effective
amount of relaxin is an amount that is effective to increase
arterial compliance by at least about 5%, at least about 10%, at
least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about 35%, at least about 40%, at least about
45%, or at least about 50%, or more, compared to the arterial
compliance in the absence of treatment with relaxin.
[0034] In some embodiments, an effective amount of relaxin is an
amount that is effective to reduce arterial stiffness by at least
about 5%, at least about 10%, at least about 15%, at least about
20%, at least about 25%, at least about 30%, at least about 35%, at
least about 40%, at least about 45%, or at least about 50%, or
more, compared to the arterial stiffness in the individual in the
absence of treatment with relaxin.
[0035] In some embodiments, an effective amount of relaxin is an
amount that is effective to increase arterial elasticity by at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 45%, or at least about 50%,
or more, compared to the arterial elasticity in the individual in
the absence of treatment with relaxin.
[0036] Disorders resulting from or associated with arterial
stiffness or reduced arterial compliance include, but are not
limited to, atherosclerosis, Type 1 diabetes, Type 2 diabetes,
coronary artery disease, scleroderma, stroke, diastolic
dysfunction, familial hypercholesterolemia, isolated systolic
hypertension, primary hypertension, secondary hypertension, left
ventricular hypertrophy, arterial stiffness associated with
long-term tobacco smoking, arterial stiffness associated with
obesity, arterial stiffness associated with age, systemic lupus
erythematosus, preeclampsia, and hypercholesterolemia. Of
particular interest in some embodiments, is arterial stiffness
associated with Type 1 diabetes, Type 2 diabetes, normal aging,
stroke, diastolic dysfunction, menopause, obesity,
hypercholesterolemia, familial hypercholesterolemia, isolated
systolic hypertension, long-term tobacco smoking, and left
ventricular hypertrophy.
[0037] An increase in arterial compliance, or a reduction in
arterial stiffness, reduces the risk that an individual will
develop a pathological condition resulting from reduced arterial
compliance.
[0038] In some embodiments, an effective amount of relaxin is an
amount that is effective to reduce the risk that an individual will
develop a pathological condition associated with or resulting from
reduced arterial compliance by at least about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at
least about 30%, at least about 35%, at least about 40%, at least
about 45%, or at least about 50%, or more, compared to the risk of
developing the condition in the absence of treatment with
relaxin.
[0039] In general, as discussed above, an effective amount of
relaxin is one that is effective to increase arterial compliance.
The term "increase" is used interchangeably herein with "stimulate"
and "promote." The Examples provide general guidance for effective
amounts used in rats. Those skilled in the art will readily be able
to determine effective amounts for use in human subjects, given the
guidance in the Examples. In general, a dose of relaxin is from
about 0.1 to 500 .mu.g/kg of body weight per day, about 6.0 to 200
.mu.g/kg of body weight per day, or about 1.0 to 100 .mu.g/kg of
body weight per day. For administration to a 70 kg person, the
dosage range would be about 7.0 .mu.g to 3.5 mg per day, about 42.0
.mu.g to 2.1 mg per day, or about 84.0 to 700 .mu.g per day. In
some embodiments, for administration to a human, an effective dose
is from about 5 .mu.g/kg body weight/day to about 50 .mu.g/kg body
weight/day, or from about 10 .mu.g/kg body weight/day to about 25
.mu.g/kg body weight/day. The amount of relaxin administered will,
of course, be dependent on the size, sex and weight of the subject
and the severity of the disease or condition, the manner and
schedule of administration, the likelihood of recurrence of the
disease, and the judgment of the prescribing physician. In each
case the daily dose may be administered over a period of time,
rather than as a single bolus, depending on the effect desired and
differences in individual circumstances.
[0040] In some embodiments, relaxin is administered to the
individual at a predetermined rate so as to maintain a serum
concentration of relaxin of from about 0.01 ng/ml to about 80
ng/ml, e.g., from about 0.01 ng/ml to about 0.05 ng/ml, from about
0.05 ng/ml to about 0.1 ng/ml, from about 0.1 ng/ml to about 0.25
ng/ml, from about 0.25 ng/ml to about 0.5 ng/ml, from about 0.5
ng/ml to about 1.0 ng/ml, from about 1.0 ng/ml to about 5 ng/ml,
from about 5 ng/ml to about 10 ng/ml, from about 10 ng/ml to about
15 ng/ml, from about 15 ng/ml to about 20 ng/ml, from about 20
ng/ml to about 25 ng/ml, from about 25 ng/ml to about 30 ng/ml,
from about 30 ng/ml to about 35 ng/ml, from about 35 ng/ml to about
40 ng/ml, from about 40 ng/ml to about 45 ng/ml, from about 45
ng/ml to about 50 ng/ml, from about 50 ng/ml to about 60 ng/ml,
from about 60 ng/ml to about 70 ng/ml, or from about 70 ng/ml to
about 80 ng/ml.
[0041] Determining Effectiveness
[0042] Whether a given relaxin formulation, or a given dosage of
relaxin is effective in increasing arterial compliance, reducing
arterial stiffness, or increasing arterial elasticity, can be
determined using any known method. Arterial stiffness may be
measured by several methods known to those of skill in the art,
including the methods discussed in the Examples.
[0043] One measure of global arterial compliance is the AC.sub.area
value, which is calculated from the diastolic decay of the aortic
pressure waveform [P(t)] using the area method (Liu et al. (1986)
Am. J. Physiol. 251:H588-H600), as described in the Example, infra.
Another measure of global arterial compliance is calculated as the
stroke volume to pulse pressure ratio (Chemla et al. (1998) Am. J.
Physiol. 274:H500-H505), as described in the Example, infra.
[0044] Local arterial compliance may be determined by measuring the
elasticity of an arterial wall at particular point using invasive
or non-invasive means. See, e.g., U.S. Pat. No. 6,267,728. Regional
compliance, which describes compliance in an arterial segment, can
be calculated from arterial volume and distensibility, and is
mainly measured with the use of pulse wave velocity. See, e.g.,
Ogawa et al., Cardiovascular Diabetology (2003) 2:10; Safar et al.,
Arch Mal Coer (2002) 95:1215-18. Other suitable methods of
measuring arterial compliance are described in the literature, and
any known method can be used. See, e.g., Cohn, J. N., "Evaluation
of Arterial Compliance", In: Hypertension Primer, Izzo, J. L. and
Black, H. R., (eds.), Pub. by Council on High Blood Pressure
Research, American Heart Association, pp. 252-253, (1993);
Finkelstein, S. M., et al., "First and Third-Order Models for
Determining Arterial Compliance", Journal of Hypertension, 10
(Suppl. 6,) S11-S14, (1992); Haidet, G. C., et al., "Effects of
Aging on Arterial Compliance in the Beagle", Clinical Research, 40,
266A, (1992); McVeigh, G. E., et al., "Assessment of Arterial
Compliance in Hypertension", Current Opinion in Nephrology and
Hypertension, 2, 82-86, (1993).
[0045] Relaxin Receptor Agonists
[0046] The instant methods involve administration of formulations
comprising a pharmaceutically active relaxin receptor agonist. As
used herein, the terms "relaxin receptor agonist" and "relaxin" are
used interchangeably to refer to biologically active (also referred
to herein as "pharmaceutically active") relaxin polypeptides from
recombinant or native (e.g., naturally occurring) sources; relaxin
polypeptide variants, such as amino acid sequence variants;
synthetic relaxin polypeptides; and non-peptide relaxin receptor
agonists, e.g., a relaxin mimetic.
[0047] Naturally occurring biologically active relaxin may be
derived from human, murine (i.e., rat or mouse), porcine, or other
mammalian sources. The term "relaxin" encompasses human H1
preprorelaxin, prorelaxin, and relaxin; H2 preprorelaxin,
prorelaxin, and relaxin; recombinant human relaxin (rhRLX); and H3
preprorelaxin, prorelaxin, and relaxin. H3 relaxin has been
described in the art. See, e.g., Sudo et al. (2003) J Biol Chem. 7;
278(10):7855-62. The amino acid sequences of human relaxin are
described in the art. For example, human relaxin amino acid
sequences are found under the following GenBank Accession Nos.:
Q3WXF3, human H3 prorelaxin; P04808, human H1 prorelaxin;
NP.sub.--604390 and NP.sub.--005050, human H2 prorelaxin; AAH05956,
human relaxin 1 preproprotein; NP.sub.--008842, human H1
preprorelaxin; etc. The term "relaxin receptor agonist" includes a
human relaxin derived from any one of the aforementioned
sequences.
[0048] The term "relaxin receptor agonist" also encompasses a
relaxin polypeptide comprising A and B chains having N- and/or
C-terminal truncations. For example, in H2 relaxin, the A chain can
be varied from A(1-24) to A(10-24) and B chain from B(.sup.-1-33)
to B(10-22); and in H1 relaxin, the A chain can be varied from
A(1-24) to A(10-24) and B chain from B(1-32) to B(10-22).
[0049] Also included within the scope of the term "relaxin receptor
agonist" are relaxin polypeptides comprising insertions,
substitutions, or deletions of one or more amino acid residues,
glycosylation variants, unglycosylated relaxin, organic and
inorganic salts, covalently modified derivatives of relaxin,
preprorelaxin, and prorelaxin. Also encompassed in the term is a
relaxin analog having an amino acid sequence which differs from a
wild-type (e.g., naturally-occurring) sequence, including, but not
limited to, relaxin analogs disclosed in U.S. Pat. No. 5,811,395,
and U.S. Pat. No. 6,200,953. Other suitable relaxins and relaxin
formulations are found in U.S. Pat. No. 5,945,402. Also encompassed
is a relaxin polypeptide modified to increase in vivo half life,
e.g., PEGylated relaxin (i.e., relaxin conjugated to a polyethylene
glycol), and the like.
[0050] Possible modifications to relaxin polypeptide amino acid
residues include the acetylation, formylation or similar protection
of free amino groups, including the N-terminal, amidation of
C-terminal groups, or the formation of esters of hydroxyl or
carboxylic groups, e.g., modification of the tryptophan (Trp)
residue at B2 by addition of a formyl group. The formyl group is a
typical example of a readily-removable protecting group. Other
possible modifications include replacement of one or more of the
natural amino-acids in the B and/or A chains with a different amino
acid (including the D-form of a natural amino-acid), including, but
not limited to, replacement of the Met moiety at B24 with
norleucine (Nle), valine (Val), alanine (Ala), glycine (Gly),
serine (Ser), or homoserine (HomoSer). Other possible modifications
include the deletion of a natural amino acid from the chain or the
addition of one or more extra amino acids to the chain. Additional
modifications include amino acid substitutions at the B/C and C/A
junctions of prorelaxin, which modifications facilitate cleavage of
the C chain from prorelaxin; and variant relaxin comprising a
non-naturally occurring C peptide, e.g., as described in U.S. Pat.
