Note: Descriptions are shown in the official language in which they were submitted.
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TREATJMENT OF HIV-ASSOCIATED DXSMORPHIA/DXSMETABOLIC SYNDROME
Fz_e_ld of the Invention
The present invention is directed to a method for
treating HIV-associated dysmorphia/dysmetabolic syndrome and
S related syndromes.
Background of the Invention
Advances in antiviral treatment of HIV infection, along
with developments in the prophylaxis and therapy of opportunistic
infections, have greatly improved the long-term health of HIV-
0 positive individuals. However, despite these advances in treating
the underlying retrovirus infection and the complications of I~.IDS,
there are still patients who have lost lean body mass, either from
malnutrition or from other causes. While several clinical studies
have associated malnutrition ~,aith decreased caloric intake,
therapies designed to increase caloric intake have not proven
effective in replacing lean body mass.
A Condition identified herein as HIV-associated
dysmorphia-dysmetabolic syndrome, or HADDS, or alternatively, fat
maldistribution syndrome, FMS, has been noted in patients
suffering from chronic HIV infection, often in conjunction with
the use of highly active combination antiretroviral therapies 'that
include an HIV protease-inhibiting agent (Carr et al, 1998; Lo et
al, 1998). It is a multisystemic, gender dimorphic disorder
associated with HIV infection which includes (Z) abnormalities of
body composition or habitus, (2) metabolic alterations, and (3)
possibly other abnormal physiology.
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The syndrome is relatively new, and, consequently, there
is not yet a standardized name for the syndrome. Names other than
those noted above include HIV-associated adipose redistribution
syndrome CHARS); protease paunch, although the syndrome may not be
directly related to protease inhibitors; HIv-related Buffalo Hump;
HIV-related breast enlargement; HIV-related symmetric lipomatosis;
HIV-associated Lipodystrophy Syndrome (HALS); and HIV-associated
Dysmorphia/Metabolic Syndrome (HARMS).
The complex syndrome, involving rapid (within weeks to
months) fat accumulation with limb wasting, facial wasting, and
concomitant hyperlipidemia and hyperinsulinemia, in the absence of
hypercortisolism, is unique in the history of medicine. Its
etiology is unknown. The bodily manifestations of the syndrome
have important public health significance. The body habitus
changes are frightening, disfiguring, and stigmatizing to
patients. These changes may lead to physical discomfort and
disability and could lead to discontinuation of otherwise
effective antiretroviral therapy or refusal to initiate medically
necessary antiretroviral therapies for fear of developing the
syndrome. This could lead to increased HIv infectivity, more
opportunistic complications, and increased medical costs.
These medical complications are occurring with increased frequency
in patients with HIV/AIDS_
It is unclear if the syndrome is drug-related. Although
it is more prevalent in HIV/AIDS patents who have received
antiretroviral medications (highly active antiretroviral therapy
with protease inhibitors), in some cases the syndrome or some of
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its manifestations have been observed in, patients who have never
received antiretrovirals (Lo et al, 1998; Carr et al, 1998).
Manifestations of the syndrome sometimes partially abate when
antiretroviral medications are discontinued or changed, but the
manifestations often do not abate completely or resolve relatively
quickly. This raises a concern that the syndrome might occur in
conjunction with immune reconstitution or autoirnmune phenomena
associated with chronic suppression of HIV-1. There is no
currently known effective medical therapy for this syndrome.
In HADDS there appears to be fat cell proliferation or
hypertrophy in several areas of the body: the omental or visceral
region, in the dorsocervical fat (which creates the "buffalo
hump"), and elsewhere, including breast fat accumulation and
occasionally multiple lipomatosis and bilateral asymmetric
lipomatosis. Muscle or lean tissue is abnormally distributed, as
there may be muscle depletion in the limbs, and abnormalities of
the genitalia resembling Peyronie's Disease. There may be other
alterations in body composition, such as cutaneous manifestations:
thinning hair, dry skin, abnormal nails, and prominent vei.ns_
This condition may or may not be associated with osteoporosis or
avascular necrosis of the hips.
