Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE TREATMENT OF
LIPODYSTROPHY
BACKGROUND
Field of the Invention
The present invention is generally directed to methods and
compositions for the treatment of lipodystrophy. More particularly, the
instant
invention is directed to combination therapy using compositions comprising
growth
hormone and one or more statin drugs in order to treat lipodystrophy.
Background of the Related Art
Lipodystrophy is a syndrome caused by a deficiency and/or destruction
of adipocytes. The disorder is characterized by a selective loss of fat and is
associated
with hypertriglyceridemia, hepatic steatosis, and severe insulin resistance
that often
results in diabetes (Rossini et al., Metabolism., 26:637-650, 1977; Reitman et
al.,
T~e~rds E~docf~iyzol. Metab., 11:410-416, 2000; Arioglu et al., Any. Intern.
tiled.
133:263-274, 2000).
Congenital generalized lipodystrophy is an autosomal recessive
disorder that is characterized by a deficiency of adipose tissue and
accompanied by a
severe resistance to insulin, leading to hyperinsulinemia, hyperglycemia, and
an
enlarged fatty liver (Seip et al., Acta Paediat~. Suppl. 413:2-28, 1996). In
addition to
this congenital disorder, lipodystrophy also may be acquired. Most
significantly, it
has been recognized that the dramatic clinical benefits of highly active
antiretroviral
therapy (HAART) are hindered by the development of HIV-lipodystrophy syndrome.
This syndrome has received various designations, including HIV-associated
dysmorphia/dysmetabolic syndrome (HADDS), the term by which it is referred to
herein below. HIV-associated adipose redistribution syndrome CHARS) is
considered
as a subset of HADDS that is chiefly characterized by visceral
lipohypertrophy.
HADDS is characterized by abnormal fat deposition, atrophy,
metabolic complications, such as, hyperlipidemia, premature atherosclerotic
lesions,
and diabetes mellitus (Carr A et al., JAcquir Defic Syvrdf°., 33:571-
576, 2003), there
may also be an associated depletion of lean body mass. This syndrome is now
commonly encountered in over 60% of patients treated for HIV infection,
particularly
CONFIRMATION COPY
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in those individuals being treated with HIV therapeutic agents, such as
protease
inhibitors as well as use of nucleoside reverse transcriptase inhibitors, such
as
stavudine. As indicated by I~ravcilc (HITS Clip. Trials, 1 (3):37-50, 2000),
to date, the
pathogenesis of HIV-lipodystrophy syndrome remains largely unexplained and
most
therapies directed at fat changes have remained unsuccessful.
Thus, while HAART has effectively prolonged the lives of individuals
with Acquired Immuno Deficiency Syndrome (AIDS), converting the disease to a
chronic, high morbidity acquired HIV-related lipodystrophy syndrome, the
incidences
of this acquired form of lipodystrophy have increased alarmingly. (Carr et
al., AIDS.
12:F51-F58, 1998; Carr et al., NEfzgl JMed. 339:1296, 1998, Carr et al.,
Lahcet,
353:2093-2099, 1999, Miller et al., Lancet, 351:871-875, 1998; Vigouroux et
al.,
Diabete Metab. 25:225-232, 1999). The abnormalities in lipid metabolism seen
in
this disorder also may lead to the increased incidence of accelerated
atherosclerosis in
HIV patients (Barbaro et al., CliyZ. The~ap. 25(9):2405-18, 2003; Sklar et
al., N Engl J
Med., 349(21):2065-7, 2003; Friis-Moller et al., NE~gl JMed., 349(21):1993-
2003,
2003). Thus, HIV-related lipodystrophy is a multifactorial syndrome, and
currently,
there is no widespread treatment for this disease.
Atherogenic dyslipidemia (AD) is a central defect of the lipodystrophy
syndrome, and also is recognized as an independent coronary heart disease risk
factor.
It is also associated with, and contributes to the pathogenesis of any of the
conditions
included in lipodystrophy syndrome, including, but not limited to
hypertriglyceridemia (HTG), insulin resistance (IR), impaired glucose
tolerance (IGT)
and type 2 diabetes mellitus (DM-2). Because of the central role of AD in the
progression of the metabolic abnormalities associated with HIV-related
lipodystrophy, an effective treatment of AD is of utmost importance. Data for
the
HIV-uninfected population suggest lipodystrophy may further include high
levels of
total cholesterol, low-density-lipoprotein (LDL) cholesterol, and
triglycerides, and
low levels of high-density-lipoprotein (HDL) cholesterol. Because of these
established risks, the National Cholesterol Education Program (NCEP) has
issued
guidelines for the treatment of AD for (non-HIV-infected) patients at risk.
In non-HIV patients, obesity is recognized as being associated with
reduced growth hormone secretion (Veldhuis et al., JCli~ Endocni~ol Metab.
80:3209-3222, 1995; Veldhuis et al., JClin Endoc~i~ol Metab. 72:51-59, 1991;
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Ghigo et al., Metabolism. 41:560-563, 1992), where growth hormone
concentrations
have been shown to vary inversely with excess weight and body fat. HIV-
lipodystrophy is different from normal obesity, because as discussed above,
the fat
deposition in HIV-lipodystrophy is redistributed and the lipodystrophic
individuals do
not tend to be overweight. Recently it was demonstrated that individuals with
HIV
lipodystrophy and increased accumulation of visceral fat also have a decreased
growth hormone secretion (Rietschel et al., JCEM 86:504-510, 2001).
Thus, introduction of HAART with protease inhibitors and nucleoside
reverse transcriptase inhibitors has greatly improved the life-expectancy of
AIDS
patients. Unfortunately, along with the increased life-expectancy, these
patients
increasingly develop secondary complications that lead to abnormal lipid
distribution
disorders. There is no doubt about the effectiveness of HAART, and it will,
therefore,
continue to be used to prolong the lives of AIDS patients. This, therefore
leads to a
need for providing additional therapies to manage the side-effects of HAART
for the
long long-term maintenance therapy of AIDS patients. Significant among these
side-
effects of the HAART that require amelioration are lipodystrophy and other
HADDS-
related metabolic dysfunctions. Lipodystrophy also is seen in non-HIV
patients.
Thus, there is a need to identify new and effective methods for the
therapeutic
intervention of both HIV-related and non-HIV related lipodystrophy.
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SUMMARY OF THE INVENTION
The present invention is directed to methods and compositions for the
treatment of lipodystrophy. In specific embodiments, the present invention
provides a
method of treating a human suffering from an abnormal lipid distribution
disorder, the
method comprising administering to the subject a growth hormone and a statin-
based
therapeutic agent. Preferably, the statin-based agent and the growth hormone
are
provided in a single pharmaceutical composition. Other embodiments contemplate
providing the statin-based agent in a first pharmaceutical composition and the
growth
hormone in a second pharmaceutical composition. Preferably, the growth hormone
is
recombinant growth hormone. However, the growth hormone may be one which has
been isolated from an animal. The statin-based drug may be any statin-based
agent
known to those of skill in the art or any analog of a statin. Preferably, the
statin-based
agent is a lovastatin or a lovastatin analog. In exemplary embodiments, the
statin-
based drug is selected from the group consisting of atorvastatin, pravastatin,
simvastatin; lovastatin, and fluvastatin.
In specific embodiments, the abnormal lipid distribution disorder is
non-HIV-related lipodystrophy. In particularly preferred embodiments, the
methods
of the invention may be used to treat an HIV-related abnormal lipid
distribution
disorder. More particularly, the HIV-related abnormal lipid distribution
disorder is
selected from the atherogenic dyslipidemia, hypertriglyceridemia, elevated
levels of
cholesterol, elevated levels of low-density-lipoprotein cholesterol, and low
levels of
high-density lipoprotein cholesterol. In other embodiments, the method is used
in the
treatment of a subject who manifests a symptom associated with diabetes
related
adiposity. More particularly, the symptom of diabetes related adiposity is
selected
from the group consisting of insulin resistance, beta-cell dysfunction, loss
of first
phase insulin secretion, impaired glucose tolerance (IGT), elevated endogenous
glucose production, excessive gluconeogenesis. In specific embodiments, the
methods of the invention are used in the treatment of a subject is suffering
from Type
2 Diabetes. The methods of the invention may advantageously involve treating
the
individual with a therapy traditionally used for the treatment of diabetes.
For
example, the method comprises administering an insulin secretagogue.
Secretagogues
used as anti-diabetic agents are well known to those of skill in the art and
include, but
are not limited to, sulphonylureas; tolbutamide; chlorpropamide; glimepiride;
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glipizide; glyburide; a meglitinides; repaglinide; pramlintide;
morphilinoguanide;
acetylcholine; a muscarinic agonist; carbachol; bethanechol; beta-L-glucose
pentaacetate; chiro-inositol; myo-inositol; GIP; GLP-l; and Extendin-4.
Derivatives,
analogs and other molecules created by rational drug design based on these
molecules
may be used as the secretagogues.
In specific embodiments, the insulin secretagogue is a non-glucose
dependent insulin secretagogue, and the combined effect of administering the
growth
hormone, statin and insulin secretagogue produces insulin release patterns
capable of
attaining glucose dependent, biphasic release characteristics with reduced
likelihood
of producing hypoglycemia.