No. 5,759,807.
[0051] Also encompassed by the term "relaxin receptor agonist" are
fusion polypeptides comprising a relaxin polypeptide and a
heterologous polypeptide. A heterologous polypeptide (e.g., a
non-relaxin polypeptide) fusion partner may be C-terminal or
N-terminal to the relaxin portion of the fusion protein.
Heterologous polypeptides include immunologically detectable
polypeptides (e.g., "epitope tags"); polypeptides capable of
generating a detectable signal (e.g., green fluorescent protein,
enzymes such as alkaline phosphatase, and others known in the art);
therapeutic polypeptides, including, but not limited to, cytokines,
chemokines, and growth factors.
[0052] All such variations or alterations in the structure of the
relaxin molecule resulting in variants are included within the
scope of this invention so long as the functional (biological)
activity of the relaxin is maintained. In general, any modification
of relaxin amino acid sequence or structure is one that does not
increase its immunogenicity in the individual being treated with
the relaxin variant. Those variants of relaxin having the described
functional activity can be readily identified using the methods
discussed herein.
Relaxin Formulations
[0053] Relaxin formulations suitable for use in the methods of the
invention are pharmaceutical formulations comprising a
therapeutically effective amount of pharmaceutically active
relaxin, and a pharmaceutically acceptable excipient. The
formulation is in some embodiments injectable and in some
embodiments designed for intravenous injection.
[0054] Any known relaxin formulation can be used in the methods of
the present invention, provided that the relaxin is
pharmaceutically active. "Pharmaceutically active" relaxin is a
form of relaxin which results in increased arterial compliance when
administered to an individual.
[0055] Relaxin may be administered as a polypeptide, or as a
polynucleotide comprising a sequence which encodes relaxin. Relaxin
suitable for use in the methods of the present invention can be
isolated from natural sources, may be chemically or enzymatically
synthesized, or produced using standard recombinant techniques
known in the art. Examples of methods of making recombinant relaxin
are found in various publications, including, e.g., U.S. Pat. Nos.
4,835,251; 5,326,694; 5,320,953; 5,464,756; and 5,759,807.
[0056] Relaxin suitable for use includes, but is not limited to,
human relaxin, recombinant human relaxin, relaxin derived from
non-human mammals, such as porcine relaxin, and any of a variety of
variants of relaxin known in the art. Relaxin, pharmaceutically
active relaxin variants, and pharmaceutical formulations comprising
relaxin are well known in the art. See, e.g., U.S. Pat. Nos.
5,451,572; 5,811,395; 5,945,402; 5,166,191; and 5,759,807, the
contents of which are incorporated by reference in their entirety
for their teachings relating to relaxin formulations, and for
teachings relating to production of relaxin. In the Examples
described herein, recombinant human relaxin (rhRLX) is identical in
amino acid sequence to the naturally occurring product of the human
H2 gene, consisting of an A chain of 24 amino acids and a B chain
of 29 amino acids.
[0057] Relaxin can be administered to an individual in the form of
a polynucleotide comprising a nucleotide sequence which encodes
relaxin. Relaxin-encoding nucleotide sequences are known in the
art, any of which can be used in the methods described herein. See,
e.g. GenBank Accession Nos. AF135824; AF076971; NM.sub.--006911;
and NM.sub.--005059. The relaxin polynucleotides and polypeptides
of the present invention can be introduced into a cell by a gene
delivery vehicle. The gene delivery vehicle may be of viral or
non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994)
1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly
(1995) Human Gene Therapy 1:185-193; and Kaplitt (1994) Nature
Genetics 6:148-153). Gene therapy vehicles for delivery of
constructs including a coding sequence of a polynucleotide of the
invention can be administered either locally or systemically. These
constructs can utilize viral or non-viral vector approaches.
Expression of such coding sequences can be induced using endogenous
mammalian or heterologous promoters. Expression of the coding
sequence can be either constitutive or regulated.
[0058] The present invention can employ recombinant retroviruses
which are constructed to carry or express a selected nucleic acid
molecule of interest. Retrovirus vectors that can be employed
include those described in EP 415 731; WO 90/07936; WO 94/03622; WO
93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO
93/10218; Vile and Hart (1993) Cancer Res. 53:3860-3864; Vile and
Hart (1993) Cancer Res. 53:962-967; Ram et al. (1993) Cancer Res.
53:83-88; Takamiya et al. (1992) J. Neurosci. Res. 33:493-503; Baba
et al. (1993) J. Neurosurg. 79:729-735; U.S. Pat. No. 4,777,127;
and EP 345,242.
[0059] Packaging cell lines suitable for use with the
above-described retroviral vector constructs may be readily
prepared (see PCT publications WO 95/30763 and WO 92/05266), and
used to create producer cell lines (also termed vector cell lines)
for the production of recombinant vector particles. Packaging cell
lines are made from human (such as HT1080 cells) or mink parent
cell lines, thereby allowing production of recombinant retroviruses
that can survive inactivation in human serum.
[0060] Gene delivery vehicles can also employ parvovirus such as
adeno-associated virus (AAV) vectors. Representative examples
include the AAV vectors disclosed by Srivastava in WO 93/09239,
Samulski et al. (1989) J. Vir. 63:3822-3828; Mendelson et al.
(1988) Virol. 166:154-165; and Flotte et al. (1993) Proc. Natl.
Acad. Sci. USA 90:10613-10617.
[0061] Also of interest are adenoviral vectors, e.g., those
described by Berkner, Biotechniques (1988) 6:616-627; Rosenfeld et
al. (1991) Science 252:431-434; WO 93/19191; Kolls et al. (1994)
Proc. Natl. Acad. Sci. USA 91:215-219; Kass-Eisler et al. (1993)
Proc. Natl. Acad. Sci. USA 90:11498-11502; WO 94/12649, WO
93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO
95/00655.
[0062] Other gene delivery vehicles and methods may be employed,
including polycationic condensed DNA linked or unlinked to killed
adenovirus alone, for example Curiel (1992) Hum. Gene Ther.
3:147-154; ligand linked DNA, for example see Wu (1989) J. Biol.
Chem. 264:16985-16987; eukaryotic cell delivery vehicles cells;
deposition of photopolymerized hydrogel materials; hand-held gene
transfer particle gun, as described in U.S. Pat. No. 5,149,655;
ionizing radiation as described in U.S. Pat. No. 5,206,152 and in
WO 92/11033; nucleic charge neutralization or fusion with cell
membranes. Additional approaches are described in Philip (1994)
Mol. Cell Biol. 14:2411-2418, and in Woffendin (1994) Proc. Natl.
Acad. Sci. 91:1581-1585.
[0063] Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in WO 90/11092 and U.S. Pat. No.
5,580,859. Uptake efficiency may be improved using biodegradable
latex beads. DNA coated latex beads are efficiently transported
into cells after endocytosis initiation by the beads. The method
may be improved further by treatment of the beads to increase
hydrophobicity and thereby facilitate disruption of the endosome
and release of the DNA into the cytoplasm. Liposomes that can act
as gene delivery vehicles are described in U.S. Pat. No. 5,422,120;
PCT Nos. WO 95/13796, WO 94/23697, and WO 91/14445; and EP No. 524
968.
[0064] Further non-viral delivery suitable for use includes
mechanical delivery systems such as the approach described in
Woffendin et al. (1994) Proc. Natl. Acad. Sci. USA 91:11581-11585.
Moreover, the coding sequence and the product of expression of such
can be delivered through deposition of photopolymerized hydrogel
materials. Other conventional methods for gene delivery that can be
used for delivery of the coding sequence include, for example, use
of hand-held gene transfer particle gun, as described in U.S. Pat.
No. 5,149,655; use of ionizing radiation for activating transferred
gene, as described in U.S. Pat. No. 5,206,152 and PCT No. WO
92/11033.
[0065] In employing relaxin for increasing arterial compliance, any
pharmaceutically acceptable mode of administration can be used.
Relaxin can be administered either alone or in combination with
other pharmaceutically acceptable excipients, including solid,
semi-solid, liquid or aerosol dosage forms, such as, for example,
tablets, capsules, powders, liquids, gels, suspensions,
suppositories, aerosols or the like. Relaxin can also be
administered in sustained or controlled release dosage forms (e.g.,
employing a slow release bioerodable delivery system), including
depot injections, osmotic pumps (such as the Alzet implant made by
Alza), pills, transdermal and transcutaneous (including
electrotransport) patches, and the like, for prolonged
administration at a predetermined rate, preferably in unit dosage
forms suitable for single administration of precise dosages.
[0066] In some embodiments, relaxin is delivered using an
implantable drug delivery system, e.g., a system that is
programmable to provide for administration of relaxin. Exemplary
programmable, implantable systems include implantable infusion
pumps. Exemplary implantable infusion pumps, or devices useful in
connection with such pumps, are described in, for example, U.S.
Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328;
6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954. A
further exemplary device that can be adapted for use in the present
invention is the Synchromed infusion pump (Medtronic).
[0067] The compositions will typically include a conventional
pharmaceutical carrier or excipient and relaxin. In addition, these
compositions may include other active agents, carriers, adjuvants,
etc. Generally, depending on the intended mode of administration,
the pharmaceutically acceptable composition will contain about 0.1%
to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of
relaxin, the remainder being suitable pharmaceutical excipients,
carriers, etc. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this art; for
example, see Remington's Pharmaceutical Sciences, Mack Publishing
Company, Easton, Pa., 15th Edition, 1995, or latest edition. The
formulations of human relaxin described in U.S. Pat. No. 5,451,572,
are non-limiting examples of suitable formulations which can be
used in the methods of the present invention.
[0068] Parenteral administration is generally characterized by
injection, either subcutaneously, intradermally, intramuscularly or
intravenously, or subcutaneously. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol or the like. In
addition, if desired, the pharmaceutical compositions to be
administered may also contain minor amounts of non-toxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents, solubility enhancers, and the like, such as for example,
sodium acetate, sorbitan monolaurate, triethanolamine oleate,
cyclodextrins, and the like.
[0069] The percentage of relaxin contained in such parenteral
compositions is highly dependent on the specific nature thereof, as
well as the needs of the subject. However, percentages of active
ingredient of 0.01% to 10% in solution are employable, and will be
higher if the composition is a solid which will be subsequently
diluted to the above percentages. In general, the composition will
comprise 0.2-2% of the relaxin in solution.