The body habitus changes are frightening and
disfiguring, and may result in psychological or physical
discomfort that lead to body image disturbance, depression, and
agoraphobia; decreased mobility from buffalo hump or truncal
obesity; ventral hernias, possibly related to rapid accumulation
of abdominal fat; and respiratory insufficiency, possibly due to
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excess accumulation of abdominal fat. These changes can also be
socially stigmatizing because they may reveal that the patient has
HIV/AIDS-
Moreover, the metabolic manifestations of the syndrome
may contribute to the development of medically serious problems,
such as sudden and severe hyperglycemia, new-onset diabetes
mellitus, difficult to treat hyperlipidemia, premature coronary
artery disease, gout, and osteoporosis with bone fractures and
avascular necrosis of the hips. Other typical metabolic or
7 physiological abnormalities may include elevated triglycerides and
cholesterol abnormalities, including elevated total cholesterol,
elevated LDL cholesterol, and'decreased HDL-cholesterol. Less
common metabolic or physiologic abnormalities appear to include
hypertension, hormonal abnormalities, or other abnormalities such
as derangement of the complement system or coagulation disorders,
leading to increased bleeding in hemophiliacs.
The changes in body composition Which may be related to
antiretroviral treatment of HIV infection are more related to the
regional distribution of fat than to the absolute amounts of fat
0 and lean tissue. The most visible regional alteration is an
increase in waist size. Kotler et al (1998) reported finding
increased visceral fat along with pelvic and perinephric fat using
a whole body MRI technique. Carr et al (1998) observed peripheral
HADDS in HIV-positive patients receiving protease inhibitors
5 which, together with the known side effects of protease
inhibitors, e.g., hvperlipidemia and diabetes mellitus, suggests
that protease inhibitors cause metabolic perturbations leading to
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insulin resistance. Carr et al (1998) documented increased
truncal fat in a majority (64%) of their protease inhibitor-
treated subjects using the technique of dual-energy X-ray
absorptiometry. They also found evidence of insulin resistance,
although clinically apparent diabetes mellitus was very uncommon.
An amino acid sequence in the catalytic site of HIV protease was
found to have a significant homology with a low density
lipoprotein receptor-like protein. These results would tend to
implicate the protease inhibitors themselves in the development of
7 this problem, but other researchers have found that the protease
inhibitors pez se axe not responsible for these symptoms.
Other visible body changes in patients which have been
reported in the literature include thinning of the skin on the
arms and legs, and increased wrinkling of the face, especially in
S the nasolabial folds. A buffalo hump (dorsocervical fat pad) may
be present, as well as enlargement of the supraclavicular fat pads
with apparent bulging. This condition also affects females, with
the most notable changes, in addition to increased waist size,
being narrowing of the hips and enlargement of the breasts.
Viraben et a1 (1997) reported a case study involving
eight patients who developed either partial or generalized
lipodystrophy after protease inhibitor therapy. In four of these
patients, either diabetes or insulin resistance was also
discovered. While two patients developed progressive loss of
subcutaneous fat from both legs, excess fat deposition in the
unaffected buttocks and abdomen gave an impression of obesity. In
six cases, a cachetic appearance was observed resulting from the
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loss of buccal, parotid and preauricular fat pads. Two patients
exhibited a generalised loss of fatty tissue from the face.
Lo et al (1998) reported the results of studies done in
eight HIV-1-positive patients referred for investigation of
buffalo hump. Only four of these patients were on triple
antiretroviral regimens that included a pxotease inhibitor. No
other signs of Cushing's Syndrome were present in the patients.
The fact that four of the patients with buffalo hump had no
history of protease-inhibitor use indicates that development of
non-Cushingoid buffalo hump is not unique to protease-inhibitor
therapy. The mechanism of further increase in triglyceride values
in patients with buffalo hump is not certain, although a possible
relation between increased triglyceride concentration and atypical
body-fat distribution should be considered. For example, central
fat accumulation may lead to the metabolic syndrome of insulin
resistance, hypertriglyceridemia and hypertension if the major
component gained is visceral fat.
Miller et al (1998) reported that some patients
experienced an increase in abdominal girth with symptoms of
0 abdominal fullness, distension, ox bloating after adding
indinavir, a protease inhibitor, to combination drug regimens for
HIV-1 infection. In several patients with these symptoms,
abdominal computed-tomography (CT) scans suggested an excess
amount of intra-abdominal fat and a relative paucity of
5 subcutaneous fat. There were no relationships among visceral fat,
hypertriglyceridemia, and hypercholesterolemia.