In certain examples, the methods of the present invention may further
comprise treating the subject with leptin.
Another aspect of the invention provides a therapeutic agent for use in
combination therapy for an abnormal lipid distribution disorder, the
composition
comprising a first composition comprising a recombinant growth hormone in a
pharmaceutically acceptable carrier, excipient or diluent; and a second
composition
comprising a statin-based drug in a pharmaceutically acceptable carrier,
excipient or
diluent. It is contemplated that the growth hormone and the statin-based drug
may be
formulated in a single formulation. Preferably, however, the growth hormone is
formulated in a separate formulation from the statin-based drug formulation.
In
specific embodiments, the growth hormone formulation and the statin-based drug
formulation may be formulated as injectable formulations. In preferred
embodiments
however, the statin-based drug formulation is formulated for oral
administration. In
specific embodiments, the therapeutic agent may be formulated into a kit which
contains the suitable implements for the administration of the various
therapeutic
components. The kit specifically may comprise lovastatin or an analog thereof
as the
statin-based drug. In specific embodiments, the statin-based drug of the
therapeutic
composition may be selected from the group consisting of atorvastatin,
pravastatin,
simvastatin, lovastatin, and fluvastatin. The compositions and methods of the
invention may employ a single statin agent or alternatively may employ two or
more
such agents.
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Other features and advantages of the invention will become apparent
from the following detailed description. It should be understood, however,
that the
detailed description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration only, because
various
changes and modifications within the spirit and scope of the invention will
become
apparent to those skilled in the art from this detailed description.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Acquired lipodystrophy (or lipohypertrophy) is a significant problem
in AIDS patients, leading to poor prognosis in individuals that are undergoing
HAART to combat-HIV infection. Other forms of lipodystrophy, e.g., obesity-
related
lipodystrophy that has attendant diabetes-related complications also provides
a
significant challenge to the current medical community. All of these
lipodystrophies
and disorders of lipid distribution in HIV and non-HIV patients may
generically be
referred to as "abnormal lipid distribution disorders." The present invention
provides
new methods for the treatment of obesity. These therapeutic methods are based
on
the novel combination of growth hormone and statin-based drugs hyperlipidemic
drugs such as LipitorTM (atorvastatin), PravacholTM (pravastatin), ZocorTM
(simvastatin), MevacorTM (lovastatin), and Lescol TM (fluvastatin. Both of
these
classes of drugs (i.e., growth hormone and statins) are well known to those of
skill in
the art and therefore the current recognition that these agents can be
combined to
achieve a therapeutically beneficial amelioration of the symptoms of
lipodystrophy.
This novel fording is exploited in the present invention to teach new methods
and
composition combinations for achieving such a therapeutic outcome. The
disorders,
methods and compositions are discussed in further detail herein below.
A. Disorders to be Treated by the Invention
As discussed herein above, the majority of HIV patients that are
receiving HAART experience HADDS, which involves the pathological accumulation
of adipose tissue in specific regional depots. The pathologic adipose tissue
accumulation of HADDS may also be associated with abnormal adipose tissue
depletion elsewhere (lipodystrophy or lipoatrophy), with or without associated
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metabolic abnormalities, premature atherosclerotic lesions, depletion of lean
body
mass, and/or other abnormal physiology. The methods of the present invention,
which comprise administering a combination of growth hormone and statin drugs,
results in the treatment of HADDS. This treatment is evidenced by a decrease,
amelioration, or correction of any of the symptoms associated with HADDS.
Thus, in certain embodiments, it is contemplated that the methods of
the invention will reduce the abnormal accumulation of adipose tissue in the
abdomen, specifically in the visceral adipose tissue compartment (Miller et
al, Lancet
21, 351(9106):871-875, 1998; Kotler et al., JAequi~ed Immun Defic Syndr 20:228-
237, 1999; I~otler et al., HIV/AIDS 1999 Annual Update, 85-92,
hiv.medscape.com,
1999; Engelson et al., Ahtivi~al Therapy 4:(Sup 2):11 [Abstract 006], 1999;
Englemson et al., Am JClin Nutf° 69(6):1162-1169, 1999) in patients
that have this
symptom. HADDS patients may also present with abnormal adipose tissue
accumulation in the dorsocervical area ("buffalo hump"), the submandibular
area
("horse collar"), the pectoral, mammary, and/or supraclavicular areas, and/or
with
subcutaneous lipomas (encapsulated benign fatty tumors, single or multiple).
It is
contemplated that the combination therapy of the invention which comprises at
least a
growth hormone composition and one statin-based therapeutic agent (e.g.,
LipitorTM,
PravacholTM, ZocorTM, MevacorTM, and Lescol TM, or other analog of lovastatin)
will
decrease, or eliminate the abnormal adipose tissue so that there is a
reduction in the
size or amount of fatty deposit at one or more of the areas selected from the
group
consisting of abdominal (visceral) fat deposits, deposits in the dorsocervical
area,
submandibular area, the pectoral, mammary, supraclavicular areas, and/or in
the
subcutaneous lipomas.
In addition to ameliorating the above abnormal adiposity, the methods
of the present invention also are directed to decreasing lipoatrophy in HADDS
patients. HADDS patients are known to develop abnormally depleted subcutaneous
adipose tissue, termed "peripheral lipodystrophy" (or lipoatrophy) at other
specific
sites. This adipose depletion is typically observed in the face (buccal,
parotid, and
periauricular fat pads), and in the subcutaneous adipose tissue surrounding
the limbs,
trunk, and/or gluteal regions. Thus, the present invention specifically
contemplates
methods of decreasing HADDS-associated subcutaneous lipid depletion by
administering a combined therapy of growth hormone and at least one statin
drug.
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Some HADDS patients also present with metabolic abnormalities (Carr
et al., AIDS 12:F51-58,1998; Carr et al., Lancet 131:1881-1883,1998; Carr et
al.,
Lancet 353(9170):2093-2099,1999; Carr et al., A~tiviral Therapy 4(Sup 2):19
[Abstract 11],1999; Lipodystrophy Rapid Report, 1999) associated with
disordered
lipid and/or glucose metabolism. Clinical manifestations may include fasting
hypertriglyceridemia, hyperlipidemia, and abnormalities of the insuliuglucose
axis
(elevated fasting insulin, elevated C-peptide, insulin resistance or reduced
insulin
sensitivity), with or without overt diabetes (Gan et al., AIDS 12:F51-58,1998;
Carr et
al., Lancet 131:1881-1883,1998; Carr et al., Lancet 353(9170):2093-2099,1999;
Carr
et al., Antivir°al Thef°apy 4(Sup 2):19 [Abstract 11],1999;
Henry et al., Lancet
351:1328, 1998; Henry et al., Lafzcet 352:1031-1032, 1998; Grunfeld, Ahtiviral
Therapy 4 (Sup 2):7 [Abstract 004], 1999). The methods of the invention are
useful
in treating one or more of these metabolic dysfunctions.
Another disorder that is specifically contemplated to be treated by the
present invention include HADDS-related coronary heart disease (CHD). There
are
preliminary reports suggesting that patients with HADDS exhibit preclinical
evidence
of increased risk for coronary heart disease (CHD). Preclinical indicators of
CHD
include increased coronary artery calcification (CAC) as quantified by
electron beam
computed tomography (EBCT), and extracoronary indicators such as increased
intima
media thickening (IMT) in the carotid artery and impaired blood flow-mediated
dilation in the brachial artery, as quantified by ultrasonography, which
signify
endothelial dysfunction that may lead to atherosclerosis and CHD. In eight
patients
with HADDS who developed increased abnormal girth with abnormally accumulated
visceral adipose tissue after initiation of HIV protease inhibitor (PI)
therapy who
underwent EBCT, I~osmiski et al., Ayztivi~al Therapy 4(Sup 2):49 [Abstract
056],
(1999) reported a mean CAC score consistent with minimal identifiable plaque
burden. There are also preliminary reports indicating that HIV patients
receiving PIs
display abnormal carotid IMT (Maggi et al., Antivinal Therapy 4(Sup 2):39
[Abstract
038], 1999) and impaired brachial flow-mediated dilation (Stein, Conference
News
Reports, AIDS Weekly via NewsRx.com (November 22, 1999), signifying
endothelial
dysfunction. The therapeutic methods of the present invention may be used to
treat
one or more of the above-discussed symptoms of HADDS-related CHD.
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Some patients with HADDS also exhibit involuntary weight loss with
depletion of lean body mass (AIDS wasting or cachexia), and possibly depletion
of
lean body mass without overt weight loss (occult wasting). The methods of the
invention may produce a useful weight gain in such individuals. Other abnormal
physiology that may be treated in patients with HADDS or lipodystrophy
syndrome
using the methods of the present invention include gout and pancreatitis
(presumably
resulting from severe hypertriglyceridemia), hepatic steatosis (possibly
reflecting
chronic lactic acidosis), hypogonadism, and possibly other hormonal
abnormalities
(Henry et al., Lancet 351:1328, 1998; Henry et al., Lafzeet 352:1031-1032,
1998;
Brinkman, AhtivifAal Therapy 4:(Sup 2):15 [Abstract 009], 1999; Lipodystrophy
Rapid
Report, 1999).