[0070] Parenteral administration may employ the implantation of a
slow-release or sustained-release system, such that a constant
level of dosage is maintained. Various matrices (e.g., polymers,
hydrophilic gels, and the like) for controlling the sustained
release, and for progressively diminishing the rate of release of
active agents such as relaxin are known in the art. See, e.g., U.S.
Pat. No. 3,845,770 (describing elementary osmotic pumps); U.S. Pat.
Nos. 3,995,651, 4,034,756 and 4,111,202 (describing miniature
osmotic pumps); U.S. Pat. Nos. 4,320,759 and 4,449,983 (describing
multichamber osmotic systems referred to as push-pull and push-melt
osmotic pumps); and U.S. Pat. No. 5,023,088 (describing osmotic
pumps patterned for the sequentially timed dispensing of various
dosage units).
[0071] Drug release devices suitable for use in administering
relaxin according to the methods of the invention may be based on
any of a variety of modes of operation. For example, the drug
release device can be based upon a diffusive system, a convective
system, or an erodible system (e.g., an erosion-based system). For
example, the drug release device can be an osmotic pump, an
electroosmotic pump, a vapor pressure pump, or osmotic bursting
matrix, e.g., where the drug is incorporated into a polymer and the
polymer provides for release of drug formulation concomitant with
degradation of a drug-impregnated polymeric material (e.g., a
biodegradable, drug-impregnated polymeric material). In other
embodiments, the drug release device is based upon an
electrodiffusion system, an electrolytic pump, an effervescent
pump, a piezoelectric pump, a hydrolytic system, etc.
[0072] Drug release devices based upon a mechanical or
electromechanical infusion pump, are also suitable for use with the
present invention. Examples of such devices include those described
in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603;
4,360,019; 4,725,852, and the like. In general, the present
treatment methods can be accomplished using any of a variety of
refillable, non-exchangeable pump systems. Osmotic pumps have been
amply described in the literature. See, e.g., WO 97/27840; and U.S.
Pat. Nos. 5,985,305 and 5,728,396.
[0073] Relaxin may be administered over a period of hours, days,
weeks, months, or years, depending on several factors, including
the degree of arterial stiffness, etc. For example, relaxin is
administered for a period of time of from about 2 hours to about 8
hours, from about 8 hours to about 12 hours, from about 12 hours to
about 24 hours, from about 24 hours to about 36 hours, from about
36 hours to about 72 hours, from about 3 days to about one week,
from about 1 week to about 2 weeks, from about 2 weeks to about 1
month, from about 1 month to about 3 months, from about 3 months to
about 6 months, from about 6 months to about 12 months, or from
about 1 year to several years. The administration may be constant,
e.g., constant infusion over a period of hours, days, weeks,
months, years, etc. Alternatively, the administration may be
intermittent, e.g., relaxin may be administered once a day over a
period of days, once an hour over a period of hours, or any other
such schedule as deemed suitable.
[0074] Formulations of relaxin may also be administered to the
respiratory tract as a nasal or pulmonary inhalation aerosol or
solution for a nebulizer, or as a microfine powder for
insufflation, alone or in combination with an inert carrier such as
lactose, or with other pharmaceutically acceptable excipients. In
such a case, the particles of the formulation may advantageously
have diameters of less than 50 micrometers, preferably less than 10
micrometers.
Combination Therapies
[0075] In some embodiments, a subject method is modified to include
administration of at least one additional therapeutic agent.
Suitable additional therapeutic agents include, but are not limited
to, estrogen receptor modulators; anti-hypertensive agents such as
calcium channel blockers, endothelin receptor antagonists,
angiotensin-I converting enzyme (ACE) inhibitors,
.alpha.-adrenergic blocking agents, vasodilators, diuretics,
.beta.-adrenergic blocking agents, renin inhibitors, and
angiotensin receptor antagonists; natriuretic peptides (e.g.,
atrial natriuretic peptide, brain natriuretic peptide, and C-type
natriuretic peptide); agents for blocking cholesterol production
(e.g. statins) and agents used to treat diabetes.
Estrogen Receptor Modulators
[0076] Suitable estrogen receptor modulators include any of a
variety of estrogen compounds, as well as Selective Estrogen
Receptor Modulators ("SERMs"). SERMs include, but are not limited
to, tamoxifen, raloxifen, droloxifene, idoxifene, lasofoxifene,
CP-336,156, GW5638, LY353581, TSE-424, LY353381, LY117081,
toremifene, fulvestrant,
4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]ph-
enyl]-2H-1-benzopyran-3-yl]phenyl-2,2-dimethylpr-opanoate,
4,4'-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and
SH646.
[0077] Suitable estrogen compounds include, but are not limited to,
mestranol, an ester of estradiol, polyestriol phosphate, estrone
sulfate, natural estrogens, synthetic estrogens, conjugated
estrogens, estradiol, estradiol sulfamates, estradiol valerate,
estradiol benzoate, ethinyl estradiol, estrone, estriol, estriol
succinate and conjugated estrogens, a conjugated equine estrogen,
estrone sulfate, 17.beta.-estradiol sulfate, 17.alpha.-estradiol
sulfate, equilin sulfate, 17.beta.-dihydroequilin sulfate,
17.alpha.-dihydroequilin sulfate, equilenin sulfate,
17.beta.-dihydroequilenin sulfate and 17.alpha.-dihydroequilenin
sulfate.
[0078] Also suitable for use are micronized forms of estrogens,
such as micronized estradiol, micronized estradiol sulfamates,
micronized estradiol valerate, micronized estradiol benzoate,
micronized estrone, or micronized estrone sulfate or mixtures
thereof, notably micronized estradiol, micronized estradiol
valerate, micronized estradiol benzoate or micronized estradiol
sulfamates.
[0079] Effective dosages of estrogens are conventional and well
known in the art. Typical approximate dosages for oral
administration are, e.g., ethinyl estradiol (0.001-0.030 mg/day),
mestranol (5-25 mcg/day), estradiol (including 17.beta. estradiol),
(0.5-6 mg/day), polyestriol phosphate (2-8 mg) and conjugated
estrogens (0.3-1.2 mg/day). Dosages for other means of delivery
will be evident to one of skill in the art. For example,
transdermal dosages will vary therefrom in accordance with the
adsorption efficacy of the vehicle employed.
[0080] Estrogen compounds can be administered by any of a variety
of conventional modes, including, e.g., oral (e.g., solutions,
suspensions, tablets, dragees, capsules or pills), parenteral
(including subcutaneous injection, or intravenous, intramuscular or
intrasternal injection or infusion techniques), inhalation spray,
transdermal, rectal, or vaginal (e.g., by vaginal rings or creams)
administration.
Anti-Hypertensive Agents
[0081] Suitable ACE inhibitor include, but are not limited to,
benazepril (Lotensin.RTM.), captopril (Capoten.RTM.), enalapril,
enalaprilat, fosinopril (Monopril.RTM.), lisinopril (Zestril.RTM.;
Prinivil.RTM.,), pentopril, quinapril (Accupril.RTM.), quinaprilat,
ramipril (Altace.RTM.), trandolapril (Mavik.RTM.), zofenopril,
moexipril (Univasc.RTM.), perindopril (Coversyl.RTM.; Aceon.RTM.),
Vasotec.RTM., cilazapril (Inhibace.RTM.).
[0082] Suitable diuretics include, but are not limited to,
acetazolamide; amiloride; bendroflumethiazide; benzthiazide;
bumetanide; chlorothiazide; chlorthalidone; cyclothiazide;
ethacrynic acid; furosemide; hydrochlorothiazide;
hydroflumethiazide; indacrinone (racemic mixture, or as either the
(+) or (-) enantiomer alone, or a manipulated ratio, e.g., 9:1 of
said enantiomers, respectively); metolazone; methyclothiazide;
muzolimine; polythiazide; quinethazone; sodium ethacrynate; sodium
nitroprusside; ticrynafen; triamterene; and trichlormethiazide.
[0083] Suitable .alpha.-adrenergic blocking agents include, e.g.,
dibenamine; phentolamine; phenoxybenzamine; prazosin;
prazosin/polythiazide (Minizide.RTM.); tolazoline; doxazosin
(Cardura); terazosin (Hytrin.RTM.); tamsulosin (Flomax.RTM.); and
alfuzosin (Uroxatral.RTM.).
[0084] Suitable .beta.-adrenergic blocking agents include, but are
not limited to, Betapace (sotalol), Blocadren (timolol), Brevibloc
(esmolol), Cartrol (carteolol), Coreg (carvedilol), Corgard
(nadolol), Inderal (propranolol), Inderal-LA (propranolol), Kerlone
(betaxolol), Levatol (penbutolol), Lopressor (metoprolol),
Normodyne (labetalol), Sectral (acebutolol), Tenormin (atenolol),
Toprol-XL (metoprolol), Trandate (labetalol), Visken (pindolol),
and Zebeta (bisoprolol).
[0085] Suitable vasodilators include, but are not limited to,
diazoxide, hydralazine (Apresoline.RTM.), minoxidil, nitroprusside
(Nipride.RTM.), sodium nitroprusside, diazoxide (Hyperstat IV),
verapamil, and nefidipine.
[0086] Suitable calcium channel blockers include, but are not
limited to, amlodipine (Norvasc.RTM.), felodipine (Plendil.RTM.),
nimodipine, isradipine, nicardipine, nifedipine (Procardia.RTM.),
bepridil (Vascor.RTM.), diltiazem (Cardiazem.RTM.), and veramapil
(Isoptin.RTM.; Calan.RTM.).
[0087] Suitable angiotensin II receptor blockers or inhibitors
include, but are not limited to, saralasin, losartan (Cozaar),
ciclosidomine, eprosartan, furosemide, irbesartan, and
valsartan.
[0088] Suitable renin inhibitors include, e.g., pepstatin and the
di- or tripeptide renin inhibitors; enalkrein, RO 42-5892 (Hoffman
LaRoche), A 65317 (Abbott), CP 80794, ES 1005, ES 8891, SQ 34017; a
compound as disclosed in U.S. Pat. No. 6,673,931; and the like.