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Additional reports of HADDS were presented at the 5th
Conference on Retroviruses and Opportunistic Infections February
1-5, 1998, at Chicago, IL. Mulligan et al (1998) compared results
obtained in patients before and after beginning an antiretroviral
regimen that included a protease inhibitor or lamivudine. No
significant changes in total or regional fat or lean body mass
were found by dual-energy X-ray absorptiometry in any group over
the short time period of about four months.
Keruly et al (1998) reported on short-term protease
7 inhibitor use in relation to hyperglycemia and diabetes. They
calculated an incidence of 0.35 per 100 person-months for severe
hyperglycemia and 0.52 for any degree of hyperglycemia. The
median time of onset after first use of a protease inhibitor
(ritonavir, saquinavir and indinavir) was 53 days. Thus, it
appears that metabolic changes are seen early in the course of PI
therapy, while the morphological changes may take longer.
However, Dong et al (1998) reviewed all cases of diabetes
associated with the use of protease inhibitors and found six cases
of hyperglycemia from across the U.S. which were related to the
0 use of protease inhibitors, and concluded that protease inhibitor-
induced hyperglycemia appears to be a rare occurrence.
Bjorntop (1996) found a similar syndrome of truncal
obesity and metabolic abnormalities, including insulin resistance
in HIV-negative subjects. This non-HIV related syndrome is known
5 as Syndrome X.
The FDA reported that there were reports of peripheral
insulin resistance due to alterations in fat metabolism in
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patients receiving protease inhibitors for treatment of their HIV
infection, and some of these diabetic patients were found to have
truncal obesity. Other patients were found to have
hypertriglyceridemia, even though this abnormality was first
reported and associated with HIV infection, in the presence or
absence of disease complications. Patients with
hypertriglyceridemia prior to starting therapy appear to have an
exacerbation of the condition while on therapy and do not return
to their previous state. In other patients, serum cholesterol
7 concentrations rise, though rarely to dangerously high levels.
Some patients developed hypertension and, in others, low serum
testosterone concentrations developed or pre-existing hypogonadism
persisted.
From the above, it is unclear if administration of
protease inhibitors results in HADDS or if the H.A.DDS results from
the antiviral effect of the protease. The changes are not seen
consistently in protease inhibitor-treated patients, and many
patients have only certain elements of the syndrome. In some
cases the syndrome or its manifestation have been observed in
0 patients who have never received antiretrovirals. The potential
for reversal of the altered fat distribution is uncertain. What
is suggested by the reports is that buffalo hump in HIV-1-infected
individuals is not necessarily associated with Cushing's Syndrome
or hyperglycemia. There is also evidence that raised triglyceride
5 concentrations may occur with visceral fat accumulation or buffalo
humps.
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As noted above, serum abnormalities occur with variable
frequency in patients with truncal obesity. Serum cortisol
concentration typically is normal, although 24-hour urinary-free
cortisol may be moderately elevated. Other studies have found no
increase in cortisol secretive. Due to alterations in fat
distribution, some patients appeared to have a phenotype
reminiscent of Cushing's Syndrome, but this was ruled out in all
reported cases.
Several studies have documented insulin resistance as a
0 result of protease inhibitor therapy, and the incidence of
clinical diabetes mellitus in patients receiving protease
inhibitors appears to be low and the severity varies.
SEROSTIM" recombinant human growth hormone (rhGH)
produced by Serono Laboratories, Inc., has recently been given
accelerated FDA approval fox treating weight loss and wasting in
patients with AIDS.
Windisch et al (1998) reported that AIDS-associated
wasting was characterized by weight loss, depletion of lean body
mass and preservation of body fat, leading to muscle weakness and
0 ~ organ failure. Although the FDA has approved recombinant growth
hormone for treating AIDS-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 AIDS have been encouraging. Post-
s marketing experience with. over 10,000 AIDS wasting patients
receiving SEROSTIM since 1995 indicates that a three-month course
was effective in 98~ of the patients.
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Schambelan et al (1996) and Krent2 et al (1993) compared
metabolic and anthropometric changes induced by pharmacological
versus physiological doses (5.0 vs. 2.5 mg, every other day) of
recombinant human growth hormone in 7.0 HIV-positive patients with
AIDS or AIDS-related complex. During treatment, insulin-like
growth factor-7. (IGF-1) levels increased significantly in the
pharmacological rhGH treatment group, whereas no significant
change was observed in IGF-1 in the group receiving a
physiological dose of rhGH. In the group treated with
0 pharmacologic 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, with concomitant decreases in fat mass and urinary
nitrogen excretion. Significant positive changes in body
composition were also observed in the pharmacological dose hGH
group, and these changes correlated with improvements in physical
function (treadmill performance). The pharmacological dose of
rhGH was associated with minor increments in fasting plasma
glucose, insulin and C-peptide concentrations, which were of
0 negligible clinical significance.