HADDS and lipodystrophy syndrome may or may not be associated with other
abnormalities, such as cutaneous abnormalities (such as thinning hair, hair
loss, hair
brittleness, dry skin, abnormal nails, ingrown toenails), disorders of the
coagulation
syndrome that result in increased bleeding in hemophiliacs, osteoporosis or
avascular
necrosis of the hips, peripheral neuropathy, nausea, fatigue, weight loss,
chronic
diarrhea, fever, mennorhagia and menstrual abnormalities, impaired sexual
dysfunction (decreased libido, erectile dysfunction), and abnormalities of the
genitalia
resembling Peyronie's Disease (Carr et al., AIDS 12:F51-58,1998; Carr et al.,
Lancet
131:1881-1883,1998; Carr et al., Lancet 353(9170):2093-2099,1999; Carr et al.,
Antivif°al Therapy 4(Sup 2):19 [Abstract 11],1999; Lipodystrophy Rapid
Report,
1999). The combined therapeutic regimen of the present invention may prove
useful
in ameliorating some or all of these HADDS-related pathologies.
In addition to lipodystrophy that manifests in HIV patients, the
methods of the present invention also may be used to treat obesity-relate
lipodystrophy in non-HIV subjects.
B. Compositions for Use in the Methods of the Invention
The methods of the present invention employ a combination of growth
hormone and statin-related drugs in order to effect treatment of
lipodystrophy. It is
contemplated that any one or more of the symptoms of lipodystrophy exemplified
herein above may be ameliorated by the use of this combination therapy. As
both
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growth hormone and statin-related agents have previously been used in the
treatment
of other disorders, those of skill in the art will readily be able to adapt
existing
compositions and regimens for use in the present invention. Simply by way of
example, the following section describes exemplary growth hormone and statin-
5 related compositions that may be used in the present invention.
a. Growth Hormone
One of the active agents used in the therapeutic methods of the present
invention is growth hormone. Preferably, the growth hormone used is human
growth
10 hormone. Human growth hormone, also known as somatotropin, is a protein
hormone
produced and secreted by the somatotropic cells of the anterior pituitary.
Secretion is
regulated by a releasing factor, i. e., the growth hormone-releasing hormone
(GHRH), and by an inhibitory factor, somatostatin. Human growth hormone plays
a
key role in somatic growth through its effects on the metabolism of proteins,
carbohydrates and lipids.
Human growth hormone is a single polypeptide chain of 191 amino
acids (Bewley et al., Irzt JPept Protein Res 4(4):281-287, 1972) having two
disulfide
bonds, one between Cys-53 and Cys-165, forming a large loop in the molecule,
and the other between Cys-182 and Cys-189, forming a small loop near the C-
terminus. The DNA sequence that confirmed the amino acid sequence was reported
by Martial et al., Science 10;205(4406):602-607, 1979. Purified hGH is a white
amorphous powder in its lyophilized form. It is readily soluble
(concentrations >10
mglL) in dilute aqueous buffers at pH greater than 7.2.
In solution, hGH exists predominantly as a monomer, with a small
fraction as dimers and higher molecular weight oligomers. Under certain
conditions,
hGH can be induced to form larger amounts of dimers, trimers and higher
oligomers.
Several derivatives of hGH are known, including naturally-occurring
derivatives,
variants and metabolic products, degradation products primarily of
biosynthetic hGH
and engineered derivatives of hGH produced by genetic methods. One example of
a
naturally-occurring derivative of hGH is GH-V, a variant of growth hormone
found
in the placenta. Other members of the gene locus are described in Chen et al.,
Ge~omics 4(4):479-497, 1989.
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11
Any derivative of hGH, including derivatives designed to be long-
lasting in the body, can be used for the purpose of the present invention as
long as it
retains the biological activity of hGH. Further it is contemplated that mixed
populations of GH derivatives also may be used.
Methionyl hGH, which was the first form of hGH to be produced
through recombinant DNA technology will be useful in the present invention.
This
compomzd is a derivative of hGH having one additional methionine residue at
its N-
terminus (Goeddel et al., Nature 281(5732):544-548, 1979). Another GH that
would
be useful is naturally-occurring variant of hGH called 20-k-hGH. This variant
of
hGH has been reported to occur in the pituitary as well as in the bloodstream
(Lewis
et al., JBaol Chem 25;253(8):2679-2687, 1978; Lewis et al., Biochem Biophys
Res
Comyn 29;92(2):511-516, 1980). This compound, which lacks the 15 amino acid
residues from Glu-32 to Gln-46, arises from an alternative splicing of the
messenger ribonucleic acid (DeNoto et al., Nueleic Acids Res 9(15):3719-3730,
1981). This compound shares many, but not all of the biological properties of
hGH.
This derivative may be used in the methods and combination therapy
compositions of
the present invention.
20-k-hGH is made in the pituitary and secreted into the blood. It
makes up about 5% of growth hormone output of adults, and about 20% of growth
hormone output of children. It has the same growth promoting activity as 22 kD
growth hormone, and has been reported to have equal to, or greater than, the
amount
of lypolytic activity as the 22 kD form. It binds to growth hormone receptors
with
equal affinity as the 22 kD growth hormone, and has one tenth the lactogenic
(prolactin-lilce) bioactivity as the 221cD hormone. Unlike 22 kD, the 20- k-
hGH has
weak anti-insulin activity.
A number of derivatives of hGH arise from proteolytic modifications
of the molecule. The primary pathway for the metabolism of hGH involves
proteolysis. The region of hGH around residues 130-150 is extremely
susceptible to
proteolysis, and several derivatives of hGH having nicks or deletions in this
region
have been described (Thorlacius-Ussing, Neuroendocrinology, 45(3):233-242,
(1987)). This region is in the large loop of hGH, and cleavage of a peptide
bond in
this region results in the generation of two chains that are connected through
the
disulfide bond at Cys-53 and Cys-165. Many of these two-chain forms are
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12
reported to have increased biological activity (Singh et al., Endocr~ivcology
94(3):883-
891, 1974). Many derivatives of human growth hormone have been generated
artificially through the use of enzymes. The enzymes trypsin and subtilisin,
as well as
others, have been used to modify hGH at various points throughout the molecule
(Lewis et al., Endocrinology 101(5):1587-1603,1977; Graff et al., JBiol Chefya
257:2365, 1982). One such derivative, called two-chain anabolic protein (2-
CAP),
was formed through the controlled proteolysis of hGH using trypsin (Becker et
al,
Abstract No. 342, 71St Annual Meeting, The Endocrine Society, Seattle, WA,
June
1989). 2-CAP was found to have biological properties very distinct from those
of the
intact hGH molecule, in that the growth-promoting activity of hGH was largely
retained and most of the effects on carbohydrate metabolism were abolished.
Asparagine and glutamine residues in proteins are susceptible to deamidation
reactions under appropriate conditions. Pituitary hGH has been shown to
undergo this
type of reaction, resulting in conversion of Asn-152 to aspartic acid and
also, to a
lesser extent, conversion of Gln-137 to glutamic acid (Lewis et al., JBiol
Chem
25;256(22):11645-11650, 1981). Deamidated hGH has been shown to have an
altered
susceptibility to proteolysis with the enzyme subtilisin, suggesting that
deamidation
may have physiological significance in directing proteolytic cleavage of hGH.
Biosynthetic hGH is known to degrade under certain storage conditions,
resulting in
deamidation at a different asparagine residue (Asn-149). This is the primary
site of
deamidation, but deamidation at Asn-152 has been observed (Beclcer et al.,
BioteclZyzol Appl Biochem 10(4):326-337, 1988). Deamidation at Gln-137 has not
been reported in biosynthetic hGH.
Methionine residues in proteins are susceptible to oxidation, primarily to the
sulfoxide. Both pituitary-derived and biosynthetic hGH undergo sulfoxidations
at
Met-14 and Met-125 (Beclcer et al., Bzotechnol Appl Biochem 10(4):326-337,
1988).
Oxidation at Met-170 has also been reported in pituitary but not biosynthetic
hGH.
Both desamide hGH and Metl4 sulfoxide hGH have been found to exhibit full
biological activity (Becker et al., Biotechhol Appl Biochem 10(4):326-337,
1988).
Truncated forms of hGH have been produced, either through the actions of
enzymes
or by genetic methods. 2-CAP, generated by the controlled actions of trypsin,
has the
first eight residues at the N-terminus of hGH removed. Other truncated
versions of
hGH have been produced by modifying the gene prior to expression in a suitable
host.
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13
The first 13 residues have been removed to yield a derivative having
distinctive
biological properties (Gertler et al., Ehdocy~inology 72118(2):720-6, 1986) in
which
the polypeptide chain is not cleaved.
The term "human growth hormone", as used in the present invention, is
intended to include the naturally-occurring derivatives, as noted above,
including,
without limitation, both the 20 kD and the 22 kD human growth hormone, GH-V,
and
other members of the growth hormone gene locus as described in Chen et al.,
Ge~omics 4(4):479-497, 1989. The term also includes functional derivatives,
fragments, variants, analogs, or salts which retain the biological activity of
growth
hormone, a.e., which act as agonists to the growth hormone receptor. In other
words,
they are capable of binding to the growth hormone receptor to initiate the
signaling
activity of the receptor.