[0089] Suitable endothelin antagonists useful in the present
invention include, but are not limited to, atrasentan (ABT-627;
Abbott Laboratories), Veletri.TM. (tezosentan; Actelion
Pharmaceuticals, Ltd.), sitaxsentan (ICOS-Texas Biotechnology),
enrasentan (GlaxoSmithKline), darusentan (LU135252; Myogen)
BMS-207940 (Bristol-Myers Squibb), BMS-193884 (Bristol-Myers
Squibb), BMS-182874 (Bristol-Myers Squibb), J-104132 (Banyu
Pharmaceutical), VML 588/Ro 61-1790 (Vanguard Medica), T-0115
(Tanabe Seiyaku), TAK-044 (Takeda), BQ-788 (Banyu Pharmaceutical),
BQ123, YM-598 (Yamanouchi Pharma), PD 145065 (Parke-Davis),
A-127722 (Abbott Laboratories), A-192621 (Abbott Laboratories),
A-182086 (Abbott Laboratories), TBC3711 (ICOS-Texas Biotechnology),
BSF208075 (Myogen), S-0139 (Shionogi), TBC2576 (Texas
Biotechnology), TBC3214 (Texas Biotechnology), PD156707
(Parke-Davis), PD180988 (Parke-Davis), ABT-546 (Abbott
Laboratories), ABT-627 (Abbott Laboratories), SB247083
(GlaxoSmithKline), SB 209670 (GlaxoSmithKline); and an endothelin
receptor antagonists discussed in the art, e.g., Davenport and
Battistini (2002) Clinical Science 103:15-35, Wu-Wong et al. (2002)
Clinical Science 103:1075-1115, and Luescher and Barton (2000)
Circulation 102:2434-2440. A suitable endothelin receptor
antagonist is TRACLEER.TM. (bosentan; manufactured by Actelion
Pharmaceuticals, Ltd.). TRACLEER.TM. is an orally active dual
endothelin receptor antagonist, and blocks the binding of
endothelin to both of its receptors endothelin receptor A and
endothelin receptor B. TRACLEER.TM. is generally administered at a
dose of 62.5 mg bid orally for 4 weeks, followed by a maintenance
dose of 125 mg bid orally.
[0090] Other suitable antihypertensive agents include, e.g.,
aminophylline; cryptenamine acetates and tannates; deserpidine;
meremethoxylline procaine; pargyline; clonidine (Catapres);
methyldopa (Aldomet); reserpine (Serpasil); guanethidine (Ismelin);
and tirmethaphan camsylate.
[0091] Statins
[0092] Suitable statins include, without limitation, products such
as Crestor, Lipitor, Lescol, Mevacor, Pravochol, Zocor and related
compounds such as those discussed, e.g., in Rev. Port. Cardio.
(2004) 23(11):1461-82; Curr Vasc Pharmacol. (2003) 3:329-33.
[0093] Therapeutic Agents for Treating Type 1 or Type 2
Diabetes
[0094] Other suitable agents for use in a combination therapy with
relaxin include therapeutic agents for treating Type 1 diabetes,
and therapeutic agents for treating Type 2 diabetes (e.g., agents
that increase insulin sensitivity).
Insulin
[0095] Therapeutic agents for treating Type 1 diabetes include any
form of insulin, as long as the insulin is biologically active,
i.e., the insulin is effective in reducing blood glucose levels in
an individual who is responsive to insulin. In some embodiments,
recombinant human insulin ("regular" insulin) or a recombinant
human insulin analog is used. In a particular embodiment, the
insulin analog is a monomeric form of insulin, e.g., human lispro.
In some instances, other forms of insulin are used alone or in
combination with recombination human insulin or each other. Insulin
that is suitable for use herein includes, but is not limited to,
regular insulin (Humulin R, Novlin R, etc.), semilente, NPH
(isophane insulin suspension; Humulin N, Novolin N, Novolin N
PenFill, NPH Ilentin II, NPH-N), lente (insulin zinc suspension;
Humulin-L, Lente Ilentin II, Lent L, Novolin L), protamine zinc
insulin (PZI), ultralente (insulin zinc suspension, extended;
Humulin U Ultralente), insuline glargine (Lantus), insulin aspart
(Novolog), acylated insulin, monomeric insulin, superactive
insulin, hepatoselective insulin, lispro (Humalog.TM.), and any
other insulin analog or derivative, and mixtures of any of the
foregoing. Commonly used mixtures include mixtures NPH and regular
insulin containing the following percentages of NPH and regular
insulin: 70%/30%, 50%/50%, 90%/10%, 80%/20%, 60%/40%, and the like.
Insulin that is suitable for use herein includes, but is not
limited to, the insulin forms disclosed in U.S. Pat. Nos.
4,992,417; 4,992,418; 5,474,978; 5,514,646; 5,504,188; 5,547,929;
5,650,486; 5,693,609; 5,700,662; 5,747,642; 5,922,675; 5,952,297;
and 6,034,054; and published PCT applications WO 00/121197; WO
90/10645; and WO 90/12814. Insulin analogs include, but are not
limited to, superactive insulin analogs, monomeric insulins, and
hepatospecific insulin analogs.
[0096] Superactive insulin analogs have increased activity over
natural human insulin. Accordingly, such insulin can be
administered in substantially smaller amounts while obtaining
substantially the same effect with respect to reducing serum
glucose levels. Superactive insulin analogs include, e.g.,
10-Aspartic Acid-B human insulin; des-pentapeptide
(B26-B30).fwdarw.Asp.sup.B10, Tyr.sup.B25-.alpha.-carboxamide human
insulin; (B26-B30).fwdarw.glu.sup.B10,
Tyr.sup.B25-.alpha.-carboxamide human insulin; destripeptide B28-30
insulin; an insulin with .gamma.-aminobutyric acid substituted for
A13Leu-A14Tyr; and further insulin analogs of the formula
des(B26-B30).fwdarw.X.sup.B10, Tyr.sup.B25-.alpha.-carboxamide
human insulin, in which X is a residue substituted at position 10
of the B chain. These insulin analogs have potencies anywhere from
11 to 20 times that of natural human insulin. All of the
above-described insulin analogs involve amino acid substitutions
along the A or B chains of natural human insulin, which increase
the potency of the compound or change other properties of the
compound. Monomeric insulin includes, but is not limited to,
lispro.
[0097] Insulin derivatives include, but are not limited to,
acylated insulin, glycosylated insulin, and the like. Examples of
acylated insulin include those disclosed in U.S. Pat. No.
5,922,675, e.g., insulin derivatized with a C.sub.6-C.sub.21 fatty
acid (e.g., myristic, pentadecylic, palmitic, heptadecylic, or
stearic acid) at an .alpha.- or .epsilon.-amino acid of glycine,
phenylalanine, or lysine.
Agents that Increase Insulin Sensitivity
[0098] In some embodiments, a subject treatment regimen for
treating an individual with Type 2 diabetes further comprises
administering an additional agent that reduces insulin resistance
(e.g., increases insulin sensitivity). Suitable agents that treat
insulin resistance include, but are not limited to, a biguanide
such as Metformin (e.g., administered in an amount of 500 mg or 850
mg three times per day), Phenformin, or a salt thereof; a
thiazolidinedione compound such as troglitazone (see, e.g., U.S.
Pat. No. 4,572,912), rosiglitazone (SmithKlineBeecham),
pioglitazone (Takeda), Glaxo-Welcome's GL-262570, englitazone
(CP-68722, Pfizer) or darglitazone (CP-86325, Pfizer, isaglitazone
(MCC-555; Mitsubishi; see, e.g., U.S. Pat. No. 5,594,016),
reglitazar (JTT-501), L-895645 (Merck), R-119702 (Sankyo/WL),
NN-2344, YM-440 (Yamanouchi), Ragaglitazar (NNC 61-0029 or DRF2725;
NovoNordisk), farglitazar (GI262570), tesaglitazar (AZ 242),
KRP-297, and the like; and combinations such as Avandamet.TM.
(rosiglitazone maleate and metformin-HCl).
Subjects Suitable for Treatment
[0099] Individuals who are suitable for treatment with a subject
method include any individual having arterial stiffness (or reduced
arterial compliance) for any reason. Such individuals include
individuals having a disorder that is associated with or results
from, reduced arterial compliance, including, but not limited to,
atherosclerosis, Type 1 diabetes, Type 2 diabetes, coronary artery
disease, scleroderma, stroke, diastolic dysfunction, familial
hypercholesterolemia, isolated systolic hypertension, primary
hypertension, secondary hypertension, left ventricular hypertrophy,
arterial stiffness associated with long-term tobacco smoking,
arterial stiffness associated with obesity, arterial stiffness
associated with age, systemic lupus erythematosus, preeclampsia,
and hypercholesterolemia.
[0100] Particularly suitable for treatment are individuals whose
measured global arterial compliance is decreased relative to a
similarly situated healthy individual. Also particularly suitable
for treatment are individuals with a measured local arterial
compliance which is decreased relative to the local arterial
compliance expected in a similarly situated healthy individual.
Individuals with a measured regional arterial compliance which is
decreased relative to that expected in a similarly healthy
individuals are also particularly suitable for treatment. In some
instances, the global, local or regional arterial compliance of an
individual at different points in time may be measured and compared
to determine whether the arterial compliance in that individual is
decreasing and approaching levels which indicate that intervention
is appropriate.
[0101] Individuals who are suitable for treatment with a subject
method include individuals who have developed, or who are at risk
of developing, age-associated arterial stiffness. Such individuals
include humans who are over the age of 50 years, e.g., humans who
are from about 50 years old to about 60 years old, from about 60
years old to about 65 years old, from about 65 years old to about
70 years old, from about 70 years old to about 75 years old, from
about 75 years old to about 80 years old, or older.
[0102] Also suitable for treatment with a subject method are
perimenopausal women, menopausal women, postmenopausal women, and
women who have ceased menstruation for non-age-related reasons,
e.g., as a result of surgery (e.g., hysterectomy, oophorectomy),
and thus have developed, or are at risk of developing, arterial
stiffness. Such women can be treated with a combination therapy
involving relaxin and estrogen. Such women can also be treated with
a combination therapy involving relaxin, estrogen, and an
anti-hypertensive agent.
[0103] Also suitable for treatment with a subject method are
individuals who have been diagnosed with Type 1 diabetes mellitus.
Also suitable for treatment with a subject method are individuals
who have been diagnosed with Type 2 diabetes mellitus. Individuals
who are insulin resistant are identified by one or more of the
following criteria: 1) a HOMA-IR value that is greater than 2.5
(based on the calculation fasting insulin (mU/ml).times.fasting
glucose (mmol/l)/22.5); 2) a fasting serum insulin level of greater
than about 20 .mu.U/mL, or greater than about 25 .mu.U/mL; 3) a
fasting serum C-peptide level of greater than about 3.5 ng/mL, or
greater than about 4.5 ng/mL.
EXAMPLES
[0104] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
Effect of Relaxin on Systemic Arterial Resistance and
Compliance
Methods
Animals
[0105] Long-Evans female rats of 12-14 weeks old were purchased
from Harlan Sprague-Dawley (Frederick, Md. USA). They were provided
PROLAB RMH 2000 diet containing 0.48% sodium (PME Feeds Inc., St.