To date, no treatment other than surgical liposuction of
the fat pad or plastic surgery has shown to be effective in
treating some features of HADDS. However, these methods do not
affect the underlying metabolic abnormalities observed in HADDS.
5 Moreover, while liposuction may decrease abnormal fat deposits,
liposuction does not restore fat to the areas from which fat has
been lost. Furthermore, these techniques present the risks of
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anesthesia and scarring. Also, it is not feasible to liposuction
visceral omental fat because of the risk of bowel perforation.
S mma of the Inve tlon
It is an object of the present izwention to overcome the
aforementioned deficiencies in the prior art.
It is another object of the present invention to treat
any type of HADDS and related syndromes, including excess
dorsocervical fat accumulation, breast fat accumulation and
0 truncal adiposity, with recombinant human growth hormone (rhGH) or
any other substance which binds to and initiates signalling of the
human growth hormone receptor or which stimulates release of or
potentiates the activity of endogenous hGH.
According to the present invention, human growth hormone
(hGH) is administered to treat HADDS and related syndromes, such
as Syndrome X (also known as Metabolic Syndrome X) and hereditary
lipodystrophy. The human growth hormone administered is
preferably recombinant human growth hormone (rhGH).
Alternatively, a substance which stimulates release of endogenous
0 growth hormone, such as growth hormone releasing hormone (GHRH) or
other substances which agonize the GHRH receptor, may be used.
Any HADDS patient can be treated by means of the present
invention.
r~ra; 1 Pct nPgcrivtion of tk~e Present Invention
5 The present invention relates to the discovery that HIV-
associated dysinorphia/dysmetabolic syndrome (HADDS) may be treated
by the administration of an effective amount of human growth
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hormone. The HADDS which may be treated in accordance with the
present invention may present itself as, for example,
dorsocervical fat pads ("buffalo hump"), visceral adiposity
("truncal obesity", "omental fat accumulation", "Crix belly" ox
"protease paunch"), abnormal breast fat accumulation, and other
abnormal fat accumulation (including single or multiple lipomas
and bilateral symmetric lipomatoses).
Human growth hormone, also known as somatotropin, is a
protein hormone produced and secreted by the somatotropic cells of
0 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.
5 Human growth hormone is a single polypeptide chain of
191 amino acids (Bewley et al, 1972) having two disulfide bands,
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
0 the amino acid sequence was reported by Martial et al (1979).
Purified hGH is a white amorphous powder in its lyophilized form.
It is readily soluble (concentrations >10 mg/L) in dilute aqueous
buffers at pH greater than 7.2.
In solution, hGH exists predominantly as a monomer, with
5 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.
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Several derivatives of hGH are known, including
naturally-occurring derivatives, variants anal 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 (1989). Any derivative of hGH,
including derivatives designed to be long-lasting in the body, can
be used fox the purpose of the present invention as long as it
0 retains the biological activity of hGH.
Methionyl hGH was the first form of hGH to be produced
through recombinant DNA technology. This compound is actually a
derivative of hGH having one additional methionine residue at its
N-terminus (Goeddel et al, 199).
.A naturally-occurring variant of hGH called 20-K-hGH has
been reported to occur in the pituitary as well as in the
bloodstream (Lewis et al, 1978; Lewis et al, 1980). This
compound, which lacks the 15 amino acid residues from Glu-32 to
Gln-46, arises from an alternative splicing of the messenger
0 ribonucleic acid (DeNoto et al, 1981). This compound shares many,
but not all of the biological properties of hGH.
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 hormo~.e, and has
been reported to have equal to or greater the amount of lipolytic
activity as the 22 kD form. It binds to growth hormone receptors
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with equal affinity as the 22 kD growth hormone, and has one tenth
the lactogenic (prolactin-like) bioactivity as the 22 kD hormone.
Unlike 22 kD, the 20-k-HGH has weak anti-insulin activity.