"Functional derivatives" as used herein covers derivatives which may be
prepared from the functional groups which occur as side chains on the residues
or the
N- or C-terminal groups, by means known in the art, and are included in the
invention as long as they remain pharmaceutically acceptable, i.e., they do
not destroy
the biological activity of hGH as described herein, i.e., the ability to bind
the hGH
receptor and initiate receptor signaling, and do not confer toxic properties
on
compositions containing the derivative. Derivatives may have chemical
moieties,
such as carbohydrate or phosphate residues, provided such a derivative retains
the
biological activity of hGH and remains pharmaceutically acceptable.
For example, derivatives may include aliphatic esters of the carboxyl groups,
amides of the carboxyl groups by reaction with ammonia or with primary or
secondary amines, N-acyl derivatives or free amino groups of the amino acid
residues formed with acyl moieties (e.g., allcanoyl or carbocyclic aroyl
groups) or O-
acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl
residues) formed
with acyl moieties. Such derivatives may also include, for example,
polyethylene
glycol side-chains which may mask antigenic sites and extend the residence of
the
molecule in body fluids.
Of particular importance is a growth hormone that has been derivatized or
combined with a complexing agent to be long lasting. For example, pegylated
versions, or growth hormones genetically engineered to exhibit long lasting
activity in
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14
the body, can be used to treat HADDS, or other abnormal lipid distribution
disorder
according to the present invention. Exemplary of hGH compositions with
increased
half life include e.g., Nutropin DepotTM a slow releasing polyactide-
coglycolide
encapsulated hGH marlceted by Alkermes/Genentech (Cook et al., J Clip
E~docrinol
Metab. 87(10):4508-14, 2002) and AlbutropinTM (HGS), an albumin fusion of hGH
that is in clinical trials (Osborn et al., Euf° JPhat~macol.;456(1-
3):149-58, 2002).
hGH that is acetylated at the N-terminus has been isolated and identified
(Lewis et al., E~rdoc~inology 104(5):1256-1265, 1979). It is not clear if
acylation
serves a regulatory role or is simply an artifact of the purification.
However, it is
expected that this the molecule exhibits anti-HADDS activity in a similar
fashion to
other hGH derivatives.
The term "salts" herein refers to both salts of carboxyl groups and to acid
addition salts of amino groups of the hGH molecule or analogs thereof. Salts
of a
carboxyl group may be formed by means known in the art and include inorganic
salts,
for example, sodium, calcium, ammonium, ferric or zinc salts, and the like,
and salts
with organic bases as those formed, for example, with amines, such as
triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid
addition
salts include, for example, salts with mineral acids, such as, for example,
hydrochloric
acid or sulfuric acid, and salts with organic acids, such as, for example,
acetic acid or
oxalic acid. Of course, any such salts must retain the biological activity of
hGH
relevant to the present invention, i.e., the ability to bind to the hGH
receptor and
initiate receptor signaling.
A "fragment" of the growth hormone according to the present invention refers
to any subset of the molecule, that is, a shorter peptide which retains the
desired
biological activity. Fragments may readily be prepared by removing amino acids
from either end of the hGH molecule and testing the resultant for its
properties as an
hGH receptor agonist. Proteases for removing one amino acid at a time from
either
the N-terminal or the C- terminal of a polypeptide are known, and so
determining
fragments which retain the desired biological activity involves only routine
experimentation.
Additionally, the polypeptide which has such hGH receptor agonist activity,
be it hGH, an analog or variant, salt, functional derivative or fragment
thereof, can
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also contain additional amino acid residues flanking the hGH polypeptide. As
long as
the resultant molecule retains the hGH receptor agonist ability of the core
polypeptide, one can determine whether any such flanking residues affect the
basic
and novel characteristics of the core peptide, i. e., its receptor agonist
characteristics,
by routine experimentation. The term "consisting essentially of', when
referring to a
specified sequence, means that additional flanking residues can be present
which do
not affect the basic and novel characteristic of the specified sequence.
A "variant" of the human growth hormone according to the present invention
refers to a molecule which is substantially similar to either the entire
peptide or a
10 fragment thereof. Variant peptides may be conveniently prepared by direct
chemical
synthesis of the variant peptide, using methods well known in the art. Of
course, a
variant human growth hormone would preferably have similar hGH receptor
binding
and signal initiating activity as hGH and which would, therefore, be expected
to have
similar anti-HADDS activity to hGH.
15 Amino acid sequence variants of the human growth hormone can be prepared
by mutations in the DNAs which encode the synthesized human growth hormone
derivatives. Such variants include, for example, deletions from, or insertions
or
substitutions of, residues within the amino acid sequence. Any combination of
deletion, insertion, and substitution may also be made to arrive at the final
construct,
provided that the final construct possesses the desired activity. Obviously,
the
mutations that will be made in the DNA encoding the variant peptide must not
alter
the reading frame and preferably will not create complementary regions that
could
produce secondary mRNA structure (see European Patent Publication No. EP
75,444,
the entire contents of which are hereby incorporated by reference).
At the genetic level, these variants ordinarily are prepared by site-directed
mutagenesis (as exemplified by Adelman et al., DNA 2(3):183-193, 1983) of
nucleotides in the DNA encoding the peptide molecule, thereby producing DNA
encoding the variant, and thereafter expressing the DNA in recombinant cell
culture.
The variants typically exhibit the same qualitative biological activity as the
non-
variant peptide.
An "analog" of human growth hormone according to the present invention
refers to a non-natural molecule which is substantially similar to either the
entire
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16
molecule or to an active fragment thereof. An analog of human growth hormone
useful in the present invention would preferably exhibit anti-HADDS activity,
at least
when administered in combination with a statin, but preferably when
administered
alone.
Examples of production of amino acid substitutions in proteins which can be
used for obtaining analogs of the hGH for use in the present invention include
any
known method steps, such as presented in U.S. Patents RE 33,653; 4,959,314;
4,588,585 and 4,737,462, to Mark et al.; 5,116,943 to Koths et al.; 4,965,195
to
Namen et al.; and 5,017,691 to Lee et al., and lysine substituted proteins
presented in
US patent 4,904,584 (Shaw et al.).
Among the substances which bind to, and initiate, signaling of the human
growth hormone receptor which may be used in accordance with the present
invention
are all of those growth hormone analogs and mimetics already known in the
literature,
such as, for example, are disclosed in U.S. Patents 5,851,992; 5,849,704;
5,849,700;
5,849,535; 5,843,453; 5,834,598; 5,688,666; 5,654,010; 5,635,604; 5,633,352;
5,597,709; and 5,534,617.
Preferably, the hGH variant or analog will have a core sequence, which is the
same as that of the native sequence or biologically active fragment thereof,
which has
an amino acid sequence having at least 70% identity to the native amino acid
sequence and retains the biological activity thereof. More preferably, such a
sequence
has at least 80% identity, at least 90% identity, or most preferably at least
95%
identity to the native sequence.
Although human growth hormone was originally obtained from pituitary
glands of cadavers, these preparations were not electorophoretically
homogeneous,
and antibodies appeared in the serum of patients treated with preparations of
the order
of 50% purity, the immunogenicity being attributed to inactive components.
Recombinant DNA technology permitted production of an unlimited supply of hGH
in a number of different systems. Purification of hGH from the culture medium
is
facilitated by the presence of only low amounts of contaminating proteins. In
fact, it
has been shown that hGH can be purified on a laboratory scale by a single
purification
step on a reversed-phase HPLC column (Hsiung et al., Biotecl~rzology 7:267,
1989).
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17
Recombinant human growth hormone, rhGH, is produced by Serono S.A., as
SEROSTIM~, which product has been given FDA approval for treating weight loss
and wasting in HIV patients. PROTROP1N~, produced by Genentech, Inc. (South
San Francisco, CA), differs slightly in structure from natural sequence hGH,
having
an additional methionine residue at the N-terminus. Recombinant hGH is
generally
marketed as vials containing hGH plus additional excipients, e.g., glycine and
mannitol, in a lyophilized form. A companion diluent vial is provided,
allowing the
patient to reconstitute the product to the desired concentration prior to
administration
of the dose. Recombinant hGH can also be marketed in other well-known manners,
such as prefilled syringes, etc.
In specific embodiments, it is contemplated that that methods
described herein will use SEROSTIM~, a recombinant human growth hormone
(rhGH) produced by Serono S.A. This product has recently been given full FDA
approval for treating wasting syndrome in patients with HIV-associated wasting
or
cachexia. Such growth hormone compositions will be useful in the methods of
the
present invention. Previously, it has been noted that many patients with overt
AIDS
wasting are able to tolerate relatively high doses (6 mg/day) of recombinant
growth
hormone (rhGH; Serono's SEROSTIM~) administered subcutaneously (s.c.), without
developing adverse effects that require dose reduction or cessation of therapy
(Schambelan et al., Ann Ihte~h Med 125(11):873-882, 1996).
It is contemplated that those of skill in the art can vary the dose of
rhGH and monitor the patient for development of adverse symptoms from the rhGH
administration. It is specifically contemplated that the subject will receive,
1 mg/day,
2 mg/day, 3 mg/day, 4 mg/day, 5 mg/day, 6 mg/day, 7 mg/day, 8 mg/day, 9
mg/day,
10 or more rhGH on a daily basis. Such therapy may be administered in a single
dose
or it may alternatively be divided into multiple doses to be administered at
set
intervals during the day. It also is contemplated that the GH composition may
be
administered at time intervals other than daily. For example, the subject may
be given
the GH therapy every other day or every week. The symptoms to be monitored to
assess the adverse effects of the GH include, but are not limited to, tissue
turgor, joint
stiffness, arthalgias, and/or paresthesias. The clinician will be able to use
such
symptoms as guidance parameters to assess whether a given dose of GH should be
adjusted (up or dowxn).
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18
Currently, those of skill in the art are conducting dose-ranging trials to
investigate the additional effective and safe doses of rhGH for patients with
HADDS.
Thus, doses of GH for use in AIDS patients are well known to those of skill in
the art.
Doses from such studies may readily be adapted for use in the methods
described
herein.
Windisch et al., Anti Phar~nzacothe~ 32(4):437-445, 1998, reported that
HIV-associated wasting was characterized by weight loss, depletion of lean
body
mass and preservation of body fat, leading to muscle weakness and organ
failure.
Although the FDA has approved recombinant growth hormone for treating HIV-
associated wasting, the adverse event profile is similar to that of other
recombinant
growth hormone products. Trials of recombinant growth hormone on the control
of
wasting in patients with HIV/AIDS have been encouraging. Post-marketing
experience with over 10,000 HIV/AIDS wasting patients receiving SEROSTIM~
since 1996 reveals that a three-month course of therapy was effective in the
majority
of patients with AIDS wasting.
Given the previous findings seen with SEROSTIM~'s effects on AIDS
wasting, it is contemplated that the therapeutic methods of the present
invention,
which contemplate a combined therapy in which rhGH is administered in
combination
with at least one statin drug, may be used periodically to control HIV-
related, or other
abnormal lipid distribution disorder. For example, such periodic therapy would
entail
treating the patient with a course of the combination therapy for a period of
1, 2, 3, 4,
5 or 6 months. Alternatively, in patients with HIV-related lipodystrophy or
HADDS,
the methods of the present invention may be used as a continuous therapy in
conjunction with HAART to control the adverse side effects of HAART that
manifest
in lipodystrophy, HADDS, and the like. It should also be understood that, to
be
useful, the treatment provided need not be absolute, provided that it is
sufficient to
carry clinical value. An agent which provides treatment to a lesser degree
than do
competitive agents may still be of value if the other agents are ineffective
for a
particular individual, if it can be used in combination with other agents to
enhance the
overall level of protection, or if it is safer than competitive agents.
During the therapy it would be advantageous to monitor the symptoms
of the patient to ensure that adverse effects from the GH are not being
experienced.
In the event that adverse effects are not seen and the combined therapy is not
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19
producing a fast enough therapeutic outcome, a more aggressive course of
therapy
may be administered wherein the dose of the rhGH and/or the statin drug is
increased.
In the event that the therapy produces side effects such as tissue turgor,
joint stiffness,
arthalgias, and/or paresthesias, the dose of the rhGH may be reduced to
alleviate the
side effects.
I~rentz et al., JAcquir Inamune Defic Syndf° 6(3):245-251, 1993,
compared metabolic and anthropometric changes induced by recombinant human
growth hormone dosed at 5.0 mg versus 2.5 mg every other day (qod) in 10
patients
with HIV/AIDS. During treatment, insulin-like growth factor-I (IGF-I) levels
increased significantly in the pharmacological rhGH treatment group receiving
5.0 mg
qod, whereas no significant change was observed in IGF-I in the group
receiving 2.5
mg qod of rhGH. In the group treated with 5.0 mg qod dose of hGH, weight loss
preceding the study was reversed in each of the four patients who completed
the
study. This weight gain was associated with increases in lean body mass and
total
body water, and with concomitant decreases in fat mass and urinary nitrogen
excretion.
In a large, randomized, placebo-controlled study, Schambelan et al.,
Ihte~°n Med 125(11):873-882, 1996, used dual X-ray absorptiometery
(DXA) scanning
to evaluate changes in body composition produced by administration of
recombinant
human growth hormone dosed at 0.1 mg/kg/day (or 4 to 6 kg per day, depending
on
patient weight) compared to placebo over a 12 weeks course of therapy. By the
end
of treatment, significant increases in lean body mass and weight are observed
in the
rhGH group, compared to the placebo group, and these increases correlated with
improvements in physical function (treadmill performance). The rhGH therapy
was
associated with minor increments in fasting plasma glucose, which were of
negligible
clinical significance.
The studies of I~rentz et al., JAcqz~if° Irnynune Defic Syhd~
6(3):245-
251, 1993 and Schambelan et al., Intern Med 125(11):873-882, 1996, may readily
be
repeated with the combination of GH/statin-based therapeutic methods of the
present
invention. Such determinations would be merely routine, and would show the
beneficial effects of the reducing, ameliorating or otherwise improving one or
more
symptoms of lipodystrophy.
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b. Statin Drugs
As discussed herein throughout, the therapeutic methods for the
present invention employ a second compound in addition to the GH. The second
active compound in the combination therapy of the present invention is a
statin-
5 related agent. By "statin-related" agent or drug the present application
refers to any
statin drug that is presently on the market, or is modified from the presently
marketed
statin drugs, and has a therapeutic effect when combined with the growth
hormone
compositions used in the invention. As such it should be understood that
analogs and
variants of preexisting statins are contemplated to be useful herein. Such
analogs or
10 variants may be produced through rational drug design techniques knomn to
those of
skill in the art. In particular, statin drugs are known as HMGCoA reductase
inhibitors. These drugs are presently in clinical use as drugs in the battle
against high
cholesterol and in the control of heart attacks, both recurrent and first
heart attacks.
These agents generally have few side effects, and help not only to lower
overall
15 cholesterol, LDL cholesterol and triglycerides, but also to increase HDL
cholesterol.
Statins are exemplified by lovastatin (CAS Registry No. 75330-75-5;
also known as mevinolin or monacolin K), and analogs of this compound have
been
described in numerous publications and patents. Exemplary statin compositions
that
are commercially available include LipitorTM (atorvastatin), PravacholTM
20 (pravastatin), ZocorTM (simvastatin), MevacorTM (lovastatin), and Lescol TM
(fluvastatin). Methods of preparing such compounds are well known to those of
skill
in the art (see e.g., U.S. Patent Nos. 6,521,762; 4,420,491; 4,342,767;
4,319,039;
4,294,846; 4,444,784; 4,582,915 and 4,820,850). As described in the foregoing
patents, statins are traditionally produced through fermentation using
organisms from
the Aspergillus genus, Mo~ascus genus, Pleu~otus genus, Cohiothy~ium genus and
the
like (see U.S. Patent No. 6,521,762 for review of such fermentation
procedures).
Moreover, formulations of statins as pharmaceutical medicament have
been described in e.g., the Physician's Desk Reference. For example, tablet
formulations of LipitorTM (atorvastatin calcium) are described at pages 2547-
2551
(Parlce-Davis, NJ.) and 2610-2613 (Pfizer, NY) of the Physician's Desk
Reference(57th Edition, 2003). These formulations are supplied as tablets of
atorvastatin calcium containing 10 mg, 20 mg, 40 mg, 50 mg, and 80 mg
atorvastatin.
The tablets are administered in doses ranging from 10 mg/day to 80 mg/day. The
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21
compositions of LipitorTM presently being used to lower cholesterol in humans
may
be used in the combined treatments of the present invention to produce a
therapeutic
amelioration of HADDS and related lipodystrophy.
PravacholTM (pravastatin sodium; Bristol-Myers Squibb, NY), is
another exemplary commercially available statin that may be used in the
combined
therapies of the present invention. PravacholTM is supplied as a 10 mg, 20 mg,
40 mg,
and 80 mg tablets. These tablets may be administered at a daily dose of
ranging from
mg/day to 80 mg/day. In exemplary treatments for hypercholesterolemia, 40
mg/day are administered as a single daily dose, with or without food. However,
it is
10 generally appreciated that this dose may be increased or lowered depending
on the
level of renal and liver function of the patient being treated. The
administration doses
and treatment guidelines for PravacholTM are discussed in further detail at
pages 1101-
1105 of the Physician's Desk Reference(57tn Edition, 2003) and may be used to
provide guidance for the use of statins in the methods of the present
invention.
ZocorTM (simvastatin; Merck & Co., Inc., NJ), is another exemplary
statin composition that may be used in the present invention. Formulations of
this
statin are described at pages 2126-2131 of the Physician's Desk Reference(57tn
Edition, 2003). The daily doses may range from 5 mg/day to 80 mg/day and those
of
skill in the art are referred to the Physician's Desk Reference for further
guidance
regarding treatment protocols that may be used and/or modified for the present
invention. It is contemplated that doses and treatment protocols that are
useful for
lowering cholesterol will also be useful in the treatment of HADDS described
in the
present application.
MevacorTM (lovastatin; Merclc & Co., Inc. NY), and Lescol TM
(fluvastatin) are other exemplary statins that are described in the
Physician's Desk
Reference(57tn Edition, 2003) at pages 2036-2041 and 2283-2287, respectively.
Those of skill in the art will readily be able to modify the above-referenced
pharmaceutical compositions that comprise various statin-related agents for
the
methods of the present invention.
For treatment protocols, those of skill may use the guidelines used for
the any of the above-referenced pharmaceutical statins. Administration of
ordinary
tablets containing statin once, twice, three or more times a day. Accordingly,
the
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22
skilled artisan may use dosages that have previously proven effective for the
above
indications as a preliminary measure of the amount of any of the above-
referenced
statins, to use in the therapeutic methods of the invention.
Oral doses of the statins are particularly contemplated. Such oral doses
may comprise the administration of between about 5 mg to about 80 mg statin
drug on
a daily basis. However, larger doses e.g., up to 200mg/day also may be used.
Thus,
the subject may receive 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg,
45
mg, 50 mg, 55 mg, f0 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100
mg, 125 mg, 150 mg, 175 mg, 200 mg or more statin drug orally. Of course it
should
be understood the subject may receive more or less of the statin. Also it
should be
understood that similar doses may be administered through other routine routes
of
administration. The statin may be delivered in a single dose or alternatively
may be
subdivided and administered in multiple doses over a given period of time.
C. Combination Tlierapies with Additional Therapeutic Agents
The methods of the invention involve the combined use of growth
hormone and statin-related compounds. However, in addition to therapies based
solely on the delivery of GH/statin combination therapy, the methods of the
present
invention also contemplate combination therapy with a third composition that
specifically targets one or more of the symptoms of lipodystrophy. In the
context of
the present invention, it is contemplated that GH/statin-based methods could
be used
similarly in conjunction with other agents for e.g., treating obesity,
diabetes and the
lilce. Such additional therapeutic compounds also may comprise compositions
that
enhance the effects of growth hormone and/or the statin-related agents.
In particular embodiments, it is contemplated that the growth
hormone/statin treatment in accordance with the present invention may be
supplemented with the administration of a substance which stimulates
production of
endogenous growth hormone either directly or indirectly by suppressing
endogenous
somatostatin secretion. It is known that human growth hormone releasing
hormone
(hGHRH) stimulates the release of hGH. Thus, the biological activity of hGH
can be
indirectly obtained by administering GHRH or a functional derivative, salt,
variant,
analog or fragment thereof which retains the biological activity of GHRH, i.
e., the
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23
ability to stimulate the release of growth hormone. Thus, for example, besides
GHRH
there may be used functional derivatives thereof in accordance with the above
definition, analogs or variants thereof, which have at least 70% sequence
identity,
more preferably 80% or 90% or, most preferably, 95% sequence identity
therewith,
yet retains the biological activity of GHRH, or a variant or analog which is a
polypeptide encoded by a DNA which hybridizes to the native DNA encoding GHRH
under moderately stringent conditions, or preferably under highly stringent
conditions, all in accordance with the definitions given hereinabove. Any of
the
GHRH or GHRH analogs or agonists known in the literature and disclosed as
simulating the release of growth hormone can be used in the present invention,
such
as those disclosed in U.S. Patents 5,792,747; 5,776,901; 5,696,089; 5,137,872;
5,767,085; 5,612,470; 5,846,936; and 5,847,066. See also Thorner et al.,
ReceyZt Pr~g
Hof°m Res., (1997), Felix et al., IntJPept Protein Res., 46(3-4):253-64
(1995),
Alba-Roth et al J Cliy~. Efzdo. Metab., 67, 1186-1189 (1988); Friend et al.,
Eu~ J
Ef~doc~inol., 137(4):377-86 (1997).
Other substances capable of promoting the release of growth hormone
i~ vivo which can be used in accordance with the present invention include
those
disclosed in U.S. Patents 5,807,985; 5,604,578; 5,795,957; 5,777,112;
5,767,118;
5,731,317; 5,726,319; 5,726,307; 5,721,251; 5,721,250, etc.
There can also be used in accordance with the present invention any
other molecule which binds to receptors on pituitary somatotrophes and
initiates
signaling of that receptor. It is known, for example, that small molecules,
sometimes
called secretagogues, have been developed which bind GHRS receptors and cause
them to initiate signaling, which signal initiation is the same as one obtains
with
natural ghrelin binding to the receptor. Such molecules are known, for
example, from
U.S. Patents 5,773,441; 5,798,337; 5,630,433; 5,767,124; and 5,723,616. See
also
Bowers et al Endocrinology, 128:2027-2035 (1991), Thorner et al., Rece~zt
Pr°og
Hof°m Res., 52:215-46 (1997), Camanni et al., F~o~t Neu~oe~doc~i~ol.
19(1):47-72,
(1998), Ankersen et al., JMed Chem., 41(19):3699-704, (1998), Smith et al.,
Science,
260(5114):1640-3 (1993) and Ghigo et al., Ho~m Res., 51 Suppl 3:9-15 (1998).
Thus,
the present invention is intended to include any substance which binds to GHRS
receptor and initiates signaling thereof so as to obtain the same ultimate
qualitative
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WO 2005/074916 PCT/EP2005/000758
24
effect as the administration of natural hGH, insofar as the treatment of HADDS
is
concerned.
In other embodiments, it is contemplated that the growth
hormone/statin-based combination therapy of the invention is supplemented with
a
therapeutic regimen that treats obesity-related diabetes. Such a therapeutic
regimen
may comprise administering an insulin secretagogue to said subject. The use of
such
secretagogues for the amelioration of diabetes is well lcnown to those of
skill in the
art. Classes of secretagogues that may be used include but are not limited to
sulphonylurea; tolbutamide; chlorpropamide; glimepiride; glipizide; glyburide;
a
meglitinides (see Physician's Desk Reference 56th Edition, page 2432 for
description
of an exemplary meglitinide pharmaceutical formulation); repaglinide;
pramlintide;
morphilinoguanide; acetylcholine; a muscarinic agonist; carbachol;
bethanechol; beta-
L-glucose pentaacetate; chiro-inositol; myo-inositol; GIP; GLP-l; and Extendin-
4.
Those of skill in the art are referred to Goodman & Gilman's The
Pharmacological
Basis of Therapeutics, Eds. Hardman et al., 9th Edition, Chapter 60 which
describes
insulin and oral hypoglycemic agents that could be used in conjunction with
the
present invention. Particularly preferred oral hypoglycemics include
sulphonylureas
(described at pages 1507-1510 of Goodman & Gilman. See also see Physician's
Deslc
reference, 56th Edition pages 717, 741, 2680, 2692, 2693, and 1086 for
descriptions of
exemplary sulphonylurea pharmaceutical formulations currently being used). It
is
contemplated that the metformin (see Physician's Desk reference, 56t~' Edition
page
1080 for description of an exemplary metformin pharmaceutical formulation) ,
phenformin or other biguanides also may be used. Thiazolidendiones, such as
ciglitazone and pioglitazone, also may prove useful in the methods of the
present
invention. Those of skill in the art also are referred to the Physician's Desk
Reference, 56th Edition pages 3275 and 1490 for descriptions of exemplary
thiazolidendione pharmaceutical formulations currently being used. Diazoxide,
an
antihypertensive agent also is known as a potent antihyperglycemic agent. It
is lilcely
that, as certain statin compounds such as LiptorTM are HMG-CoA reductase
inhibitors,
additional such inhibitors will be identified in such additional inhibitors
may be used
in the present invention to produce an additional therapeutic effect against
lipodystrophy in accordance with the present invention. Such additional agents
may
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or may not be analogs of lovastatin, as long as they act as inhibitors of HMG-
CoA
reductase in a manner similar to lovastatin analogs.
Leptin (Zhang et al., Nature 372:425, 1994) is a protein hormone with
important effects in regulating body weight, metabolism and reproductive
function
5 (see e.g., U.S. Patent No. 5,935,810). The protein is approximately ~16 kDa
in mass
and encoded by the obese (ob) gene. Leptin is expressed predominantly by
adipocytes but leptin also is secreted by cells in the epithelium of the
stomach and in
the placenta. Leptin receptors are highly expressed in areas of the
hypothalamus
known to be important in regulating body weight, as well as in T lymphocytes
and
10 vascular endothelial cells. Studies with obese and non-obese humans have
shown a
strong positive correlation of serum leptin concentrations with percentage of
body fat,
and also that there was a higher concentration of ob mRNA in fat from obese
compared to thin subjects. It appears that as adipocytes increase in size due
to
accumulation of triglyceride, they synthesize more and more leptin. Daily
injections
15 of recombinant mouse or human leptin into ob/ob mice (i.e., the obese mouse
mutants
that are unable to synthesize leptin) led to a dramatic reduction in food
intake within a
few days, and to roughly a 50% reduction in body weight within a month.
Moreover,
when leptin is given to normal mice, they lose weight, show profound depletion
of
adipose tissue and manifest increases in lean mass. It has been shown that
treatment
20 with leptin promotes lipolysis in adipose tissue, but has no apparent
effect on lean
tissue. Given these results, it is contemplated that the rhGH/statin-based
therapies of
the present invention may advantageously be supplemented with a therapeutic
regimen that provides the subject with leptin.
To achieve the appropriate therapeutic outcome in the combination
25 therapies contemplated herein, be it a decrease in girth of the area at
which the
abnormal fat deposition has taken place, an improvement in the appearance of
the area
of the body where the fat has become depleted, a reduction in hypoglycemia, an
increase in insulin secretion or production, or other parameter, one would
generally
administer to the subject the GH and the statin-related composition and at
least one
other therapeutic agent (third therapeutic agent). These compositions would be
provided in a combined amount effective to produce the desired therapeutic
outcome.
This process may involve administering the rGH therapy, the statin-based
therapeutic
composition and the third therapeutic composition at the same time. This may
be
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26
achieved by administering a single composition or pharmacological formulation
that
includes all of the active agents, or by administering to the subject three
distinct
compositions or formulations, at the same time, wherein one composition
includes the
rhGH, the second compositions includes the statin-related agent, and the third
composition includes the third therapeutic agent.
Alternatively, the rhGH treatment may precede or follow statin-based
therapy and/or the third agent treatment by intervals ranging from minutes to
weeks.
In embodiments where two or more of the therapeutic compositions are
administered
separately, one would generally ensure that a significant period of time did
not expire
between the time of each delivery, such that the statin-based agent and rhGH
and/or
the third agent would still be able to exert an advantageously combined effect
on the
cell. In such instances, it is contemplated that one would administer all
three
compositions within about 12-24 hours of each other and, more preferably,
within
about 6-12 hours of each other, with a delay time of only about 12 hours being
most
preferred. In some situations, it may be desirable to extend the time period
for
treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to
several
weeks (l, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
D. Pharmaceutical Compositions
Pharmaceutical compositions for administration according to the
present invention can comprise at least one formulation of human growth
hormone
according to the present invention in a pharmaceutically acceptable form
optionally
combined with a pharmaceutically acceptable carrier. These compositions can be
administered by any means that achieve their intended purposes. Amounts and
regimens for the administration of a composition according to the present
invention
can be determined readily by those with ordinary skill in the art for treating
HADDS,
or other abnormal lipid distribution disorder. As discussed above, those of
skill in the
art could initially employ amounts and regimens of GH currently being used in
a
medical context. To this effect, those skilled in the art are specifically
referred to
each of the entries in the Physician's Desk Reference, 56t~' Edition, at pages
2818-
2820 (GENOTROPINO), 3215-3215 (GEREF~), 1930-1934 (HUMATROPEO),
2419-2421 (NORDITROPIN~), 1417-1425 (NUTROPIN~), 3225-3226
(SAIZENO), and 3229-3231 (SEROSTIM~) each incorporated herein by reference.
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27
Each of these entries in the Physician's Desk Reference provide exemplary
guidance
as to types of formulations, routes of administration and treatment regimens
that may
be used in administering GH. Any of the protocols, formulations, routes of
administration and the like described therein can readily be modified for use
in the
present invention.
Compositions within the scope of this invention include all
compositions comprising at least one human growth hormone or derivative,
analog, or
variant thereof according to the present invention in an amount effective to
achieve its
intended purpose. Similarly, as the therapeutic methods of the present
invention
contemplate a combination therapy in which statin-based agents are
administered in
addition to the human growth hormone-based therapy, the pharmaceutical
compositions of the invention also contemplate all compositions comprising at
least
one statin-based therapeutic agent, or analog thereof in an amount effective
to achieve
the amelioration of one or more of the symptoms of lipodystrophy when
administered
in combination with the human growth hormone.
While individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the art. Typical
dosages of
the growth hormone comprise about 0.01 to about 0.1 mg/kg body weight per day,
which will usually amount to about 1-6 mg/day, subcutaneously for e.g., 5 to
12
weeks. Of course, those of skill in the art may choose a treatment regimen
that lasts
longer, e.g., up to 48 weeks. When administered to AIDS patients, the hGH anti-
HADDS therapy may be administered concomitantly with other AIDS therapies or
other therapies designed to alleviate the symptoms of lipodystrophy as
discussed
herein above. Since supraphysiologic doses of hGH (> 5 mg/day) have been
safely
administered to AIDS wasting patients continuously on a daily basis as s.c.
injections
for periods of two to four years, it is contemplated that the combined
therapies which
employ growth hormone and at least one statin agent also may be effective over
such
periods. While continuous, daily administration is contemplated, it may be
desirable
to ceases the combined therapy when the symptoms of lipodystrophy are
alleviated.
Of course, the therapy may be reinitiated in the event that abnormal adipose
tissue re-
accumulates.
It is understood that the suitable dose of a composition according to the
present invention will depend upon the age, health and weight of the
recipient, kind of
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28
concurrent treatment, if any, frequency of treatment, and the nature of the
effect
desired. However, the most preferred dosage can be tailored to the individual
subject,
as is understood and determinable by one of skill in the art, without undue
experimentation. This typically involves adjustment of a standard dose, e.g.,
reduction of the dose if the patient has a low body weight.
As discussed above, the total dose required for each treatment may be
administered in multiple doses or in a single dose. The compositions may be
administered alone or in conjunction with other therapeutics directed to the
disease or
directed to other symptoms thereof.
As is apparent from the disclosure presented herein, in a broad aspect
the present application contemplates clinical application of a combination
therapy
comprising a first composition that contains a growth hormone formulation, and
a
second composition that contains a statin-based drug. Therefore, the
compositions
should be formulated into suitable pharmaceutical composition $, i. e., in a
form
appropriate for in vivo applications in such combination therapies. Generally,
this
will entail preparing compositions that are essentially free of pyrogens, as
well as
other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to
render delivery vectors stable and allow for uptake by target cells. Buffers
also will
be employed when recombinant cells are introduced into a patient. Aqueous
compositions of the present invention comprise an effective amount of each of
the
therapeutic agents being used, dissolved or dispersed in a pharmaceutically
acceptable
carrier or aqueous medium. Such compositions also are referred to as inocula.
The
phrase "pharmaceutically or pharmacologically acceptable" refer to molecular
entities
and compositions that do not produce adverse, allergic, or other untoward
reactions
when administered to an animal or a human. As used herein, "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents
and the
like. The use of such media and agents for pharmaceutically active substances
is well
known in the art. Except insofar as any conventional media or agent is
incompatible
with the therapeutic compositions, its use in therapeutic compositions is
contemplated. Supplementary active ingredients also can be incorporated into
the
compositions.
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29
The active compositions of the present invention include classic
pharmaceutical preparations of growth hormone which have been discussed herein
as
well as those known to those of skill in the art. Statins, such as, e.g.,
LipitorTM and
the like, also are known to those of skill in the art. Administration of these
compositions according to the present invention will be via any common route
so long
as the target tissue is available via that route. Most commonly, these
compositions are
formulated for oral administration. However, other conventional routes of
administration, e.g., by subcutaneous, intravenous, intradermal, intramusclar,
intramamm ~ y, intraperitoneal, intrathecal, intraocular, retrobulbar,
intrapulmonary
(e.g., term release), aerosol, sublingual, nasal, anal, vaginal, or
transdermal delivery,
or by surgical implantation at a particular site also may be used particularly
when oral
administration is problematic. The treatment may consist of a single dose or a
plurality of doses over a period of time.
The active compounds may be prepared for administration as solutions
of free base or pharmacologically acceptable salts in water suitably mixed
with a
surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared
in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary conditions of storage and use, these preparations contain a
preservative to
prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. In all cases the
form must be
sterile and must be fluid to the extent that easy syringability exists. It
must be stable
under the conditions of manufacture and storage and must be preserved against
the
contaminating action of microorganisms, such as bacteria and fungi. The
carrier can
be a solvent or dispersion medium containing, for example, water, ethanol,
polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like),
suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained,
for example, by the use of a coating, such as lecithin, by the maintenance of
the
required particle size in the case of dispersion and by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial an antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be preferable to
include
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isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
of the
injectable compositions can be brought about by the use in the compositions of
agents
delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active
5 compounds in the required amount in the appropriate solvent with various of
the other
ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various sterilized
active
ingredients into a sterile vehicle which contains the basic dispersion medium
and the
required other ingredients from those enumerated above. In the case of sterile
10 powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum-drying and freeze-drying techniques which yield a
powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-
filtered solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and
15 all solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic
and absorption delaying agents and the like. The use of such media and agents
for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, its
use in the
therapeutic compositions is contemplated. Supplementary active ingredients
also can
20 be incorporated into the compositions.
For oral administration the therapeutic agents of the present invention
may be incorporated with excipients and used in the form of non-ingestible
mouthwashes and dentifrices. A mouthwash may be prepared incorporating the
active
ingredient in the required amount in an appropriate solvent, such as a sodium
borate
25 solution (Dobell's Solution). Alternatively, the active ingredient may be
incorporated
into an antiseptic wash containing sodium borate, glycerin and potassium
bicarbonate.
The active ingredient may also be dispersed in dentifrices, including: gels,
pastes,
powders and slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include water, binders,
abrasives,
30 flavoring agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts include the acid
addition salts
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31
(formed with the free amino groups of the protein) and which are formed with
inorganic acids such as, for example, hydrochloric or phosphoric acids, or
such
organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the
free carboxyl groups also can be derived from inorganic bases such as, for
example,
sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic
bases
as isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically
effective. The formulations are easily administered in a variety of dosage
forms such
as injectable solutions, drug release capsules and the like. For parenteral
administration in an aqueous solution, for example, the solution should be
suitably
buffered if necessary and the liquid diluent first rendered isotonic with
sufficient
saline or glucose. These particular aqueous solutions are especially suitable
for
intravenous, intramuscular, subcutaneous and intraperitoneal administration.
"Unit dose" is defined as a discrete amount of a therapeutic
composition dispersed in a suitable carrier. Examples of preferred doses of
the
growth hormone and the statin have been discussed above. Parenteral
administration
of one or both of the therapeutic compounds may be carried out with an initial
bolus
followed by continuous infusion to maintain therapeutic circulating levels of
drug
product. Those of ordinary skill in the art will readily optimize effective
dosages and
administration regimens as determined by good medical practice and the
clinical
condition of the individual patient.
The frequency of dosing will depend on the pharmacolcinetic
parameters of the agents and the routes of administration. The optimal
pharmaceutical formulation will be determined by one of skill in the art
depending on
the route of administration and the desired dosage. See for example
Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co, Easton PA 18042) pp
1435
1712, incorporated herein by reference. Such formulations may influence the
physical state, stability, rate of ih vivo release and rate of in vivo
clearance of the
administered agents. Depending on the route of administration, a suitable dose
may
be calculated according to body weight, body surface areas or organ size.
Further
refinement of the calculations necessary to determine the appropriate
treatment dose
is routinely made by those of ordinary skill in the art without undue
experimentation,
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32
especially in light of the dosage information and assays disclosed herein as
well as the
pharmacokinetic data observed in animals or human clinical trials.
Appropriate dosages may be ascertained through the use of established
assays for determining blood levels in conjunction with relevant dose response
data.
The final dosage regimen will be determined by the attending physician,
considering
factors which modify the action of drugs, e.g., the drug's specific activity,
severity of
the damage and the responsiveness of the patient, the age, condition, body
weight, sex
and diet of the patient, the severity of any infection, time of administration
and other
clinical factors. As studies are conducted, further information will emerge
regarding
appropriate dosage levels and duration of treatment for specific diseases and
conditions.
In certain embodiments, the growth hormone or other protein may be
administered using gene therapy embodiments that employ viral delivery, the
unit
dose may be calculated in terms of the dose of viral particles being
administered.
Viral doses include a particular number of virus particles or plaque forming
units
(pfu). For embodiments involving adenovirus, particular unit doses include
103, 104,
105, 106, 10~, 108, 109, 101°, 1011, 1012, 1013 or 1014 pfu. Particle
doses may be
somewhat higher (10 to 100-fold) due to the presence of infection defective
particles.
It will be appreciated that the pharmaceutical compositions and
treatment methods of the invention may be useful in fields of human medicine
and
veterinary medicine. Thus the subject to be treated may be a mammal,
preferably
human or other animal. For veterinary purposes, subjects include for example,
farm
animals including cows, sheep, pigs, horses and goats, companion animals such
as
dogs and cats, exotic and/or zoo animals, laboratory animals including mice
rats,
rabbits, guinea pigs and hamsters; and poultry such as chickens, turkey ducks
and
geese.
E. Examples
The following examples) is included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of slcill in
the art that
the techniques disclosed in the examples) that follows represent techniques
discovered by the inventor to function well in the practice of the invention,
and thus
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33
can be considered to constitute preferred modes for its practice. However,
those of
skill in the art should, in light of the present disclosure, appreciate that
many changes
ca.n be made in the specific embodiments which are disclosed and still obtain
a like or
similar result without departing from the spirit and scope of the invention.
AIDS patients with a history of long-term use (an average of 12
months) of HAART that manifest symptoms of HADDS, including buffalo humps,
central adiposity and peripheral muscle wasting associated with fatigue, along
with
elevated levels of plasma triglycerides and/or cholesterol are selected for
the study.
Therapy with rhGH (SEROSTIM~) is initiated in all patients at a dose
of e.g., 4 mg/day subcutaneously (other concentrations, e.g., 6 mg/day could
be used).
At the same time, the patients are treated with a statin in a dosage amount of
40
mg/day, which is administered orally. This dosage amount is based on the
average
dose of statin drugs commonly administered for antilipemic purposes. The
patients
are maintained on this regimen for three months and monitored every two weeks
for
improvements of fat maldistribution. In parallel therapies, patients are
treated with
just rhGH and just statin. After 3 months notable improvements in fat
maldistribution, with 25-75% reduction in buffalo hump syndrome and abdominal
girth, but no change in peripheral lipodystrophy is seen in the combined
treatment
with rhGH and statin. Individuals receiving rhGH or statin alone do not
experience as
dramatic an improvement in fat maldistribution as seen with the combined
therapy.
Weights were stable, and there were no consistent changes in total body fat
and blood
lipids, despite 5-10% gain in fat-free mass.
An effective treatment with the methods of the present invention is any
notable reduction in the size of, and firmness of, the buffalo hump and
truncal
adiposity. Preferably, the reduction in size and firmness of buffalo hum and
truncal
adiposity that is seen with the combined rhGH/statin treatment is greater than
that
seen in patients with HADD S that are treated with rhGH alone (Tortes et al.,
Abstract
32164: 12th World AIDS Conference, Geneva [Abstract 32164], 1998; Tortes,
Abstract 675: 6th Conference on Retroviruses and Opportunistic Infections
[Abstract
675], 1999; Wanke et al., AIDS 13(15):2099-2103, 1999; Mauss et al., Antiviral
TlZeyapy 4(Sup 2):27 [Abstract 018], 1999; Engleson et al., Ahtiviral Therapy
4:(Sup
2):11 [Abstract 006], 1999; Engleson et al., Am J Clin Nutr 69(6):1162-1169,
1999;
Milano et al., Ahtiviral Therapy 4(Sup 2):41 [Abstract 042], 1999). Tortes,
Abstract
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WO 2005/074916 PCT/EP2005/000758
34
675: 6tn Conference on Retroviruses and Opportunistic Infections [Abstract
675],
1999, has reported that, by four months, rhGH therapy dosed at 4~to 6 mg/day
significantly reduces the size and firmness of buffalo humps, and reduces
truncal
adiposity, with no change in peripheral lipodystrophy, while fat free mass
increased 5
to 10%. There were no significant or consistent changes in body weight, total
body
fat, or blood lipids during the treatment period.
Collectively, these clinical studies cited above demonstrate that
therapy with rhGH (SEROSTIM~), administered subcutaneously, in doses ranging
from 3 to 6 mg per day for 12 to 24 weeks significantly reduces abnormally
accumulated fat, compared to baseline. Specifically, SEROSTIM~ (rhGH) has been
shown to reduce abdominal girth (Wanke et al., AIDS 13(15):2099-2103, 1999),
visceral adiposity (Engleson et al., Antivi~al Therapy 4:(Sup 2):l 1 [Abstract
006],
1999; Engleson et al., Am J Clih Nutr 69(6):1162-1169, 1999; Mauss et al.,
AIDS
12(Sup 4):145, 1990, buffalo hump (Torres, Abstract 32164: l2tn World AIDS
Conference, Geneva [Abstract 32164], 1998; Torres et al., Abstract 675: 6tn
Conference on Retroviruses and Opportunistic Infections [Abstract 675], 1999),
and
solitary lipomas (Milano et al., Antivi~al Therapy 4(Sup 2):41 [Abstract 042],
1999).
Therapy with rhGH (SEROSTIM~) also increased lean body mass and body cell
mass as quantified by biolectrical impedance analysis (Wanke et al., AIDS
13(15):2099-2103, 1999; Engleson et al., A~ztivi~al Therapy 4:(Sup 2):11
[Abstract
006], 1999; Engleson et al., Am J Clih Nuts 69(6):1162-1169, 1999). The use of
the
statin drug in combination with rhGH is expected to be more effective in
reducing the
symptoms of lipodystrophy. Further, it is contemplated that the statin may be
effective in reducing the dosage and frequency of rhGH administration needed
to
produce the therapeutic effects.
Collective side effects of rhGH administration include swelling of the
fingers or paresthesia due to tissue turgor, a few transient elevations of
fasting glucose
and triglycerides. It is contemplated that the use of the statin-based
therapeutic agent
may ameliorate these side effects of rhGH therapy. At 12 weeks, total
cholesterol and
fasting triglycerides dropped significantly, while HDL cholesterol and glucose
increased, but none of these changes were deemed clinically significant
(Engleson et
al., Autivir~al Therapy 4:(Sup 2):l 1 [Abstract 006], 1999; Engleson et al.,
Am JClin
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WO 2005/074916 PCT/EP2005/000758
Nutr~ 69(6):1162-1169, 1999). No additional episodes of hypertension or
elevated
pancreatic enzymes have been reported.
All of the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the present
5 disclosure. While the compositions and methods of this invention have been
described in terms of preferred embodiments, it will be apparent to those of
skill in
the art that variations may be applied to the compositions and/or methods and
in the
steps or in the sequence of steps of the method described herein without
departing
from the concept, spirit and scope of the invention. More specifically, it
will be
10 apparent that certain agents which are both chemically and physiologically
related
may be substituted for the agents described herein while the same or similar
results
would be achieved. All such similar substitutes and modifications apparent to
those
skilled in the art are deemed to be within the spirit, scope and concept of
the invention
as defined by the appended claims.
15 The references cited herein throughout, to the extent that they provide
exemplary procedural or other details supplementary to those set forth herein,
are all
specifically incorporated herein by reference.