Louis, Mo. USA) and water ad libitum. The rats were maintained on a
12 h light/dark cycle. The Institutional Animal Care and Use
Committee of the Magee-Womens Research Institute approved all
animal procedures.
Surgical Preparation
[0106] Briefly, the rats were habituated to Nalgene metabolism
cages for one week (VWR Scientific Products), followed by further
habituation to a harness/7.5 cm spring assembly for another week
while in the metabolism cage (Harvard Apparatus, Holliston, Mass.
USA). The animals were fitted to the harness under isofluorane
anesthesia. After this habituation period, the rats were
anesthetized with 60 mg/kg ketamine i.m. and 21 mg/kg pentobarbital
i.p., and placed in the prone position on a heating pad. After
application of 70% ethanol and betadine to all exposed skin areas,
ampicillin was administered s.c. (0.2 ml of a 125 mg/ml solution)
and atropine was also administered s.c. (0.075 ml of a 0.4 mg/ml
solution). Next, a sterile tygon catheter (18 in long, 0.015 in ID,
0.0300D) connected to a syringe containing Ringer's solution, as
well as a sterile thermodilution microprobe (22 cm long, F #1.5;
Columbus Instruments, Columbus, Ohio USA) were threaded through the
spring. The tygon catheter was subsequently threaded through the
hole in the harness and then tunneled subcutaneously from the
midpoint between the shoulder blades out the small incision behind
the ear using an 18-gauge trocar.
[0107] The thermodilution catheter was also threaded through the
harness assembly and then tunneled subcutaneously from the midpoint
between the scapulae out the skin incision in the left costal
margin. The spring was then reattached to the harness. The rat was
repositioned on the back. A 1.0 cm skin incision was made in the
left inguinal region. The external iliac artery was isolated and
prepared for catheterization. The thermocouple was then tunneled
subcutaneously exiting at the inguinal incision. The thermocouple
was next inserted into the external iliac artery being directed
rostrally, so that it passed easily into the internal iliac artery
and subsequently into the aorta. At the 4.0 cm, the thermocouple
lay approximately 1.0 cm below the left renal artery. Next, a
horizontal 2.0 cm incision was made over the trachea, 1.0 cm above
the cricoid notch.
[0108] Through this incision, a large subcutaneous pocket was
dissected in the neck and above the left shoulder. The right
jugular vein and carotid artery were then isolated and prepared for
catheterization, the latter facilitated by placing a small roll of
gauze under the neck to elevate this deep structure. Using the
18-gauge trocar, the tygon catheter was tunneled subcutaneously
from the small incision behind the right ear out the incision in
the neck. The tygon catheter was implanted in the right jugular
vein 3.0 cm, thereby placing the catheter tip at the confluence of
the anterior vena cava and the right atrial appendage. The
battery/transmitter of a sterile mouse pressure catheter
(TA11PA-C20; ca. F #1.2; Data Sciences International, St. Paul,
Minn. USA) was inserted in the subcutaneous pocket. The mouse
pressure catheter was then implanted in the right carotid artery
2.8 cm, thereby placing the catheter tip at the confluence of the
right carotid artery and aortic arch. All wounds were closed with
4-0 silk or autoclips. After instilling 0.05 ml of a heparin
solution into the jugular catheter and plugging it with a straight
pin, the rat was placed in the metabolism cage and given ampicillin
by drinking water for 2 days (100 mg/50 ml with 2 tablespoons of
dextrose). The spring and catheters that exit the cage top were
secured.
[0109] Terbutrol was given s.c. for post-operative analgesia as
soon as the rats were recovered sufficiently from the anesthesia.
For low dose administration of recombinant human relaxin (4.0
.mu.g/h rhRLX) for 10 days, two Alzet model 2002 osmotic minipumps
(Durect Corporation, Curpertino, Calif. USA) were inserted
subcutaneously in the back of the animal under isoflurane
anesthesia. For high dose administration for 10 days (25 .mu.g/h),
one Alzet model 2ML2 osmotic minipump was implanted. After
completion of the measurement for the last time point, the rat was
anesthetized with 60 mg/kg pentobarbital i.v. Blood was obtained
from the abdominal aorta for rhRLX levels, osmolality and
hematocrit. The position of the jugular catheter relative to the
right atrium, the placement of the pressure catheter relative to
the aortic arch, and the position of the thermocouple relative to
the left renal artery were recorded.
In Vivo Studies: Hemodynamics and Systemic Arterial Mechanical
Properties
[0110] Time control studies were first performed in 5 rats, in
order to document the stability of systemic hemodynamics over a 17
day period after surgery. Measurements were recorded on days 4-5,
7-8, 9-10, 13-14, and 16-17 after surgery. The low and high dose
rhRLX protocols entailed 6 and 7 rats, respectively. In addition,
the vehicle for rhRLX (20 mM sodium acetate, pH 5.0) was
administered to another 6 rats. After 2 baseline measurements of
systemic hemodynamics on days 5 and 7 after surgery, either low or
high dose rhRLX or vehicle was administered by osmotic minipump.
Systemic hemodynamics were again assessed on days 3, 6, 8 and 10
after initiation of rhRLX or vehicle infusion.
[0111] Each measurement consisted of 4 to 6 recordings of cardiac
output and blood pressure waveforms that were obtained when the rat
was either sleeping or resting. At least 10 min was allowed between
recordings. These measurements were obtained between 9 am and 3
pm.
[0112] Cardiac Output.
[0113] To measure cardiac output, we used the thermodilution
technique. Osborn et al. (1986) Am. J. Physiol. 251:H1365-H1372.
Ringer's solution of known volume and temperature was injected into
the anterior vena cava using the Micro Injector 400 (Columbus
Instruments). The cardiac output was calculated from the change in
blood temperature (Cardiotherm 400R, Columbus Instruments). The
cardiac output as determined by the Cardiotherm 400R was calculated
as:
[0114] CO=[(B.sub.T-I.sub.T)*V.sub.I]/.intg.B.sub.T(t) where,
B.sub.T is the blood temperature (recorded by the thermocouple
implanted in the abdominal aorta), I.sub.T is the injectate
temperature (room temperature), V.sub.I is the injectate volume
(150 .mu.L), and B.sub.T(t) is the blood temperature as a function
of time.
[0115] Blood Pressure.
[0116] Instantaneous aortic pressure was recorded using a blood
pressure telemetry system (Data Sciences International, St. Paul,
Minn. USA). Mills et al. (2000) J. Appl. Physiol. 88:1537-1544. The
aortic pressure was recorded by a pressure catheter implanted in
the aortic arch via the right carotid artery and transmitted to an
external receiver. Steady-state aortic pressure was digitized
online using a PC-based data acquisition system with 16 bit
resolution and 2000 Hz sampling rate and stored as text files for
off-line analysis. Each measurement consisted of a 30 second
sampling duration.
[0117] Aortic pressure analysis. Analysis of the acquired data and
calculation of global AC was performed by a custom computer program
developed using MATLAB software (MathWorks Inc., Natick, Mass.
USA). Briefly, individual beats were selected (3-15 cycles) from
the 10 seconds of the aortic pressure recording, immediately
preceding the measurement of cardiac output. The ensemble was
averaged as described by Burattini et al. ((1985) Comput. Biomed.
Res. 18:303-312) to yield a single representative beat for each
trial. The mean arterial pressure (MAP), peak systolic pressure
(P.sub.s), and end diastolic pressure (P.sub.d) were calculated
from this averaged beat. Pulse pressure (PP) was calculated as
P.sub.s-P.sub.d. Systemic vascular resistance (SVR) was calculated
by dividing the MAP by CO.
[0118] Global Arterial Compliance.
[0119] Two measures of global arterial compliance were calculated.
The first (AC.sub.area) was calculated from the diastolic decay of
the aortic pressure waveform [P(t)] using the area method (2):
AC.sub.area=A.sub.d[SVR(P.sub.1-P.sub.2)] where P.sub.1 and P.sub.2
are the pressures at the beginning and end of the diastolic decay
curve, respectively, and A.sub.d is the area under the P(t)
waveform over this region. The second measure of global arterial
compliance was calculated as the stroke volume-to-pulse pressure
ratio (Chemla et al. (1998) Am. J. Physiol. 274:H500-H505). Stroke
volume was defined as CO/HR.
In Vitro Studies: Arterial Passive Mechanics
[0120] Nonpregnant female rats were administered rhRLX (4 .mu.g/h)
or vehicle by osmotic minipump for 5 days. A kidney was removed and
placed in ice-cold HEPES buffered physiological saline solution
(PSS, a modified Kreb's buffer). The HEPES-physiologic saline
solution was composed of (in mmol/L): sodium chloride 142,
potassium chloride 4.7, magnesium sulfate 1.17, calcium chloride
2.5, potassium phosphate 1.18, HEPES 10, glucose 5.5, and was pH
7.4 at 37.degree. C. A stereo dissecting microscope, fine forceps
and iridectomy scissors were used to isolate interlobar arteries as
described by Gandley et al. ((2001) Am. J. Physiol. 280:R1-R7)
(unpressurized inner diameter, 100-200 .mu.m). An arterial segment
was then transferred to an isobaric arteriograph (Living Systems
Instrumentation, Burlington, Vt. USA) and mounted on 2 glass
micro-cannulae suspended in the chamber. After the residual blood
was flushed from the lumen of the artery, the distal cannula was
occluded to prevent flow. The proximal cannula was attached to a
pressure transducer, a pressure servo-controller and a peristaltic
pump. The servocontroller maintained a selected intraluminal
pressure that was changed in a stepwise manner. An electronic
dimension analyzing system obtained arterial diameter measures.
[0121] The vessels were incubated in the bath with 10.sup.-4 M
papaverine and 10.sup.-2 M EGTA in calcium-free HEPES PSS. After a
30 min equilibration period, transmural pressure was increased in
14 steps beginning at 0 mmHg up to 150 mmHg. Inner and outer
diameters as well as wall thickness were measured following each
pressure step when the vessel had reached a steady state. Midwall
radius (R.sub.m) and circumferential wall stress (.sigma.) were
calculated from these data as described before (Cholley et al.
(2001) J. Appl. Physiol. 90:2427-2438). Vessel wall elastic
properties were quantified in terms of the incremental elastic
modulus (E.sub.inc), which was calculated from the .sigma.-R.sub.m
relationship (Pagani et al. (1979) Circ. Res. 44:420-429).
Serum Measurements
[0122] Serum osmolality was measured using a freezing-point
depression instrumentation osmometer (Model 3 MO; Advanced
Instruments, Needham Heights, Mass. USA). The levels of rhRLX in
serum were measured by a quantitative sandwich immunoassay as
previously described (Jeyabalan et al. (2003) Circ Res.