A number of derivatives of hGH arise from prvteolytic
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 deletior~s in this
region have been described (Thorlacius-Ussing, 1987). This region
0 is in the large loop of hGH, and cleavage of a peptide bond there
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 reported to have increased biological activity (Singh et
al, 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,
1977; Graff et al, 1982). One such derivative, called two-chain
anabolic protein (2-CAP), was Formed through the controlled
0 proteolysis of hGH using trypsin (Becker et al, 1989). 2-CRP 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.
5 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,
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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, 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 (Asn-149). This is the
primary site of deamidation, but deamidation at Asn-152 is also
7 seen (Becker et al, 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
(Becker et al, 1988). Oxidation at Met-170 has also been reported
in pituitary but not biosynthetic hGH_ Both desamide hGH and Met-
14 sulfoxide hGH have been found to exhibit full biological
activity (Becker et al, 1988).
Truncated forms of hGH have been produced, either
0 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. The first 13 residues have been
5 removed to yield a derivative having distinctive biological
properties (Gertler et al, 1985) in which the polypeptide chain is
not cleaved.
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Although human growth hormone was originally obtaizzed
from pituitary glands of cadavers, these preparations were not
electorphoretically homogeneous, and antibodies appeared in the
serum of patients treated with preparations of the order of 500
purity, the immunogezzicity being attributed to inactive
components. Recombinant DNA technology permitted production of an
unlimited supply of hGkl 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 (1989).
Recombinant human growth hormone, rhGH, is produced
by Serono Laboratories, Inc., as SEROSTIM" which product has
been given accelerated FDA approval for treating weight loss and
wasting in AIDS patients. PROTROPIN" 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.
After intravenous administration, the elimination of hGH
is described by first-order kinetics with a serum half-life of 7.2-
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30 minutes in both animals and humans (Moors et al, 1988;
Hendricks et al, 1985). Traditionally, intramuscular injection
has been the method of choice as the preferred route of delivery.
In humans, absorption of exogenous hGH appears to be more rapid
from the intramuscular site, with a time to maximum concentration
of two to three hours, compared to four to six hours after
subcutaneous administration. The disappearance phase from serum
has been reported to range from 12-20 hours for intramuscular
administration, and 20-24 hours after subcutaneous administration
(Albertsson-Wikland et al, 1986; Jorgensen et al, 1987). In
general, no significant differences have been observed in the
pharmacokinetics or biological activities of recombinant natural
sequence hGH, recombinant N-methion.yl-hGH, or pituitary-derived
material in humans (Moors et al, 1988; Jorgensson et al, 1988).
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 (1989). The term also includes functional derivatives,
fragments, variants, analogs, or salts which retain the biological
activity of growth hormone, i.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 fux~,ctioxnal groups which
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occur as side chains on the residues or the N- or C-terminal
groups, by means known. in the art, and axe 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 hG8 receptor and
initiate receptor signalling, and do not confer toxic properties
on compositions containing it. Derivatives may have chemical
moieties, such as carbohydrate or phosphate residues, provided
such a derivative retains the biological activity of hGH and
0 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 amine groups of the amino acid residues formed
5 with acyl moieties (e.g., alkanoyl 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
p 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 the
S body, can be used to treat HADDS according to the present
invention.
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HGH that is acetylated at the N-terminus has been
isolated and identified (Levels et al, 1979). It is not clear if
acylation serves a regulatory role or is simply an artifact o~ the
purification. However, it is expected that this the molecule
exhibits anti-HADDS activity in a similar fashion to other hGH
derivatives.
The term "derivatives" is intended tv include only those
derivatives that do not change one amino acid to another of the
twenty commonly-occurring natural amino acids.
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 adds, such as, for example,
hydrochloric acid or sulfuric acid, and salts with organic acids,
0 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 signalling.
A "fragment" of the growth hormone according to the
5 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
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either end of the hGH molecule and testing the resultant fox
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 also contain
0 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,
5 by routine experimentation. The team "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. This term does
not comprehend substitutions, deletions or additions within the
0 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 fragment thereof.
Variant peptides may be conveniently prepared by direct chemical
5 synthesis of the variant peptide, using methods well known in the
art. Of course, a variant human growth: hormone would have similar
hGH receptor binding and signal initiating activity as hGH and
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which would, therefore, be expected to have similar anti-HADDS
activity to hGH.
Amino acid sequence variants o~ 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
0 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 (cf. European latent Publication No. EP 75,444, the
S entire contents of which axe hereby incorporated by reference).
At the genetic level, these variants ordinarily axe
prepared by site-directed mutageneis (as exemplified by Adelman et
al, 1983) of nucleotides in the DNA encoding the peptide molecule,
thereby producing DNA encoding the variant, and thereafter
0 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 molecule or to an
active fragment thereof. An analog of human growth hormone useful
in the present invention would exhibit anti-HADDS activity.
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The types of substitutions which may be made in the
human growth hormone according to the present invention may be
based on analysis of the frequencies of amino acid changes between
a homologous protein of different species. Based upon such
analysis, conservative substitutions may be defined herein as
exchanges within one of the following rive groups:
I. Small, aliphatic, nonpolar or slightly polar
residues:
Ala, Ser, Thr, Pro, Gly
II. Polar, negatively-charged residues and their
amides:
Asp, Asn, Glu, Gln
III. Polar, positively-charged residues:
His, Arg, Lys
IV. Large, aliphatic non-polar residues:
Met, Leu, Ile, Val, Cys
V_ Large aromatic residues:
Phe, Try, Trp
Within the foregoing groups, the following substitutions
are considered to be "highly conservative":
Asp/Glu
His/Arg/Lys
Phe/Tyr/Trp
Met/Leu/Ile/Val
Semi-conservative substitutions are defined to be
exchanges between two of groups (I)-(IV) above which are limited
to supergroup (A), comprising (I), (II), and (III) above, or to
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supergroup (B), comprising (IV) and (V) above. Substitutions axe
not limited to the genetically encoded or even the naturally-
occurring amino acids. When the epitope is prepared by peptide
synthesis, the desired amino acid may be used directly.
Alternatively, a genetically encoded amino acid may be modified by
reacting it with an organic derivatizing agent that is capable of
reacting with selected side chains or terminal residues.
Cysteinyl residues most commonly are reacted with alpha
haloacetates (and corresponding amines), such as chloroacetic acid
0 or chloroacetamide, to give carboxylmethyl or carboxyamidomethyl
derivatives. Cysteinyl residues also axe derivatized by reaction
with bromotrifluoroacetone, alpha-bromo-beta-(5-
imidazoyl)propionic acid, chloroacetyl phosphate, N-
alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl
.5 disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,
or chloro-7-nitrobenao-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with
diethylprocarbonate at pH 5.5-7.0 because this agent is relatively
specific fox the histidyl side chain. Parabromophenacyl bromide
;0 is also useful; the reaction is preferably performed in 0.1 M
sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
:5 residues. Other suitable reagents for derivatizing alpha-amino
acid-containing residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
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trinitrobenzenesulfonic acid; O-methyliosurea; 2,4-pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal; 2,3-
butanedione; and ninhydrin. Derivati~ation of arginine residues
requires that the reaction be performed in alkaline conditions
because of the high pKa of the guanidine functional group.
Furthermore, these reagents may react with the groups of lysine,
as well as the arginine epsilon-amino group.
p The specific modification of tyrosyl residues per se
has been studied extensively, with particular.interest in
introducing spectral labels into tyrosyl residues by reaction with
aromatic diazonium compounds or tetranitromethane. Most commonly,.
N-acetylimidazole and tetranitromethane are used to form O-acetyl
5 tyfosyl species and e-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are
selectively modified by reaction with carbodiimides (R'N-C-N-R')
such as 1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or 1-
ethyl-3-(4-azonia-9:,4-dimethylpentyl)carbodiimide. Furthermore,
0 aspartyl and glutamyl residues axe converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl anal aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic
5 conditions. Either form of these residues falls within the scope
of this invention.
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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
signalling 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; 5,533,352; 5,597,709; and 5,534,617.
Preferably, the hG~-i 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
0 preferably, such a sequence has at least 80% identity, at least
90% identity, or most preferably at least 95% identity to the
native sequence.
The term "sequence identity" as used herein means that
the sequences axe compared as follows. The sequences are aligned
using Version 9 of the Genetic Computing Group's GAP (global
alignment program), using the default (BLOSUM62) matrix. (values -4
to +11) with a gap open penalty of -12 (for the first null of a
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gap) and a gap extension penalty of -4 (per each additional
consecutive null in the gap>. lifter alignment, percentage
identity is calculated by expressing the number of matches as a
percentage of the number of amino acids in the claimed sequence.
Analogs or variants in accordance with the present
invention may also be determined in accordance with the following
procedure- The DNA of the native sequence is known to the prior
art and is found in the literature (Martial et al, 1979).
Polypeptides encoded by any nucleic acid, such as DNA or RNA,
p which hybridizes to the complement of the native DNA or RNA under
highly stringent or moderately stringent conditions, as long as
that polypeptide maintains the biological activity of the native
sequence, are also considered to be within the scope of the
present invention.
Stringency conditions are a function of the temperature
used in the hybridization experiment, the molarity o~ the
monovalent cations and the percentage of formamide in the
hybridization solution. To determine the degree of stringency
involved with any given set of conditions, one first uses the
0 equation of Meinkoth et al. (1984) for determining the stability
of hybrids of 100% identity expressed as melting temperature Tm of
the DNA-DNA hybrid:
Tcn = 81.5°C + 16.6 (LogM) ~ 0.41 (mGC) - 0.61 (% ~orm) - 500/I~
where M is the molarity of monovalent cations, °sGC is the
S percentage of G and C nucleotides in the DNA, % form is the
percentage of formamide in the hybridization solution, and L is
the length of the hybrid in base pairs. For each 2°C that the Tm
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is reduced from that calculated for a 100% identity hybrid, the
amount of mismatch permitted is increased by about lo. Thus, if
the Tm used for any given hybridization experiment at the
specified salt and formamide concentrations is 10°C below the Tm
calculated for a 100% hybrid according to equation of Meinkoth,
hybridization will occur even if there is up to about l0%
mismatch.
As used herein, highly stringent conditions are those
which are tolerant of up to about 15~ sequence divergence, while
moderately stringent conditions are those which axe tolerant of up
to about 20% sequence divergence. Without limitation, examples of
highly stringent (7.2-15°C below the calculated Tm of the hybrid)
and moderately (15-20°C below the calculated Tm of the hybrid)
conditions use a wash solution of 2 X SSC (standard saline
citrate) and 0.5% SDS at the appropriate temperature below the
calculated Tm of the hybrid. The ultimate stringency of the
conditions is primarily due to the washing conditions,
particularly if the hybridization conditions used are those which
allow less stable hybrids to form along with stable hybrids. The
0 wash conditions at higher stringency then remove the less stable
hybrids. A common hybridization condition that can be used with
the highly stringent to moderately stringent wash conditions
described above is hybridization in a solution of 6 X SSC (or 6 X
SSPE), S X Denhardt's reagent, 0.5a~SDS, 100 ~Cg/ml denatured,.
fragmented salmon sperm DNA at a temperature approximatEly 20° to
25°C below the Tm. If mixed probes are used, it is preferable to
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use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel,
1987-1998).
while the present invention provides recombinant methods
for making the human growth hormone derivatives, these derivatives
may also be made by conventional protein synthesis methods which
are well known to those skilled in the art.
The growth hormone treatment in accordance w~.th the
present invention may be accomplished either by administration of.
exogenous growth hormone or by administration of a substance which
stimulates production of endogenous growth hormone either directly
or indirectly by supressing 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 ability
to stimulate the release of growth hormone. Thus, fox example,
besides GHRH there may be used functional derivatives thereof in
accordance with the above definition, analogs or variants thereof,
0 which have at least 70% sequence identity, more preferably 80% or
90% or, most preferably, 95% sequence identity therewith, yet
retains the biological activity o~ GHRH, or a variant or analog
which is a polypeptide encoded by a DNA which hybridises to the
native DNA encoding GHRH under moderately stringent conditions, or
5 preferably under highly stringent conditions, all in accordance
with the definitions given hereinabove. L~ny of the GHRH or GHRH
analogs or agonists known in the literature and disclosed as
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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 (1997), Felix et
al (1995), Alba-Roth et al (1988), Friend et al (1997).
Other substances capable of promoting the release of
growth hormone in vivo which can be used in accordance with the
present invention include those disclosed in U.S. patents
5,807,985; 5,804,578; 5,795,957; 5,777,112; 5,767,118; 5,731,317;
0 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 the hGH receptor and
initiates signalling of that receptor. It is known, fox example,
that small. molecules, sometimes called secretagogues, have been
5 developed which bind hGH receptors and cause them to aggregate and
initiate signalling, which signal initiation is the same as one
obtains with natural hGH binding to the receptor. Such molecules
axe known, for example, from U.S. patents 5,773,441; 5,798,337;
5,830,433; 5,767,124; and 5,723,616. See also Bowers et al
0 (1991), Thorner et al (1997), Camanni et al (1998), Ankersen et al
(1998), Smith et al (1993) and Ghigo et al (1998). Thus, the
present invention is intended to include any substance which binds
to hGH receptor and initiates signalling thereof so as to obtain
the same ultimate qualitative effect as the administration of
5 natural hGH, insofar as the treatment of HADDS is concerned.
Pharmaceutical. compositions for administration according
to the present invention can comprise at least one human growth
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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.
Fox example, administration can be by parenteral, such
as subcutaneous, intravenous, intramuscular, intraperitoneal,
0 aerosol, or transdermal routes. The dosage administered depends
upon the age, health and weight of the recipient, type of previous
or concurrent treatment, if any, frequency of the treatment and
the nature of the effect desired.
Compositions within the scope o~ this invention include
all, composition 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.
while individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the
art. Typical dosages 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 5-30 weeks. When administered to AIDS
patients, the hGH anti-HADDS therapy may be administered
concomitantly with other AIDS therapies.
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
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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.
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 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.
The total dose required fox 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.
p In addition to the compounds of the invention, a
pharmaceutical composition may contain suitable pharmaceutically
acceptable carriers, such as excipients, carriers and/or
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically.
Experimez~tal
Eight AIDS patients, six male and two female, with a
history of long-term use (an average of 12 months) of highly
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active antiretroviral therapy (HAART), including protease
inhibitors (6 indinavir, 2 ritonavir/saquinavir) developed HADDS,
including buffalo humps, central adiposity and peripheral muscle
wasting associated with fatigue, along with elevated levels of
plasma triglycerides +/- cholesterol.
Therapy with rhGH (SEROSTIM.) was initiated in all
patients at a dose of 5 mg/day subcutaneously. Four patients
completed three months of rhGH and had notable improvements in fat
maldistr~.bution, with 25-75% reduction in buffalo hump syndrome
and abdominal girth, but no change in peripheral hipodystrophy.
weights were stable, and there were no consistent changes in total
body fat and blood lipids, despite 5-10% gain in fat-free mass.
One patient discontinued rhGH due to carpal tunnel syndrome and
had recurrence of HADDS. Three patients have had over six months
of therapy. One patient was lost to follow-up after six weeks of
therapy, and one patient has received fewer than eight weeks of
therapy. Yet, at last observation, all had notable reductions in
the size of and firmness of the buffalo hump and truncal
adiposity. These experiments establish that rhGH is effective in
treating H.ADDS, including reduction of buffalo humps and truncal
adiposity_
Having now fully described this invention, it will be
appreciated by those skilled in the art that the same can be
performed within a wide range of equivalent parameters,
concentrations and conditions without departing from the spirit
and scope of the invention and without undue experimentation.
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While this invention has been described in connection
with specific embodiments thereof, it will be understood that it
is capable of further modifications. This application is intended
to cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth as follows in the scope of the appended
0 claims.
All references cited herein, including journal articles
or abstracts, published or unpublished U.S. or foreign patent
application, issued U.S. or foreign patents or any other
references, are entirely incorporated by reference herein,
5 including all data, tables, figures and text presented in the
cited references. Additionally, the entire contents of the
references cited within the references cited herein are also
entirely incorporated by reference.
Reference to known method steps, conventional methods
0 steps, known methods or conventional methods is not any way an
admission that any aspect, description or embodiment of the
present invention is disclosed, taught or suggested in the
relevant art_
The foregoing description of the specific embodiments
5 will so fully reveal the general nature of the invention that
others can, by applying knowledge within the skill of the art
(including the contents of the references cited herein), readily
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modify and/or adapt for various application such specific
embodiments, without undue experimentation, without departing from
the general concept of the present invention. Therefore, such
adaptations and modifications are intended to be within the
meaning an range of equivalents of the disclosed embodiments,
based on the teaching and guidance presented herein. It is to be
understood that the phraseology or terminology herein is for the
purpose of description and not of limitation, such that the
terminology or phraseology of the present specification is to be
0 interpreted by the skilled artisan in light of the teachings and
guidance presented herein, in combination with the knowledge of
one of ordinary skill in the art.
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