93(12):1249-57).
Preparation of rhRLX
[0123] Two model 2002 osmotic minipumps (Durect Corporation,
Cupertino, Calif. USA) were used to deliver the rhRLX for 10 days
at the dose of 4 .mu.g/h which yielded concentrations of
circulating relaxin similar to those measured during early to
midgestation in rats, i.e., 10-20 ng/ml when pregnancy-induced
renal vasodilation is maximal in this species. One model 2ML2
osmotic minipump was used to deliver rhRLX at the dose of 25
.mu.g/h for 10 days which we expected to produce concentrations of
circulating hormone comparable to those recorded during mid to late
gestation when further increases in CO and decreases in SVR are
observed in this species. The rhRLX (Connetics, Palo Alto, Calif.
USA) provided as a 5.0 mg/ml solution in 20 mM sodium acetate, pH
5.0 was diluted in the same buffer.
Statistical Analysis
[0124] Data are presented as means+SEM. One or two-factor
repeated-measures ANOVA (Zar (1984) Biostatistical Analysis,
Englewood Cliffs, N.J.: Prentice Hall) was used to compare mean
values among various groups. If significant main effects or
interactions were observed, comparisons between groups were
performed using Fisher's LSD or Dunnett's test. The student's
paired `t` test was used to compare the composite mean values
during infusion of rhRLX (i.e., values averaged over all time
points during rhRLX infusion) with baseline. Least squares
regression analysis was performed on .sigma.-R.sub.m and
E.sub.inc-R.sub.m relationships. Analysis of excess variance (or
extra sum of squares) was used to compare these relationships
between vehicle and relaxin-treated groups. P<0.05 was taken to
be significant.
Results
In Vivo Studies
[0125] Time Control.
[0126] The stability of systemic arterial hemodynamics and load
over a 17-day period after surgery in control rats (Table 1). Heart
rate declined significantly due to a training effect as previously
reported (Conrad and Russ (1992) Am. J. Physiol. 31:R472-477).
Stroke volume reciprocally increased, such that CO was unchanged.
All other variables did not change significantly over the 17-day
period after surgery, thus this conscious rat model can be used to
obtain meaningful data under the experimental conditions described
next (Table 1).
TABLE-US-00001 TABLE 1 Time Control Rats Time After .DELTA..sub.T
CO HR* SV* AC.sub.area SVR MAP Surgery (days) (.degree. C.)
(mL/min) (bpm) (mL) (.mu.l/mmHg) (mmHg s/mL) (mmHg) 4-5 0.37 .+-.
0.01 119 .+-. 3 428 .+-. 7 0.28 .+-. 0.01 6.8 .+-. 0.3 57 .+-. 2
107.6 .+-. 0.8 7-8 0.37 .+-. 0.01 121 .+-. 3 378 .+-. 8 0.32 .+-.
0.01 7.2 .+-. 0.3 55 .+-. 2 107.3 .+-. 1.5 9-10 0.38 .+-. 0.01 120
.+-. 4 382 .+-. 8 0.31 .+-. 0.01 6.9 .+-. 0.3 55 .+-. 2 108.1 .+-.
1.5 13-14 0.35 .+-. 0.01 115 .+-. 4 354 .+-. 5 0.32 .+-. 0.01 7.3
.+-. 0.3 58 .+-. 2 107.0 .+-. 1.5 16-17 0.32 .+-. 0.02 122 .+-. 3
349 .+-. 7 0.36 .+-. 0.01 8.0 .+-. 0.4 52 .+-. 2 103.7 .+-. 2.2
Mean.+-.SEM. N=5 rats. .DELTA..sub.T, change in blood temperature
after injection of Ringer's solution into the right heart; CO,
cardiac output; HR, heart rate; SV, stroke volume; AC.sub.area,
global arterial compliance calculated using area method; SVR,
systemic vascular resistance; MAP, mean arterial pressure.
*P<0.05 by single factor repeated measures ANOVA.
[0127] Rats Administered Vehicle (for rhRLX).
[0128] These results were derived from 3 rats administered the
vehicle for rhRLX at the infusion rate of 1 .mu.l/h, and from
another 3 rats administered the vehicle for rhRLX at the infusion
rate of 5 .mu.l/h. (These correspond to the flow rates for the low
and high dose administration of rhRLX, respectively.) The results
were comparable, and therefore, combined. FIGS. 1 and 2 depict the
percent change from baseline for systemic hemodynamics and other
variables. Similar to the time control studies, there was a
significant decrease in heart rate, which was offset by an
insignificant rise in stroke volume, such that CO remained
unchanged. All other variables remained relatively constant.
Combining all of the time points during administration of vehicle
yielded overall changes in CO, global AC, and SVR of -1.4+1.3,
2.2+4.6, and 0.4+3.4% of baseline, respectively (all PNS vs.
baseline). As expected, there was no measurable rhRLX in the serum,
and the osmolality was 309+6 mOsm/kg water.
[0129] Rats Administered Low Dose rhRLX (4 .mu.g/h).
[0130] The absolute values for systemic hemodynamics and other
parameters are shown in Table 2, while FIGS. 1 and 2 show the
temporal pattern of percentage change from baseline.
TABLE-US-00002 TABLE 2 Rats Administered Low Dose rhRLX (4 .mu.g/h)
Days After .DELTA..sub.T CO* HR SV* AC.sub.area* SVR* MAP Minipump
(.degree. C.) (mL/min) (bpm) (mL) (.mu.l/mmHg) (mmHg s/mL) (mmHg)
Baseline 0.34 .+-. 0.02 128 .+-. 2 417 .+-. 6 0.31 .+-. 0.01 6.2
.+-. 0.3 51 .+-. 2 111.6 .+-. 3.7 2-3 0.31 .+-. 0.01 151 .+-. 6 436
.+-. 9 0.35 .+-. 0.01 7.2 .+-. 0.3 43 .+-. 3 110.2 .+-. 4.9 6 0.31
.+-. 0.01 149 .+-. 7 419 .+-. 6 0.36 .+-. 0.02 7.1 .+-. 0.5 46 .+-.
3 116.9 .+-. 3.8 8 0.31 .+-. 0.01 159 .+-. 7 427 .+-. 11 0.37 .+-.
0.02 7.4 .+-. 0.5 44 .+-. 2 112.1 .+-. 2.8 10 0.31 .+-. 0.01 153
.+-. 5 418 .+-. 10 0.37 .+-. 0.01 7.7 .+-. 0.5 44 .+-. 2 115.2 .+-.
5.8
Mean.+-.SEM. N=6 rats. Two baseline measurements were made on days
5 and 7 after surgery. These results were averaged for each rat.
For abbreviations, see Table 1.
[0131] Low dose rhRLX significantly increased CO relative to
baseline and to vehicle infusion (FIG. 1A). The infusion of rhRLX
prevented the decline normally observed in HR (c.f. vehicle, FIG.
1B), and the hormone significantly increased SV (FIG. 1C). Thus,
increases in both SV and HR combined to raise the CO relative to
vehicle-infused rats. Systemic vascular resistance fell
significantly relative to baseline and vehicle infusion (FIG. 2A),
while MAP remained unchanged (FIG. 2B).
[0132] Global AC significantly increased relative to baseline and
vehicle infusion (FIG. 2C). There was no significant change in
pulse pressure; however, the ratio of stroke volume-to-pulse
pressure, another index of arterial compliance, increased
significantly during the infusion of low dose rhRLX relative to
baseline and to vehicle infusion (FIG. 2D). The time course of
changes in variables that showed a significant change with low dose
rhRLX administration (i.e., significant F value for relaxin and/or
interaction), was further examined. By post hoc pairwise
comparisons of data at different time points (Fisher's LSD). Both
CO and SV were significantly higher than baseline at day 3. While
SV continued to increase until day 8 (P<0.05, day 8 vs. day 3)
(FIG. 1C), there were no significant changes with time in CO beyond
day 3 (FIG. 1A). This was a result of a small, but insignificant,
fall in HR from day 3 to day 8 (FIG. 1B).
[0133] SVR and both measures of global AC were significantly
altered at day 3; thereafter there were no further significant
changes (FIG. 2). In general, maximal changes in arterial
hemodynamics and mechanical properties following low dose rhRLX
administration were observed at the earliest time point examined
(day 3), with no further temporal alterations. Combining all of the
time points during administration of low dose rhRLX yielded an
overall increase in CO and global AC of 19.2+4.8 and 21.4+3.6%
above baseline, respectively, and an overall decrease in SVR of
15.5+2.4% below baseline (all P<0.01 vs. baseline). Serum rhRLX
and osmolality were 14+2 ng/ml and 284+2 mOsm/kg water,
respectively. The latter significantly decreased compared to
vehicle infusion.
[0134] Rats Administered High Dose rhRLX (25 .mu.g/h).
[0135] The absolute values for systemic hemodynamics and other
variables are presented in Table 3, and FIGS. 1 and 2 portray the
percent change from baseline. The results for the high dose
infusion were comparable to the low dose administration in
direction, but somewhat less in magnitude.
TABLE-US-00003 TABLE 3 Rats Administered High Dose rhRLX (25
.mu.g/h) Days After .DELTA..sub.T CO* HR SV AC.sub.area* SVR* MAP
Minipump (.degree. C.) (mL/min) (bpm) (mL) (.mu.l/mmHg) (mmHg s/mL)
(mmHg) Baseline 0.37 .+-. 0.02 129 .+-. 6 438 .+-. 10 0.30 .+-.
0.02 7.7 .+-. 0.7 53 .+-. 3 111.6 .+-. 3.7 2-3 0.33 .+-. 0.02 141
.+-. 7 454 .+-. 13 0.31 .+-. 0.02 8.5 .+-. 0.8 49 .+-. 4 110.2 .+-.
4.9 6 0.35 .+-. 0.02 147 .+-. 4 432 .+-. 10 0.34 .+-. 0.01 8.1 .+-.
0.4 48 .+-. 2 116.9 .+-. 3.8 8 0.35 .+-. 0.03 150 .+-. 5 451 .+-. 9
0.33 .+-. 0.01 9.4 .+-. 0.7 45 .+-. 2 112.1 .+-. 2.8 10 0.34 .+-.
0.02 146 .+-. 9 442 .+-. 6 0.33 .+-. 0.02 9.2 .+-. 1.0 48 .+-. 3
115.2 .+-. 5.8
Mean.+-.SEM. N=7 rats. Two baseline measurements were made on days
5 and 7 after surgery. These results were averaged for each rat.
For abbreviations, see Table 1.
[0136] The temporal analysis of changes in individual variables
with high dose rhRLX was performed in a manner similar to that for
the low dose rhRLX. Once again, CO (FIG. 1A), SV (FIG. 1C), SVR
(FIG. 2A), and global AC (SV/PP method) (FIG. 2D) were maximally
altered by the earliest time point examined (day 3), with no
further significant changes thereafter. The temporal response of
global AC as calculated by the area method (FIG. 2C) deviated
slightly from this general pattern--AC.sub.area at day 6 was not
different from that at baseline. This is likely an aberrant
measurement because the second measure of global AC at all time
points (FIG. 2D) and AC.sub.area at days 3, 8, and 10 (FIG. 2C)
were significantly higher than baseline. Combining all of the time
points during administration of high dose rhRLX yielded an overall
increase in CO and global AC of 14.1+3.2 and 15.6+4.7% above
baseline, respectively, and an overall decrease in SVR of 9.7+2.4%
below baseline (all P<0.02). Serum relaxin and osmolality were
36+3 ng/ml and 287+1 mOsm/kg water, respectively. The latter
significantly decreased compared to vehicle infusion.
[0137] Arterial Pressure Waveforms.
[0138] Representative arterial waveforms from a single rat at
baseline and after administration of rhRLX are depicted in FIG. 3A.
They illustrate that the mouse pressure catheter (TA11PA-C20)
provides high fidelity recordings necessary for determining global
AC. Ensemble average arterial pressure waveforms, derived using the
methodology proposed by Burattini et al. (supra) are shown in FIG.
3B for the 3 groups of rats on day 10 of infusion. As discussed
above, SV significantly increased and SVR significantly decreased
following rhRLX administration (Tables 2 and 3). If these were the
only alterations, one would expect to see a clear change in
pressure waveform morphology: increased pulse pressure and hastened
diastolic decay of arterial pressure. However, as illustrated in
FIG. 3B, rhRLX administration did not significantly affect pressure
waveform morphology, as indicated by unchanged pulse pressure and
diastolic decay. This invariant pressure waveform morphology, in
the presence of increased SV and decreased SVR, is consistent with
a simultaneous increase in global AC.
In Vitro Studies
[0139] Arterial Passive Mechanics.
[0140] These in vitro experiments were performed to examine the
effects of rhRLX administration on passive (i.e., in the absence of
active smooth muscle tone) mechanical properties of vascular wall.
As mentioned before (Methods section), primary measurements
consisted of vessel inner and outer diameters at various levels of
intraluminal pressure. Circumferential wall stress (.sigma.) and
midwall radius (R.sub.m) were calculated from these primary
measurements and .sigma.-R.sub.m relationship was used to quantify
vessel wall elastic behavior (e.g., incremental elastic modulus,
E.sub.inc). .sigma.-R.sub.m (FIG. 4A) and E.sub.inc-R.sub.m (FIG.
4B) relationships for small renal arteries were significantly
different between the two groups (P<0.001 by analysis of excess
variance) such that .sigma. and E.sub.inc were smaller for a given
R.sub.m in the relaxin-treated group. In contrast, the unstressed
R.sub.m, R.sub.mo (i.e., R.sub.m at .sigma.=0), was not different
between the two groups (relaxin-treated: 105.+-.5 .mu.m;
vehicle-treated: 98.+-.6 .mu.m). Thus, the R.sub.m axis can be
considered as circumferential wall strain. These data indicate that
relaxin treatment significantly reduced vessel wall stiffness
(E.sub.inc) at matched R.sub.m (strain) values. This reduced
passive wall stiffness contributes to the increased global AC seen
in conscious animals with relaxin treatment (vide supra).
Example 2
Effects of Relaxin on Systemic Arterial Hemodynamics and Mechanical
Properties in Conscious Rats: Sex Dependency and Dose Response
Methods
Animals
[0141] Long-Evans male and female rats of 12-14 weeks were
purchased from Harlan Sprague-Dawley (Frederick, Md. USA). They
were provided PROLAB RMH 2000 diet containing 0.48% sodium (PME
Feeds Inc., St. Louis, Mo. USA) and water ad libitum. The rats were
maintained on a 12:12-h light-dark cycle. This investigation
conforms with the Guide for the Care and Use of Laboratory Animals
published by the US National Institute of Health (NIH Publication
No. 85-23, revised 1996).
Administration of Recombinant Human Relaxin (rhRLX)
[0142] The rhRLX (BAS, Palo Alto, Calif. USA) was provided as a 5.0
mg/ml solution in a buffer (20 mM sodium acetate, pH 5.0). It was
diluted as necessary in the same buffer. For the low dose infusion
protocol, two model 2002 osmotic minipumps (Durect Corporation,
Cupertino, Calif. USA) were used to deliver the rhRLX for 10 days
at the dose of 4 .mu.g/h. This dose was designed to yield
concentrations of circulating relaxin similar to those measured
during early to midgestation in rats, i.e., 10-20 ng/ml (Sherwood O
D, Endocrinol Rev 25(2): 205-234, 2004). For the high dose infusion
protocol, one model 2ML2 osmotic minipump was used to deliver rhRLX
at 50 .mu.g/h for 6 days, which was expected to produce serum
concentrations comparable to those recorded during late gestation
(Sherwood O D, Endocrinol Rev 25(2): 205-234, 2004) when further
increases in CO and decreases in SVR are observed in this species
(Gilson et al., Am J Physiol 32: H1911-H1918, 1992; Slangen et al.,
Am J Physiol 270: H1779-1784, 1996). Finally, in a third protocol,
rhRLX was administered by i.v. bolus over 3 min (13.44 ml) followed
by a continuous i.v. infusion for 4 hours.
Surgical Preparation
[0143] As in Example 1, rats were anesthetized with 60 mg/kg
ketamine i.m. and 21 mg/kg pentobarbital i.p. They were then
instrumented, using sterile technique, as follows: (i) a tygon
catheter implanted in the right jugular vein with the tip lying at
the junction of the anterior vena cava and right atrium, (ii) a
thermodilution microprobe (36 cm long, F#1.5; Columbus Instruments,
Columbus, Ohio USA) implanted in the abdominal aorta via the left
femoral artery with the tip lying 1.0 cm below the left renal
artery, and (iii) a mouse pressure catheter (TA11PA-C20, F#1.2;
Data Science International, St. Paul, Minn. USA) implanted in the
right carotid artery with the tip lying at the junction of the
right carotid artery and aortic arch. For the acute administration
of rhRLX, another tygon catheter was implanted in the inferior vena
cava via the left femoral vein such that the tip lay 1.0 cm below
the right renal artery.
[0144] After instilling 0.05 ml of a heparin solution into the
jugular catheter and plugging it with a straight pin, rats were
given ampicillin by drinking water for 2 days (100 mg/50 ml with 2
tablespoons of dextrose). Terbutrol was given s.c. for
post-operative analgesia.
[0145] For chronic administration of low dose recombinant human
relaxin (4.0 .mu.g/h rhRLX) in the male rats for 10 days, two Alzet
model 2002 osmotic minipumps (Durect Corporation, Curpertino,
Calif. USA) were inserted subcutaneously in the back of the animal
under isoflurane anesthesia. For chronic high dose administration
in the female rats for 6 days (80 .mu.g/h), one Alzet model 2ML2
osmotic minipump was implanted. High dose rhRLX was also
administered to another group of female rats acutely by intravenous
bolus over 3 min (13.4 .mu.g/ml) followed by a continuous infusion
for 4 h.
[0146] After completion of the measurement for the last time point,
rats were anesthetized with 60 mg/kg pentobarbital i.v. Blood was
obtained from the abdominal, aorta for measurements of plasma rhRLX
levels. The position of the jugular catheter relative to the right
atrium, the placement of the pressure catheter relative to the
aortic arch, and the position of the thermocouple relative to the
left renal artery were recorded.
Hemodynamics and Systemic Arterial Mechanical Properties
[0147] The low and high dose rhRLX protocols entailed 7 male and 9
female rats, respectively. After two baseline measurements of
systemic hemodynamics on days 5 and 7 after surgery, either low or
high dose rhRLX was administered by osmotic minipump. Systemic
hemodynamics were again assessed on days 3, 6, 8 and 10 after
initiation of relaxin infusion for the low dose male rats and days
3 and 6 for the high dose female rats. Each measurement consisted
of 4 to 8 recordings of cardiac output and blood pressure waveforms
obtained when the rat was either sleeping or resting. Seven to 10
minutes were allowed between recordings. These measurements were
obtained between 9 AM and 3 PM.
[0148] For acute administration of high dose rhRLX, 5 female rats
were used. Baseline measurements of systemic hemodynamics were
obtained followed by intravenous infusion of high dose rhRLX for 4
hours. Systemic hemodynamics were assessed continuously during the
4 hour infusion.
[0149] We used the thermodilution technique (Osborn et al., Am J
Physiol 251: H1365-H1372, 1986) to measure cardiac output.
Instantaneous aortic pressure waveforms were recorded using a blood
pressure telemetry system (Data Sciences International, St. Paul,
Minn. USA) (Mills et al., J Appl Physiol 88: 1537-1544, 2000). The
aortic pressure recorded by the pressure catheter implanted in the
aortic arch was transmitted to an external receiver. Steady-state
aortic pressure was digitized online using a PC-based data
acquisition system with 16 bit resolution and 2000 Hz sampling rate
and stored as text files for off-line analysis. Each measurement
consisted of a 30 second sampling duration.
[0150] Analysis of the acquired data and calculation of global AC
was performed using a custom computer program developed using
Matlab software (MathWorks Inc., Natick, Mass. USA). Briefly,
individual beats were selected (3-15 cycles) from the 10 seconds of
the aortic pressure recording, immediately preceding the
measurement of cardiac output. The ensemble was averaged as
described by Burattini et al. (2) to yield a single representative
beat for each trial. The mean arterial pressure (MAP), peak
systolic pressure (P.sub.s), and end diastolic pressure (P.sub.d)
were calculated from this averaged beat. Pulse pressure (PP) was
calculated as P.sub.s-P.sub.d. Systemic vascular resistance (SVR)
was calculated by dividing the MAP by CO.
[0151] Two measures of global arterial compliance were calculated.
The first (AC.sub.area) was calculated from the diastolic decay of
the aortic pressure waveform [P(t)] using the area method (18):
AC.sub.area=A.sub.d/[SVR(P.sub.1-P.sub.2)]
where P.sub.1 and P.sub.2 are the pressures at the beginning and
end of the diastolic decay curve, respectively, and A.sub.d is the
area under the P(t) waveform over this region. The second measure
of global arterial compliance was calculated as the stroke
volume-to-pulse pressure ratio, SV/PP (Chemla et al., Am J Physiol
274: H500-H505, 1998). Stroke volume was defined as CO/HR.
Serum Measurements
[0152] Serum osmolality was measured using a freezing-point
depression instrumentation osmometer (Model 3 MO; Advanced
Instruments, Needham Heights, Mass. USA). The levels of rhRLX in
serum were measured by a quantitative sandwich immunoassay as
previously described (Jeyabalan et al., Circ Res 93: 1249-1257,
2003).
Statistical Analysis
[0153] Data are presented as means.+-.SEM. Data from a previous
study (Conrad et al., Endocrinology 145(7): 3289-3296, 2004;
Example 1) wherein low and medium doses of rhRLX were administered
to female rats are included for comparison. Two-factor repeated
measures ANOVA (Zar J H, Biostatistical Analysis. Englewood Cliffs:
Prentice Hall, 1984) was used to compare mean values between low
dose male and female rats at various time points. The same analysis
was performed to compare mean values among low, medium, and high
doses of rhRLX in female rats at various time points. One-factor
repeated measures ANOVA (Conrad et al., Endocrinology 145(7):
3289-3296, 2004) was used to compare mean values at various time
points following initiation of high dose rhRLX acute infusion to
baseline values. If significant main effects or interactions were
observed, pairwise comparisons between groups were performed using
Fisher's LSD test. The student's paired `t` test was used to
compare the composite mean values (defined later) during chronic
infusion of rhRLX with baseline. P<0.05 was taken to be
significant. Finally, linear regression was used to analyze the
relationships between the magnitudes of the change in each arterial
property of individual rats in response to relaxin infusion and the
baseline values of that property. Group differences in the linear
regression parameters were examined using ANCOVA, implemented as
multiple linear regression with dummy variables (Gujarati D, Am
Statistician 24: 18-22, 1970).
Results
[0154] Male Rats Administered Low Dose rhRLX (4 .mu.g/h).
[0155] The temporal patterns of several systemic hemodynamic
variables, expressed as a percentage of baseline values, are
illustrated in FIG. 5 and absolute values of these variables are
presented in Table 4. For the purpose of comparison, the data from
our previous study (Conrad et al., Endocrinology 145(7): 3289-3296,
2004) examining the effects of rhRLX infusion at 4 .mu.g/h in
female rats are also presented in FIG. 5. Low dose rhRLX
significantly increased CO relative to baseline in male rats. There
was a slight (.about.6%), but statistically significant, rise in HR
in the relaxin-treated male rats (FIG. 5A). However, there was a
greater rise in SV (FIG. 5B) indicating that the elevation in CO
resulted primarily from an increase in SV, and to a lesser degree
from a rise in HR. Mean arterial pressure was not significantly
changed during rhRLX infusion (FIG. 5D). At the final time point
(i.e., day 10 after the onset of rhRLX infusion), there was no
statistically significant difference between the effects of rhRLX
administration on systemic hemodynamics in the male and female
rats.
[0156] The temporal effects of rhRLX infusion on systemic arterial
properties in male rats, expressed as a percentage of baseline
values, are depicted in FIG. 6. Once again, absolute values for
these variables are presented in Table 4 and data from female rats
are also shown in FIG. 6 for comparison. Systemic vascular
resistance fell significantly relative to baseline (FIG. 6A), while
both measures of arterial compliance (AC.sub.area and SV/PP) were
significantly increased (FIGS. 2B and 2C). At the final time point
(i.e., day 10 after the onset of rhRLX infusion), the changes in
arterial properties were not statistically different between male
and female rats.
TABLE-US-00004 TABLE 4 Male Rats Administered Low Dose rhRLX (4
.mu.g/h) Days after .DELTA..sub.T CO* HR* SV* AC.sub.area* SVR* MAP
minipump (.degree. C.) (mL/min) (bpm) (mL) (.mu.l/mmHg) (mmHg s/mL)
(mmHg) Baseline 0.32 .+-. 0.03 148 .+-. 9 416 .+-. 12 0.36 .+-.
0.03 7.5 .+-. 0.6 49 .+-. 3 115.8 .+-. 2.4 3 0.31 .+-. 0.01 160
.+-. 6 440 .+-. 8 0.36 .+-. 0.01 7.6 .+-. 0.5 46 .+-. 1 121.3 .+-.
3.6 6 0.29 .+-. 0.02 169 .+-. 4 441 .+-. 6 0.38 .+-. 0.01 7.9 .+-.
0.3 43 .+-. 1 119.9 .+-. 1.7 8 0.27 .+-. 0.02 178 .+-. 6 445 .+-. 4
0.40 .+-. 0.01 8.7 .+-. 0.4 41 .+-. 1 118.8 .+-. 2.0 10 0.28 .+-.
0.02 183 .+-. 11 442 .+-. 5 0.41 .+-. 0.02 8.8 .+-. 0.5 41 .+-. 2
121.6 .+-. 2.6
Mean.+-.SEM. N=7 rats. .DELTA..sub.T, change in blood temperature
after injection of Ringer's solution into the right heart; CO,
cardiac output; HR, heart rate; SV, stroke volume; AC.sub.area,
global arterial compliance calculated using area method; SVR,
systemic vascular resistance; MAP, mean arterial pressure.
*P<0.05 by single factor repeated measures ANOVA.
[0157] We calculated a composite mean change from baseline for each
variable by averaging values at all successive time points during
the infusion of rhRLX that were characterized by a significant
change from baseline and were not significantly different from each
other (i.e., the plateau phase). This yielded overall increases in
CO and global AC of 20.5.+-.4.2% and 19.4.+-.6.9% from baseline,
respectively, and an overall decrease in SVR of 12.7.+-.3.9% from
baseline (all P<0.05 vs. baseline). There was no statistical
difference between these results in male rats and those reported
for female rats (Example 1). Serum rhRLX was 17.7.+-.1.1 ng/ml, a
value similar to that previously observed in female rats
administered the same rhRLX regimen, 14.0.+-.2.0 ng/ml.
[0158] Female Rats Administered High Dose rhRLX (50 .mu.g/h).
[0159] Absolute values of systemic hemodynamics and arterial
properties are listed in Table 5 and their temporal patterns
following the initiation of rhRLX infusion are depicted in FIGS. 3
and 4. For the purpose of comparison, data from Example 1 examining
the effects of low (serum concentration=14.+-.2 ng/ml) and medium
(serum concentration=36.+-.3 ng/ml) dose rhRLX infusion in female
rats are also shown in FIGS. 3 and 4. Low and medium dose rhRLX
infusion significantly increased CO, mainly by increasing SV. Both
doses also significantly reduced SVR and increased AC (Conrad et
al., Endocrinology 145(7): 3289-3296, 2004). These alterations were
all observed by the earliest time point studied--3 days after the
onset of rhRLX administration. Serum rhRLX concentration for the
high dose infusion in the present study was 71.5.+-.1.6 ng/ml.
However, there was no change from baseline in any of the systemic
hemodynamics or arterial properties (FIGS. 3 and 4). Thus, the
effects of rhRLX on systemic hemodynamics and arterial properties
are apparently biphasic.
TABLE-US-00005 TABLE 5 Female Rats Administered High Dose rhRLX (50
.mu.g/h) Days after .DELTA..sub.T CO HR SV AC.sub.area SVR MAP*
minipump (.degree. C.) (mL/min) (bpm) (mL) (.mu.l/mmHg) (mmHg s/mL)
(mmHg) Baseline 0.34 .+-. 0.02 134 .+-. 5 425 .+-. 14 0.32 .+-.
0.02 7.0 .+-. 0.4 53 .+-. 2 115.9 .+-. 2.7 3 0.32 .+-. 0.02 141
.+-. 6 438 .+-. 6 0.32 .+-. 0.01 7.2 .+-. 0.3 52 .+-. 2 120.2 .+-.
3.3 6 0.31 .+-. 0.01 146 .+-. 6 437 .+-. 8 0.33 .+-. 0.02 7.4 .+-.
0.4 51 .+-. 3 121.1 .+-. 2.8
Mean.+-.SEM. N=8 rats. .DELTA..sub.T, change in blood temperature
after injection of Ringer's solution into the right heart; CO,
cardiac output; HR, heart rate; SV, stroke volume; AC.sub.area,
global arterial compliance calculated using area method; SVR,
systemic vascular resistance; MAP, mean arterial pressure.
*P<0.05 by single factor repeated measures ANOVA.
[0160] To determine whether there would be significant alterations
in systemic hemodynamics and arterial properties in response to
high dose rhRLX treatment at a time point earlier than 3 days after
the onset of rhRLX administration, an additional 5 female,
conscious rats were treated with acute i.v. infusion of rhRLX over
4 hours. Serum rhRLX concentration was 64.1.+-.1.0 ng/ml. The
temporal effects of short-term, high dose rhRLX infusion on
systemic hemodynamics and arterial properties in female rats,
expressed as a percentage of baseline values, are depicted in FIGS.
5 and 6. Heart rate was significantly increased (.about.13%) at
both the 2 and 4 hour time points (FIG. 9A). This increase in HR
was offset by a decrease (although statistically insignificant) in
SV (FIG. 9B), resulting in no significant change in CO (FIG. 9C).
There was a small (.about.8%), but statistically significant,
increase in MAP (FIG. 9D). There were no statistically significant
changes from baseline in any of the systemic arterial properties
(FIG. 9).
[0161] The above-described data suggests that the magnitude of the
change in arterial properties of individual rats (male or female)
in response to infusion of low dose rhRLX was dependent on the
baseline value of that particular property. To validate this trend,
the relationship between baseline values of SVR, AC.sub.area and
SV/PP and their respective composite percentage changes from
baseline during rhRLX infusion was analyzed. Linear regression
analysis revealed that the effect of rhRLX infusion (i.e., the
percent change from baseline) on SVR (FIG. 9A) and AC, as measured
by both AC.sub.area (FIG. 9B) and SV/PP (FIG. 9C), were all highly
dependent on their baseline values. Specifically, rats with low AC
at baseline were characterized by a greater increase in AC in
response to relaxin treatment. Similarly, rats that had high SVR at
baseline responded to relaxin with a greater decrease in SVR.
Further analysis (ANCOVA) indicated that these linear relationships
were not different between male and female rats.
[0162] The results above show that relaxin elicits similar effects
on systemic hemodynamics and arterial properties in both male and
female rats even though relaxin is traditionally considered to be a
female hormone and is not believed to circulate in male rats
(Sherwood O D, Endocrinol Rev 25(2): 205-234, 2004).
[0163] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *