U.S. patent application number 13/805190 was filed with the patent office on 2013-06-06 for glycoproteins having lipid mobilizing properties an therapeutic uses thereof.
The applicant listed for this patent is Steven Russell, Michael J. Tisdale. Invention is credited to Steven Russell, Michael J. Tisdale.
Application Number | 20130143797 13/805190 |
Document ID | / |
Family ID | 44584798 |
Filed Date | 2013-06-06 |
United States Patent
Application |
20130143797 |
Kind Code |
A1 |
Tisdale; Michael J. ; et
al. |
June 6, 2013 |
Glycoproteins Having Lipid Mobilizing Properties an Therapeutic
Uses Thereof
Abstract
The invention provides formulations and methods for ameliorating
symptoms associated with metabolic disorders, such as cachexia,
hypoglycemia, obesity, diabetes, and the like by administering
Zn-.alpha..sub.2-glycoproteins or a functional fragment thereof,
alone or in combination with additional agents, such as .beta.
adrenergin receptor agonists, .beta. adrenergin receptor
antagonists, and/or glycemic control agents.
Inventors: |
Tisdale; Michael J.;
(Claverdon, GB) ; Russell; Steven; (Wedensbury,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tisdale; Michael J.
Russell; Steven |
Claverdon
Wedensbury |
|
GB
GB |
|
|
Family ID: |
44584798 |
Appl. No.: |
13/805190 |
Filed: |
June 27, 2011 |
PCT Filed: |
June 27, 2011 |
PCT NO: |
PCT/GB11/00966 |
371 Date: |
February 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61358596 |
Jun 25, 2010 |
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61384652 |
Sep 20, 2010 |
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61420677 |
Dec 7, 2010 |
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Current U.S.
Class: |
514/4.8 ;
514/6.5; 514/7.4 |
Current CPC
Class: |
A61K 38/17 20130101;
A61K 38/1741 20130101; A61K 9/0043 20130101; A61P 31/18 20180101;
A61P 21/04 20180101; A61P 19/02 20180101; A61P 3/04 20180101; A61K
45/06 20130101; A61K 9/0014 20130101; A61P 7/00 20180101; A61P 3/10
20180101; A61P 31/04 20180101; A61P 21/00 20180101; A61P 11/00
20180101; A61K 31/137 20130101; A61P 9/04 20180101; A61K 38/26
20130101; A61P 35/00 20180101; A61P 43/00 20180101; A61P 13/12
20180101; C07K 14/473 20130101; A61P 3/06 20180101; A23L 33/17
20160801; A61P 5/50 20180101; A61K 31/138 20130101; A61K 47/60
20170801; A61K 9/0053 20130101; A61K 38/1709 20130101; A61K 38/28
20130101; A61K 47/61 20170801; A61P 3/00 20180101; A61K 38/17
20130101; A61K 2300/00 20130101; A61K 31/137 20130101; A61K 2300/00
20130101; A61K 38/1709 20130101; A61K 2300/00 20130101; A61K 38/26
20130101; A61K 2300/00 20130101; A61K 38/28 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/4.8 ;
514/7.4; 514/6.5 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 31/138 20060101 A61K031/138; A61K 45/06 20060101
A61K045/06 |
Claims
1-145. (canceled)
146. A formulation comprising a zinc-.alpha..sub.2-glycoprotein
(ZAG), a ZAG variant, a modified ZAG, or a functional fragment
thereof.
147. The formulation of claim 146, wherein the ZAG is
mammalian.
148. The formulation of claim 147, wherein the ZAG is human.
149. The formulation of claim 148, wherein the ZAG consists of the
amino acid sequence set forth in SEQ ID NO: 1.
150. The formulation of claim 149, wherein the ZAG is conjugated to
a non-protein polymer.
151. The formulation of claim 150, wherein the ZAG is sialylated,
PEGylated or modified to increase solubility or stability.
152. The formulation of claim 146, wherein the ZAG is recombinant
or synthetic.
153. The formulation of claim 146, wherein the modified ZAG
consists of the wild-type ZAG amino acid sequence with one or more
mutations to the amino acid sequence selected from deletions,
additions or conservative substitutions.
154. The formulation of claim 146, wherein the ZAG further
comprises one or more of a leader sequence and a trailing
sequence.
155. The formulation of claim 150, wherein the ZAG is
glycosylated.
156. The formulation of claim 155, wherein the ZAG is glycosylated
as a result of a posttranslational modification.
157. The formulation of claim 146, wherein the functional fragment
is generated by proteolysis chemical degradation, or
folding-domain-preserving fragmentation.
158. The formulation of claim 146, wherein the formulation
comprises at least 5, 10, 25, 50, 100 mg of ZAG.
159. The formulation of claim 146, further comprising a
pharmaceutically acceptable carrier.
160. The formulation of claim 146, further comprising one or more
agents selected from the group consisting of a .beta.3 agonist and
.beta.-adrenergic receptor (.beta.-AR) antagonist.
161. The formulation of claim 160, wherein the .beta.-AR antagonist
is selected from the group consisting of a .beta.2-adrenergic
receptor (.beta.2-AR) antagonist, a .beta.1-adrenergic receptor
(.beta.1-AR) antagonist, and a .beta.-adrenergic receptor
(.beta.3-AR) antagonist.
162. The fonnulation of claim 160, wherein the .beta.3 agonist is
selected from the group consisting of epinephrine (adrenaline),
norepinephrine (noradrenaline), isoprotenerol, isoprenaline,
propranolol, alprenolol, arotinolol, bucindolol, carazolol,
carteolol, clenbuterol, denopamine, fenoterol, nadolol, octopamine,
oxyprenolol, pindolol, [(cyano)pindolol], salbuterol, salmeterol,
teratolol, tecradine, trimetoquinolol, 3'-iodotrimetoquinolol,
3',5'-iodotrimetoquinolol, Amibegron, Solabegron, Nebivolol,
AD-9677, AJ-9677, AZ-002, CGP-12177, CL-316243, CL-317413,
BRL-37344, BRL-35135, BRL-26830, BRL-28410, BRL-33725, BRL-37344,
BRL-35113, BMS-194449, BMS-196085, BMS-201620, BMS-210285,
BMS-187257, BMS-187413, the CONH2 substitution of SO3H of
BMS-187413, the racemates of BMS-181413, CGP-20712A, CGP-12177,
CP-114271, CP-331679, CP-331684, CP-209129, FR-165914, FR-149175,
ICI-118551, ICI-201651, ICI-198157, ICI-D7114, LY-377604,
LY-368842, KTO-7924, LY-362884, LY-750355, LY-749372, LY-79771,
LY-104119, L-771047, L-755507, L-749372, L-750355, L-760087,
L-766892, L-746646, L-757793, L-770644, L-760081, L-796568,
L-748328, L-748337, Ro-16-8714, Ro-40-2148, (-)-RO-363, SB-215691,
SB-220648, SB-226552, SB-229432, SB-251023, SB-236923, SB-246982,
SR-58894A, SR-58611, SR-58878, SR-59062, SM-11044, SM-350300,
ZD-7114, ZD-2079, ZD-9969, ZM-215001, and ZM-215967.
163. The formulation of claim 161, wherein the .beta.3-AR
antagonist is SR59230A.
164. The formulation of claim 160, wherein the .beta.3-AR
antagonist is selected from the group consisting of propranolol,
(-)-propranolol, (+)-propranolol, practolol, (-)-practolol,
(+)-practolol, CGP-20712A, ICI-118551, (-)-bupranolol, acebutolol,
atenolol, betaxolol, bisoprolol, esmolol, nebivolol, metoprolol,
acebutolol, carteolol, penbutolol, pindolol, carvedilol, labetalol,
levobunolol, metipranolol, nadolol, sotalol, and timolol.
165. The formulation of claim 146, further comprising a glycemic
reducing agent selected from insulin, glucagon-like peptide-1
(GLP-1), or analogs thereof.
166. The formulation of claim 146, wherein the formulation is in
the form of a liquid, paste, powder, emulsion, suspension, or
solid.
167. The formulation of claim 146, wherein the formulation is in a
form suitable for oral administration as a spray, tablet,
lyophilized powder, semi-solid gel, sublingual dose, fast-melt
dose, buccal dose, liquid dose, lozenge, film, chewed dose,
taste-masked dose, or effervescent dose.
168. The formulation of claim 146, wherein the formulation is in a
form suitable for intranasal or pulmonary administration, including
spray, liquid, paste, emulsion, suspension, lyophilized powder, or
semi-solid gel.
169. The forumlation of claim 146, wherein the formulation is in a
form suitable for topical administration, including spray, liquid,
paste, emulsion, suspension, lyophilized powder, cream, ointment or
semi-solid gel.
170. The formulation of claim 146, wherein the formulation is
suitable for injection via autoinjectors, pump devices, prefilled
syringes, and needle free technologies.
171. The formulation of claim 146, further comprising one or more
excipients selected from the group consisting of phosphate, Tris,
arginine, glycine, Tween 80, sucrose, trehalose, mannitol, casein
proteins, and derivatives thereof.
172. The formulation of claim 146, wherein the ZAG, ZAG variant,
modified ZAG, or functional fragment thereof is present as
polymeric.
173. A foodstuff additive or nutritional supplement comprising the
formulation of claim 146.
174. A foodstuff or nutritional supplement comprising a consumable
carrier in combination with the formulation of claim 146.
175. A method for delivering a zinc-.alpha..sub.2-glycoprotein
(ZAG) to a mammalian subject, the method comprising delivering to
the subject by oral administration the formulation of claim
146.
176. A method for increasing a subject's endogenous level of a
zinc-.alpha..sub.2-glycoprotein (ZAG), the method comprising
administering to the subject the formulation of claim 146.
177. A method of ameliorating symptoms of cachexia in a subject
comprising administering to the subject in need of such treatment a
therapeutically effective dosage of an inhibitor of a polypeptide
having the sequence as shown in SEQ ID NO: 1, wherein there is
amelioration of symptoms associated with cachexia following
treatment.
178. A method of treating a subject to bring about a reduction in
weight loss comprising administering to the subject in need of such
treatment a therapeutically effective dosage of an inhibitor of the
polypeptide having the sequence as shown in SEQ ID NO: 1 in alone
or in combination with one or more agents selected from the group
consisting of a .beta.adrenergic receptor (.beta.3-AR) antagonist
and a .beta.3-adrenergic receptor (.beta.3-AR) antagonist.
179. A method of ameliorating symptoms of diabetes or obesity in a
mammalian subject comprising administering to the subject in need
of such treatment a therapeutically effective dosage of a
formulation of claim 146 in combination with a glycemic reducing
agent selected from insulin, glucagon-like peptide-1 (GLP-1), or
analogs thereof in any sequence or simultaneously.
180. A method of monitoring zinc-.alpha..sub.2-glycoprotein (ZAG)
activity in a mammalian subject, comprising: a) orally
administering the subject the formulation of claim 146; and b)
detecting the level of ZAG activity; thereby monitoring ZAG
activity in the subject.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates generally to medicinal
formulations and supplements, and more particularly, to
formulations and methods for altering the metabolism of a subject,
as well as ameliorating disorders such as cachexia, obesity,
diabetes and insulin resistance.
[0003] 2. Background Information
[0004] The prevalence of obesity in adults, children and
adolescents has increased rapidly over the past 30 years in the
United States and globally and continues to rise. Obesity is
classically defined based on the percentage of body fat or, more
recently, the body mass index (BMI), also called Quetlet index
(National Task Force on the Prevention and Treatment of Obesity,
Arch. Intern. Med., 160: 898-904 (2000); Khaodhiar, L. et al.,
Clin. Cornerstone, 2: 17-31 (1999)). The BMI is defined as the
ratio of weight (kg) divided by height (in meters) squared.
[0005] Overweight and obesity are associated with increasing the
risk of developing many chronic diseases of aging seen in the U.S.
Such co-morbidities include type 2 diabetes mellitus, hypertension,
coronary heart diseases and dyslipidemia, gallstones and
cholecystectomy, osteoarthritis, cancer (of the breast, colon,
endometrial, prostate, and gallbladder), and sleep apnea. It is
estimated that there are around 325,000 deaths annually that are
attributable to obesity. The key to reducing the severity of the
diseases is to lose weight effectively. Although about 30 to 40%
claim to be trying to lose weight or maintain lost weight, current
therapies appear not to be working. Besides dietary manipulation,
pharmacological management and in extreme cases, surgery, are
sanctioned adjunctive therapies to treat overweight and obese
patients (Expert Panel, National Institute of Health, Heart, Lung,
and Blood Institute, 1-42 (June 1998); Bray, G. A., Contemporary
Diagnosis and Management of Obesity, 246-273 (1998)). Drugs have
side effects, and surgery, although effective, is a drastic measure
and reserved for morbidly obese.
[0006] Cachexia is wasting of both adipose and skeletal muscle mass
caused by disease. It occurs in many conditions and is common with
many cancers when remission or control fails. Patients with
advanced cancer, AIDS, and some other major chronic progressive
diseases may appear cachectic. Cachexia can occur in people who are
eating enough, but who cannot absorb the nutrients. While cachexia
may be mediated by certain cytokines, especially tumor necrosis
factor-.alpha., IL-1b, and IL-6, which are produced by tumor cells
and host cells in the tissue mass, there is currently no widely
accepted treatment for cachexia.
[0007] Diabetes mellitus is a major cause of morbidity and
mortality. Chronically elevated blood glucose leads to debilitating
complications: nephropathy, often necessitating dialysis or renal
transplant; peripheral neuropathy; retinopathy leading to
blindness; ulceration of the legs and feet, leading to amputation;
fatty liver disease, sometimes progressing to cirrhosis; and
vulnerability to coronary artery disease and myocardial
infarction.
[0008] There are two primary types of diabetes. Type I, or
insulin-dependent diabetes mellitus (IDDM) is due to autoimmune
destruction of insulin-producing beta cells in the pancreatic
islets. The onset of this disease is usually in childhood or
adolescence. Treatment consists primarily of multiple daily
injections of insulin, combined with frequent testing of blood
glucose levels to guide adjustment of insulin doses, because excess
insulin can cause hypoglycemia and consequent impairment of brain
and other functions. Increasing scrutiny is being given to the role
of insulin resistance to the genesis, progression, and therapeutic
management of this type of diabetic disease.
[0009] Type II, or noninsulin-dependent diabetes mellitus (NIDDM)
typically develops in adulthood. NIDDM is associated with
resistance of glucose-utilizing tissues like adipose tissue,
muscle, and liver, to the actions of insulin. Initially, the
pancreatic islet beta cells compensate by secreting excess insulin.
Eventual islet failure results in decompensation and chronic
hyperglycemia. Conversely, moderate islet insufficiency can precede
or coincide with peripheral insulin resistance. There are several
classes of drugs that are useful for treatment of NIDDM: 1) insulin
releasers, which directly stimulate insulin release, carrying the
risk of hypoglycemia; 2) prandial insulin releasers, which
potentiate glucose-induced insulin secretion, and must be taken
before each meal; 3) biguanides, including metformin, which
attenuate hepatic gluconeogenesis (which is paradoxically elevated
in diabetes); 4) insulin sensitizers, for example the
thiazolidinedione derivatives rosiglitazone and pioglitazone, which
improve peripheral responsiveness to insulin, but which have side
effects like weight gain, edema, and occasional liver toxicity; 5)
insulin injections, which are often necessary in the later stages
of NIDDM when the islets have failed under chronic
hyperstimulation.
[0010] Insulin resistance can also occur without marked
hyperglycemia, and is generally associated with atherosclerosis,
obesity, hyperlipidemia, and essential hypertension. This cluster
of abnormalities constitutes the "metabolic syndrome" or "insulin
resistance syndrome". Insulin resistance is also associated with
fatty liver, which can progress to chronic inflammation (NASH;
"nonalcoholic steatohepatitis"), fibrosis, and cirrhosis.
Cumulatively, insulin resistance syndromes, including but not
limited to diabetes, underlie many of the major causes of morbidity
and death of people over age 40.
[0011] Despite the existence of such drugs, diabetes remains a
major and growing public health problem. Late stage complications
of diabetes consume a large proportion of national health care
resources. There is a need for new orally active therapeutic agents
which effectively address the primary defects of insulin resistance
and islet failure with fewer or milder side effects than existing
drugs.
[0012] Zinc-.alpha..sub.2-glycoprotein (ZAG) has been identified as
a lipid mobilizing factor (LMF) with the potential to induce fat
loss in cancer cacehxia. ZAG was shown to induce lipolysis in white
adipocytes by interaction with a .beta.3-adrenergic receptor, while
in vivo it increased expression of uncoupling protein-1 (UCP-1) in
brown adipose tissue (BAT), and induced loss of body fat. In
addition to some tumors, ZAG is also produced by white adipose
tissue (WAT) and BAT and its expression is upregulated in cachexia.
In contrast ZAG expression in adipose tissue of obese humans was
only 30% of that found in non-obese subjects. This suggests that
loss of ZAG expression in WAT could account for some of the
features of obesity. Certainly inactivation of both ZAG alleles in
mice led to an increase in body weight which was more pronounced
when the animals were fed a high fat diet. The lipolytic response
to various agents was significantly decreased in adipocytes from
ZAG deficient animals.
[0013] To date studies on the lipid mobilizing effect of ZAG have
been carried out in both mice and rats using human and murine ZAG.
The studies indicate that ZAG is evolutionarily conserved and
exhibits cross-species activity, e.g., murine ZAG exhibiting
substantially the same activity in humans and vice-versa.
[0014] There remains a lack of effective and safe alternatives for
altering metabolism and treatment of metabolic diseases, such as
obesity, diabetes and cachexia. There is therefore a need for new
formulations for such uses.
SUMMARY OF THE INVENTION
[0015] The present invention is based in part on the finding that
Zinc-.alpha..sub.2-glycoprotein has an effect on body weight and
insulin responsiveness in adult obese hyperglycemic (ob/ob) mice
and mature Wistar rats, and that anti-ZAG antibodies prevent weight
loss in cachexia situations. Such a finding is useful in methods
for moderating body weight, improving insulin responsiveness or
ameliorating the symptoms associated with cachexia or diseases
associated with muscle wasting.
[0016] In one embodiment the present invention provides a
formulation comprising a zinc-.alpha..sub.2-glycoprotein (ZAG), a
ZAG variant, a modified ZAG, or a functional fragment thereof In
one aspect, the ZAG is mammalian, e.g., human, and may include the
amino acid sequence set forth in SEQ ID NO: 1. The ZAG peptide may
conjugated to a non-protein polymer. The ZAG peptide may be
sialylated, PEGylated or modified to increase solubility or
stability. The ZAG peptide may be recombinant or synthetic. In
various aspects, the ZAG peptide may be modified ZAG and include
the wild-type ZAG amino acid sequence with one or more mutations to
the amino acid sequence selected from deletions, additions or
conservative substitutions. In various aspects, the ZAG peptide may
include one or more of a leader sequence and a trailing sequence.
The ZAG peptide may also be glycosylated, e.g., as a result of a
posttranslational modification. Additionally, in various
embodiments the formulation may further include a pharmaceutically
acceptable carrier. The formulation may also include one or more
agents including a .beta.3 agonist and .beta.-adrenergic receptor
(.beta.-AR) antagonist, such as a .beta.2-adrenergic receptor
(.beta.2-AR) antagonist, a .beta.1-adrenergic receptor (.beta.1-AR)
antagonist, and a .beta.3-adrenergic receptor (.beta.3-AR)
antagonist. In some aspects, the formulation of claim 1 may further
include a glucagon-like peptide-1 (GLP-1) or an analog thereof.
[0017] In another embodiment, the invention provides a foodstuff
additive or nutritional supplement including the formulation of the
invention as described herein.
[0018] In another embodiment, the invention provides a method for
delivering a formulation to a mammalian subject, the method
including administering to the mammalian subject the formulation as
described herein.
[0019] In another embodiment, the invention provides a method for
delivering a zinc-.alpha..sub.2-glycoprotein (ZAG) to a mammalian
subject, the method including delivering to the subject by oral
administration the formulation as described herein.
[0020] In another embodiment, the invention provides a method for
orally delivering a zinc-.alpha..sub.2-glycoprotein (ZAG) to a
mammalian subject in mega doses similar to that of mega dosed oral
insulin requiring systemic absorption of administered ZAG as
described herein.
[0021] In another embodiment, the invention provides a method for
orally delivering a zinc-.alpha..sub.2-glycoprotein (ZAG) to a
mammalian subject in surprisingly effective low doses similar to
that of intravenous administration of ZAG and in formulations
surprisingly not requiring systemic absorption of administered ZAG
as described herein.
[0022] In another embodiment, the invention provides a method for
increasing a subject's endogenous level of a
zinc-.alpha..sub.2-glycoprotein (ZAG), the method including
administering to the subject the formulation as described
herein.
[0023] The present invention further provides a method of
ameliorating symptoms of cachexia in a subject. The method includes
administering to the subject in need of such treatment a
therapeutically effective dosage of an inhibitor of the biological
activity of a polypeptide having the sequence as shown in SEQ ID
NO: 1, resulting in an amelioration of symptoms associated with
cachexia following treatment. In one embodiment, the inhibitor is a
monoclonal antibody that binds a polypeptide that comprises a
sequence at least 80% homologous to the polypeptide having the
sequence as shown in SEQ ID NO: 1. In another embodiment, the
treatment includes daily administration for 10 days. In another
embodiment, the inhibitor is administered daily, every other day,
every 2 days, or every 3 days, for up to 10 days or longer. In
another embodiment, the antibody is administered twice daily. The
antibody may be administered intravenously, subcutaneously,
sublingually, intranasally, orally, or via inhalation. In another
embodiment, the inhibitor is administered in combination with one
or more agents selected from the group consisting of a
.beta.3-adrenergic receptor (.beta.3-AR) antagonist. In one
embodiment, the .beta.3-AR antagonist is SR59230A. In another
embodiment, the antibody is glycosylated. In another embodiment,
the agent that inhibits the homologous polypeptide is a
non-antibody agent, for example but not limited to, an aptamer.
[0024] In another aspect, the present invention provides a method
of treating a subject to bring about reduction in weight loss. The
method includes administering to the subject in need of such
treatment a therapeutically effective dosage of an inhibitor of the
polypeptide having the sequence as shown in SEQ ID NO: 1 in
combination with one or more agents selected from the group
consisting of a .beta.3-adrenergic receptor (.beta.3-AR)
antagonist. In one embodiment, the inhibitor is a monoclonal
antibody that binds a polypeptide that comprises a sequence at
least 80% homologous to the polypeptide having the sequence as
shown in SEQ ID NO: 1. In another embodiment, the .beta.3-AR
antagonist is SR59230A. In another embodiment, the antibody is
glycosylated. In another embodiment, the agent that inhibits the
homologous polypeptide is a non-antibody agent, for example but not
limited to, an aptamer.
[0025] In another aspect, the present invention provides a
pharmaceutical composition comprising an antibody, or functional
fragment thereof, that binds the polypeptide having the sequence as
shown in SEQ ID NO: 1 and an agent selected from the group
consisting of a .beta.3-adrenergic receptor (.beta.3-AR) antagonist
and a .beta.3 antagonist. In one embodiment, the .beta.3-AR
antagonist is SR59230A. In another embodiment, the antibody is
glycosylated.
[0026] This disclosure provides materials and methods for
supplementing a human or animal diet. In one aspect, the instant
disclosure provides nutritional supplement formulations. A
nutritional supplement formulation of the invention can include
zinc-.alpha.2-glycoprotein (ZAG) or a functional fragment thereof.
The disclosure also provides materials such as kits that include
one or more nutritional supplement formulations, such as
nutritional supplement formulations that include ZAG or functional
fragments thereof In another aspect, the disclosure provides a
method of delivery of an orally administered therapeutic agent,
including administering a .beta.3 agonist in combination with the
orally administered therapeutic agent.
[0027] The formulations, kits, and methods herein can be useful for
improving a human's health and/or to promote weight loss, or
independent of weight loss, improve insulin resistance and reduce
hyperglycemia. These formulations, kits and methods may therefore
find use in the treatment of diseases associated with obesity
and/or hyperglycemia.
[0028] In another aspect, the invention provides a food stuff that
includes the formulation of the invention in combination with a
consumable carrier. Exemplary consumable carriers include, but are
not limited to cookies, brownies, crackers, breakfast bars, energy
bars, cereals, cakes, breads, beverages, meat products, and meat
substitute products.
[0029] In another aspect, the invention provides a method of
supplementing a human diet.
[0030] The method includes ingesting formulation that includes
zinc-.alpha..sub.2-glycoprotein (ZAG) or a functional fragment
thereof. In one embodiment, the ZAG is mammalian, such as the human
ZAG polypeptide having the sequence as shown in SEQ ID NO: 1, or a
fragment thereof. The method may be performed daily for 10 days. In
another embodiment, the formulation is ingested daily, every other
day, every 2 days, or every 3 days, for up to 10 days or longer. In
another embodiment, the formulation is ingested twice daily. In
another embodiment, the formulation is ingested in combination with
one or more agents selected from the group consisting of a
.beta.3-adrenergic receptor (.beta.3-AR) agonist and a .beta.AR
agonist and a .beta.3-AR antagonist. In one embodiment, the
.beta.3-AR antagonist is SR59230A. In another embodiment,
.beta.3-AR agonist is AMNI-BRL37344 (BRL37344). In another
embodiment, the formulation is ingested or delivered in combination
with one or more agents used to improve glycemic control whether
sequentially in any order or in parallel. In one embodiment, the
glycemic control agent is insulin or any derivative or analog
thereof. In another embodiment, the glycemic control agent is a
glucagon-like peptide-1 (GLP-1) or any derivative or analog
thereof.
[0031] In another aspect, the invention provides a method of
delivery of an orally administered therapeutic agent, wherein the
therapeutic agent is delivered in combination with a .beta.3
agonist. In another embodiment, the .beta.3 agonist and the
therapeutic agent are delivered simultaneously. In yet another
embodiment, the .beta.3 agonist is administered prior to or
following administration of the therapeutic agent. In certain
embodiments, the therapeutic agent is ZAG. In other embodiments,
the therapeutic agent includes atrial natriuretic peptides, brain
natriuretic peptides, platelet aggregation inhibitors,
streptokinase, heparin, urokinase, renin inhibitors, insulin,
antibiotics, and sleep inducing peptide.
[0032] In a further aspect, the present invention provides a method
of treating a subject to bring about a weight reduction or
reduction in obesity. The method includes administering to the
subject in need of such treatment a nutritional supplement
formulation that includes a therapeutically effective dosage of a
polypeptide having the sequence as shown in SEQ ID NO: 1 or a
fragment thereof.
[0033] In another embodiment, the invention provides a method of
monitoring zinc-.alpha..sub.2-glycoprotein (ZAG) activity in a
mammalian subject. The method includes: a) orally administering the
subject the formulation of the invention; and b) detecting the
level of ZAG activity; thereby monitoring ZAG activity in the
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1A is a pictorial diagram showing characterization of
ZAG and its effect on lipolysis and body weight of ob/ob mice.
Coomassie staining after 12% SDS-PAGE showing total proteins in 293
cell media and ZAG purified as described.
[0035] FIG. 1B is a pictorial diagram showing the results of a
Western blot showing expression of ZAG in culture medium and
purified ZAG.
[0036] FIG. 1C is a graphical diagram showing ZAG mRNA levels in
adipose tissue and liver tissue in MAC16 mice undergoing weight
loss. P<0.01.
[0037] FIG. 1D is a graphical diagram showing the results of
lipolysis in epididymal adipocytes from non-obese (.box-solid.) and
ob/ob mice (.quadrature.) in response to isoprenaline (Iso) and
ZAG. Differences from non-obese mice are shown as *p<0.05,
**p<0.01 and ***p<0.001.
[0038] FIG. 1E is a graphical diagram showing the results of
lipolysis in adipocytes from epididymal (ep), subcutaneous (s.c.)
and visceral (vis) deposits from obese (ob/ob) and non-obese (non
ob) mice with either no treatment (.box-solid.), isoprenaline (10
.mu.M) (.quadrature.) or ZAG (0.46 .mu.M) (). Differences from
epididymal adipocytes are shown as **p<0.01.
[0039] FIG. 1F is a graphical diagram showing the effect of ZAG
(.box-solid.) on body weight of ob/ob mice in comparison with PBS
(.diamond-solid.) as described in the methods. Differences in
weight form time zero and PBS controls are shown as
***p<0.001.
[0040] FIG. 1G is a graphical diagram showing the effect of ZAG
(.quadrature.) on body temperature of the mice shown in e in
comparison with PBS controls (.box-solid.). Differences from
control are shown as ***p<0.001.
[0041] FIG. 2A is a graphical diagram showing glucose tolerance of
ob/ob mice treated with ZAG. Plasma glucose levels in ob/ob mice in
the fed state either treated with ZAG (.box-solid.) or PBS
(.diamond-solid.) for 3 days after i.v. administration of glucose
(2 g/kg). p<0.001 from PBS. Blood samples were removed from the
tail vein at intervals after glucose administration and used for
the measurement of glucose and insulin.
[0042] FIG. 2B is a graphical diagram showing plasma insulin levels
in ob/ob mice treated with ZAG after oral administration of glucose
(1 g/kg). p<0.001 from PBS.
[0043] FIG. 2C is a graphical diagram showing glucose uptake into
epididymal (ep), visceral (vis) and subcutaneous (s.c.) adipocytes
of ob/ob mice treated with ZAG for 5 days in the presence of 0
(.box-solid.), 1(.quadrature.) or 10 nM insulin (). Differences in
the presence of ZAG are indicated as ***p<0.001.
[0044] FIG. 2D is a graphical diagram showing uptake of
2-deoxy-D-glucose into gastrocnemius muscle of ob/ob mice treated
with either ZAG or PBS for 5 days in the absence or presence of
insulin (100 nM). Differences in the presence of insulin are shown
as *p<0.05 or **p<0.01, while differences in the presence of
ZAG are shown as ***p<0.001.
[0045] FIG. 2E is a pictorial diagram showing the effect of ZAG on
the expression of GLUT4 glucose transporter in skeletal musclein of
ob/ob mice. After treatment of ob/ob mice for 5 days skeletal
muscle was removed and Western blotted for expression of GLUT4.
[0046] FIG. 3A is a graphical diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and used for the measurement of protein synthesis.
Differences from PBS controls, or non-obese animals are shown as
***p<0.001.
[0047] FIG. 3B is a graphical diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and used for the measurement of protein degradation.
Differences from PBS controls, or non-obese animals are shown as
***p<0.001.
[0048] FIG. 3C is a graphical diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and used for the measurement of chymotrypsin-like enzyme
activity. Differences from PBS controls, or non-obese animals are
shown as ***p<0.001.
[0049] FIG. 3D is a pictorial diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and Western blotted for expression of 20S-proteasome
.alpha.-subunits.
[0050] FIG. 3E is a pictorial diagram showing the effect of ZAG on
signaling pathwasy in skeletal muscle of ob/ob mice. After
treatment of ob/ob mice for 5 days skeletal muscle was removed and
Western blotted for expression of p42.
[0051] FIG. 3F is a pictorial diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and Western blotted for expression of myosin.
[0052] FIG. 3G is a pictorial diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob mice.
After treatment of ob/ob mice for 5 days skeletal muscle was
removed and Western blotted for expression of actin as a
control.
[0053] FIG. 4A is a pictorial diagram showing the effect of ZAG on
catabolic signaling pathways in skeletal muscle by Western blotting
of phospho PKR in gastrocnemius muscle of ob/ob mice after
treatment with either PBS or ZAG for 5 days. The total forms of the
proteins serve as loading controls. Differences from PBS controls
are shown as ***p<0.001 while differences from non-obese mice
are shown as # p<0.001.
[0054] FIG. 4B is a pictorial diagram showing the effect of ZAG on
catabolic signaling pathways in skeletal muscle by Western blotting
of phospho eIF2a in gastrocnemius muscle of ob/ob mice after
treatment with either PBS or ZAG for 5 days. The total forms of the
proteins serve as loading controls. Differences from PBS controls
are shown as ***p<0.001 while differences from non-obese mice
are shown as # p<0.001.
[0055] FIG. 4C is a pictorial diagram showing the effect of ZAG on
catabolic signaling pathways in skeletal muscle by Western blotting
of phospho PLA.sub.2 in gastrocnemius muscle of ob/ob mice after
treatment with either PBS or ZAG for 5 days. The total forms of the
proteins serve as loading controls. Differences from PBS controls
are shown as ***p<0.001 while differences from non-obese mice
are shown as # p<0.001.
[0056] FIG. 4D is a pictorial diagram showing the effect of ZAG on
catabolic signaling pathways in skeletal muscle by Western blotting
of phospho p38MAPK in gastrocnemius muscle of ob/ob mice after
treatment with either PBS or ZAG for 5 days. The total forms of the
proteins serve as loading controls. Differences from PBS controls
are shown as *** p<0.001 while differences from non-obese mice
are shown as # p<0.001.
[0057] FIG. 4E is a graphical diagram showing the effect of ZAG on
catabolic signaling pathways in skeletal muscle by activity of
caspase-3 (.box-solid.) and caspase-8 (.quadrature.) in
gastrocnemius muscle of ob/ob mice after treatment with either PBS
or ZAG for 5 days.
[0058] FIG. 5A is a pictorial diagram showing expression of HSL in
response to ZAG. Western blots show expression of phospho HSL in
adipocytes of non-obese mice 3 h after no treatment (Con), or
treatment with isoprenaline (10 mM) or ZAG (0.46 .mu.M) alone, or
in the presence of PD98059 (25 .mu.M) after 5 days treatment with
ZAG.
[0059] FIG. 5B is a pictorial diagram showing expression of HSL by
immunoblotting in epididymal (ep) adipocytes after 5 days treatment
with ZAG.
[0060] FIG. 5C is a pictorial diagram showing expression of HSL by
immunoblotting in subcutaneous (sc) adipocytes after 5 days
treatment with ZAG.
[0061] FIG. 5D is a pictorial diagram showing expression of HSL by
immunoblotting in visceral (vis) adipocytes after 5 days treatment
with ZAG.
[0062] FIG. 5E is a pictorial diagram showing expression of ATGL in
epididymal adipocytes after 5 days treatment with ZAG.
[0063] FIG. 5F is a pictorial diagram showing expression of ATGL in
subcutaneous adipocytes after 5 days treatment with ZAG.
[0064] FIG. 5G is a pictorial diagram showing expression of ATGL in
visceral adipocytes after 5 days treatment with ZAG.
[0065] FIG. 5H is a pictorial diagram showing expression of pERK in
epididymal adipocytes after 5 days treatment with ZAG.
[0066] FIG. 5I is a pictorial diagram showing expression of pERK in
subcutaneous adipocytes after 5 days treatment with ZAG.
[0067] FIG. 5J is a pictorial diagram showing expression of pERK in
visceral adipocytes after 5 days treatment with ZAG.
[0068] FIG. 5K is a graphical diagram showing the response of
adipocytes from epididymal (ep), subcutaneous (sc) and visceral
(vis) deposits from ob/ob mice treated with either PBS or ZAG for 5
days to the lipolytic effect of BRL37344. Differences from PBS
controls are indicated as ***p<0.01, while differences in the
presence of PD98059 is shown as # p<0.001.
[0069] FIG. 6A is a pictorial diagram showing the Effect of
treatment of ob/ob mice for 5 days with ZAG on the expression of
ZAG in WAT. Western blot showing expression of ZAG in ep, sc, and
vis adipocytes. Day 0 represents the day the adipocytes were
removed from the mice.
[0070] FIG. 6B is a pictorial diagram showing expression of ZAG in
epididymal adipocytes that were suspended in RMPI medium as
described in methods. The samples were then taken out at daily
intervals and Western blotted for ZAG expression. Day 0 represents
the day the adipocytes were removed from the mice.
[0071] FIG. 6C is a pictorial diagram showing expression of HSL in
epididymal adipocytes that were suspended in RMPI medium as
described in methods. The samples were then taken out at daily
intervals and Western blotted for HSL expression. Day 0 represents
the day the adipocytes were removed from the mice.
[0072] FIG. 6D is a pictorial diagram showing expression of UCP1 in
BAT removed from mice. Differences from PBS treated mice are shown
as ***p<0.001.
[0073] FIG. 6E is a pictorial diagram showing expression of UCP3 in
BAT removed from mice. Differences from PBS treated mice are shown
as ***p<0.001.
[0074] FIG. 6F is a pictorial diagram showing expression of UCP3 in
gastrocnemius muscle removed from mice. Differences from PBS
treated mice are shown as ***p<0.001.
[0075] FIG. 7A is a graphical diagram showing weight loss of the
ob/ob mice during the 21 day study. ZAG was injected at days 1, 4,
5, 8, 13, 16, 18, and 19; PBS was injected at the same time
points.
[0076] FIG. 7B is a graphical diagram showing weight change (g) of
the ob/ob mice (weight 80-90 g) during treatment with ZAG.
[0077] FIG. 7C is a graphical diagram showing increased body
temperature of the ob/ob mice during the 21 day study. ZAG was
injected at days 1, 4, 5, 8, 13, 16, 18, and 19; PBS was injected
at the same time points.
[0078] FIG. 8A is a graphical diagram showing a progressive
decrease in urinary glucose excretion during the first 5 days of
treatment.
[0079] FIG. 8B is a graphical diagram showing a progressive
decrease in urinary glucose excretion during the 21 day study.
[0080] FIG. 9 is a graphical diagram showing glycerol release
stimulated by isoprenaline (iso) isolated adipocytes which have
been in culture up to 5 days from ob mice treated with and without
ZAG.
[0081] FIG. 10 is a pictorial diagram showing the complete amino
acid sequence (SEQ ID NO: 1) of the human plasma
Zn-.alpha..sub.2-glycoprotein, as published by T. Araki et al.
(1988) "Complete amino acid sequence of human plasma
Zn-.alpha..sub.2-glycoprotein and its homology to
histocompatibility antigens."
[0082] FIG. 11 is a graphical diagram showing lipolytic activity of
human ZAG in isolated rat epididymal adipocytes, compared with
isoprenaline (10 .mu.M) in the absence or presence of SR59230A (10
.mu.M) or anti-ZAG antibody (1:1000) (IgG). Each value is an
average of 5 separate studies. Differences from control are shown
as b, p<0.01 or c, p<0.001, while differences from ZAG alone
are indicated as e, p<0.01 or f, p<0.001.
[0083] FIG. 12A is a graphical diagram showing the effect of daily
i.v. administration of ither ZAG (50 .mu.g/100 g b.w.) in 100 .mu.l
PBS (.box-solid.) or PBS alone (.diamond-solid.) on body weight of
male Wistar rats over a 10 day period. The protocol for the
experiment is given in the methods section.
[0084] FIG. 12B is a graphical diagram showing the body temperature
of male Wistar rats administered either ZAG (.box-solid.) or PBS
(.diamond-solid.) as described in FIG. 12A.
[0085] FIG. 12C is a graphical diagram showing the uptake of
2-deoxy-D-glucose into epididymal adipocytes of male Wistar rats
after 10 days treatment with either ZAG (open box) or PBS (closed
box) for 10 days, as shown in FIG. 12A, in the absence or presence
of insulin (60 .mu.U/ml).
[0086] FIG. 12D is a graphical diagram showing glucose uptake into
gastrocnemius muscle and BAT of male Wistar rats after 10 days
treatment with either ZAG or PBS, in the absence or presence of
insulin (60 .mu.U/ml). Differences between ZAG and PBS treated
animals are shown as a, p<0.05, b, p<0.01 or c, p<0.001,
while differences in the presence of insulin are shown as or f,
p<0.001.
[0087] FIG. 12E is a graphical diagraph showing tissue Rg in ob/ob
mice administered ZAG. c, p<0.001 from PBS.
[0088] FIGS. 13A-13C are pictorial diagrams of Western blots
showing expression of GLUT4 in BAT (FIG. 13A) and WAT (FIG. 13B)
and gastrocnemius muscle (FIG. 13C) of male Wistar rats treated
with either PBS or ZAG for 10 days as shown in FIG. 12. Differences
between ZAG and PBS treated animals are shown as c, p<0.001.
[0089] FIGS. 14A and 14B are pictorial diagrams of Western blots
showing expression of UCP1 and UCP3 in BAT (FIG. 14A) and WAT (FIG.
14B) of male Wistar rats treated with either PBS or ZAG for 10 days
as shown in FIG. 12. Differences between ZAG and PBS treated
animals are shown as c, p<0.001.
[0090] FIGS. 15A and 15B are pictorial diagrams of Western blots
showing expression of ATGL (FIG. 15A) and HSL (FIG. 15B) in
epididymal adipose tissue of male Wistar rats treated with either
PBS or ZAG for 10 days as shown in FIG. 12. Differences between ZAG
and PBS treated animals are shown as c, p<0.001.
[0091] FIGS. 16A-16C are pictorial diagrams of Western blots
showing expression of ZAG in gastrocnemius muscle (FIG. 16A), WAT
(FIG. 16B) and BAT (FIG. 16C). Tissues were excised from male
Wistar rats treated with either PBS or ZAG for 10 days as shown in
FIG. 12. Differences between ZAG and PBS treated animals are shown
as c, p<0.001.
[0092] FIGS. 17A and 17B are pictorial diagrams of Western blots
showing expression of phosphorylated and total forms of pPKR (FIG.
17A) and peIF2.alpha. (FIG. 17B) in gastrocnemius muscle of male
Wistar rats treated with either PBS or ZAG for 10 days as shown in
FIG. 12. The densitometric analysis is the ratio of the phosphor to
total forms, expressed as a percentage of the value for rats
treated with PBS.
[0093] FIGS. 18A and 18B is a graphical diagram showing
phenylalanine release (FIG. 18A) and protein synthesis (FIG. 18B)
in C2C12 myotubes treated with and without ZAG for 4 h in the
presence of various concentrations of glucose. Statistically
significant c, P<0.001 from control; f, P<0.001 from glucose
alone.
[0094] FIG. 19 is a graphical diagram showing pheylalanine release
in C2C12 myotubes treated with and without ZAG in the presence of
various concentrations of glucose and with and without SR59230A.
Statistically significant b, P<0.01 and c, P<0.001 from
control; e, P<0.05 and f, P<0.001 from glucose alone.
[0095] FIG. 20 is a graphical diagram showing protein synthesis in
C2C12 myotubes treated with and without ZAG in the presence of
various concentrations of glucose and with and without SR59230A.
Statistically significant b, P<0.01 and c, P<0.001 from
control; e, P<0.05 and f, P<0.001 from glucose alone; I,
P<0.001 from glucose+SR.
[0096] FIG. 21 is a graphical diagram showing ROS activity in C2C12
myotubes treated with various concentrations of glucose with and
without ZAG. Statistically significant c, P<0.001 from control
f, P<0.001 from glucose alone.
[0097] FIG. 22A is a pictorial diagram of a Western blot showing
pPKR in C2C12 myotubes treated with glucose with and without ZAG.
Statistically significant c, P<0.001 from control f, P<0.001
from glucose alone.
[0098] FIG. 22B is a pictorial diagram of a Western blot showing
peIF2a in C2C12 myotubes treated with glucose with and without ZAG.
Statistically significant c, P<0.001 from control f, P<0.001
from glucose alone.
[0099] FIGS. 23A and 23B are graphical diagrams showing the results
of an insulin tolerance test in ob/ob mice treated with and without
ZAG. Statistically significant b, P<0.05 and c, P<0.001 from
with ZAG.
[0100] FIG. 24 is a graphical diagram showing the oxidation of
D4U-.sup.14C glucose] to .sup.14CO.sub.2 in ob/ob mice.
[0101] FIG. 25 is a graphical diagram showing production of
.sup.14CO.sub.2 from [.sup.14C carboxy] triolein in ob/ob mice.
[0102] FIG. 26 is a graphical diagram showing reduction in weight
loss in mice administered anti-ZAG, as compared to mice
administered BRL37344 (cachexia model).
[0103] FIG. 27 is a graphical diagram showing glucose tolerance in
ob/ob mice treated with the .beta.3 agonist, BRL37344 in the
absence and presence of an anti-ZAG antibody.
[0104] FIG. 28 is a graphical diagram showing the results of
lipolysis in epididymal murine adipocytes in response to
isoprenaline (Iso), ZAG, and an anti-ZAG antibody.
[0105] FIG. 29 is a graphical diagram showing weight change in
ob/ob mice treated with BRL in the absence or presence of anti-ZAG
where BRL was added either 24 h prior to anti-ZAG ab or at the same
time.
[0106] FIG. 30 is a graphical diagram showing decreased proteolysis
and increased muscle synthesis in ZAG treated ob/ob mice.
[0107] FIG. 31 is a graphical diagram showing weight change in
ob/ob mice treated with and without ZAG.
[0108] FIG. 32 is a graphical diagram showing body temperature in
ob/ob mice treated with and without ZAG.
[0109] FIG. 33 is a graphical diagram showing urine glucose levels
in ob/ob mice treated pith and without ZAG.
[0110] FIG. 34 is a pictorial diagram of a Western blot showing ZAG
in ob/ob mice following oral administration. Treatment with rhZAG
administered orally causes an increase in endogenously expressed
murine ZAG in plasma.
[0111] FIG. 35 is a pictorial diagram of a Western blot showing ZAG
expression in WAT from ob/ob mice treated with and without human
ZAG (p.o.). Treatment with rhZAG administered orally causes an
increase in endogenously expressed murine ZAG in WAT.
[0112] FIG. 36 is a graphical diagram showing weight change in
ob/ob mice treated with and without ZAG (p.o.) in the absence or
presence of propranolol, a gereral .beta.-AR antagonist.
Propranolol was increased from 20 to 40 mg/kg on day 3, after which
change in weight loss altered from the negative slope of the
ZAG-treated animals to the positive slope of the untreated
animals.
[0113] FIG. 37 is a graphical diagram showing change in body
temperature in ob/ob mice treated with and without ZAG (p.o.) in
the absence or presence of propranolol. Propranolol was increased
from 20 to 40 mg/kg on day 3, after which body temperature of the
ZAG+Prop animals tracked that of untreated animals.
[0114] FIG. 38 is a pictorial diagram of a Western blot showing ZAG
using anti-mouse ZAG in mouse serum from mice treated with and
without ZAG in the absence or presence of propranonol. Endogenous
murine ZAG increases with treatment by orally administered rhZAG,
and such increase is blocked by propranolol.
[0115] FIG. 39 is a pictorial diagram of a Western blot showing ZAG
using anti-human ZAG against mouse serum from mice treated with and
without ZAG in the absence or presence of propranonol. Human ZAG is
not detected in mouse serum with or without propranolol.
[0116] FIG. 40 is a graphical diagram showing glucose levels during
a glucose tolerance test in ob/ob mice treated with and without ZAG
(p.o.) in the absence or presence of propranolol.
[0117] FIG. 41 is a pictorial diagram of a Western blot showing ZAG
in ob/ob mice following oral administration. Treatment with rhZAG
administered orally causes an increase in endogenously expressed
murine ZAG in plasma.
[0118] FIG. 42 is a pictorial diagram of a Western blot showing ZAG
expression in WAT from ob/ob mice treated with and without human
ZAG (p.o.).
[0119] FIG. 43 is a pictorial diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob
mice.
[0120] FIG. 44 is a pictorial diagram showing the effect of ZAG on
signaling pathwasy in skeletal muscle of ob/ob mice.
[0121] FIG. 45 is a pictorial diagram showing the effect of ZAG on
protein synthesis and degradation in skeletal muscle of ob/ob
mice.
[0122] FIG. 46 is a pictorial diagram of a 14C ZAG autoradiograph
showing stomach and plasma levels of ZAG from ob/ob mouse treated
p.o., the samples being taken 24-hours post treatment.
[0123] FIG. 47 is a graphical diagram showing weight change in
ob/ob mice treated with and without 50 ug ZAG (p.o./gavage).
[0124] FIG. 48 is a graphical diagram showing glucose urine levels
in ob/ob mice treated with and without 50 ug ZAG (p.o./gavage).
[0125] FIG. 49 is a pictorial diagram of a Western blot. Anti-ZAG
Diminishes Affects Caused by BRL37344 in vivo: Western blot of UCP3
in BAT of ob/ob mice treated with and without BRL in the absence or
presence of Anti-ZAG. Treatment of ob/ob mice with BRL37344 causes
an increase of UCP3 in BAT, an effect which is blocked by the
administration of anti-ZAG antibodies.
[0126] FIG. 50 is a pictorial diagram of a Western blot. ZAG
administered orally to ob/ob mice causes an up-regulation of
endogenous murine ZAG in plasma and in WAT. Western blot of mouse
ZAG in p.o. rhZAG-dosed samples of plasma(top) and WAT
(bottom).
[0127] FIG. 51 is a pictorial diagram of a Western blot.
Propranolol blocks the increase in murine serum ZAG due to
treatment with rhZAG p.o., but the administered human ZAG is not
found in plasma. Western blot of ZAG using Anti-mouse ZAG in mouse
serum from Mice treated with and without ZAG in the absence or
presence of propranonol (top). Human ZAG is not detected in mouse
serum. Western blot of ZAG using Anti-human ZAG in mouse serum from
Mice treated with and without ZAG in the absence or presence of
propranonol (bottom).
[0128] FIG. 52A is a graphical diagram showing the effect of ZAG
concentration on cyclic AMP production in CHO cells transfected
with .beta.1-AR (.box-solid.) .beta.2-AR (.quadrature.) and
.beta.3-AR (hashed). FIG. 52B is a graphical diagram showing effect
of isoprenaline (10 .mu.M) on cyclic AMP production in .beta.1-
(.box-solid.), .beta.2- (.quadrature.) and .beta.3-AR (hashed)
transfected CHO cells in the absence or presence of SR59230A (10
.mu.M). Differences from basal levels of cyclic AMP are indicated
as either a, p<0.05 or c, p<0.001.
[0129] FIG. 52C is a graphical diagram showing mRNA levels.
Expression of .beta.1-, .beta.2- and .beta.3-AR in CHO-K1 cells
transfected with the respective human genes as absolute numbers of
.beta.-AR mRNA molecules/.mu.g of total RNA, measured by RT-real
time PCR (closed boxes) in comparison with expression of GAPDH in
the same sample (open boxes). FIG. 52D is a graphical diagram
showing cyclic AMP production in CHO-KI cells transfected with
human .beta.1-, .beta.2- and .beta.3-AR in response to forskolin
(20 .mu.M). (closed boxes) in relation to basal levels (open
boxes).
[0130] FIGS. 52E, 52F and 52G is a graphical diagram showing
specific binding of ZAG to CHO-K1 cells transfected with human
.beta.1- (FIG. 52E), .beta.2- (FIG. 52F) and .beta.3-AR (FIG. 52G)
in the absence (.box-solid.) or presence (.diamond-solid.) of 100
uM non-labelled ZAG Binding of similar concentrations of ZAG frozen
and thawed(X) is also indicated.
[0131] FIG. 52H is a graphical diagram showing lipolytic activity
of ZAG (0.58 .mu.M), either fresh (open), or frozen and thawed once
(dashed), in comparison with isoprenaline (Iso; 10 .mu.M) (solid)
in murine epididymal adipocytes. Differences from control are shown
as c, p<0.001, while differences between fresh and frozen ZAG
and in the presence of SR59230A are shown as f, p<0.001.
[0132] FIG. 53A-53C are a graphical diagrams showing the effect of
propanolol on body weight (53A), body temperature (53B) and urinary
glucose excretion (53C) in ob/ob mice treated with ZAG. Animals
were divided into 4 groups (n=5 per group) to receive daily
administration of ZAG (50 mg, iv) (.box-solid.) ZAG+propanolol (40
mgkg.sup.-1, po) (.tangle-solidup.), while controls received either
PBS (.diamond-solid.) or PBS and propanolol (X). Differences from
PBS are shown as c, p<0.001, while differences from ZAG alone
are shown as f, p<0.001.
[0133] FIG. 53D is a pictorial diagram of liver histology after 60
days comparing control-treated and ZAG--treated example
sections.
[0134] FIG. 54A are a graphical diagrams showing total areas under
the glucose curves (AUC) in arbitrary units and plasma glucose
levels during a glucose tolerance test 3 days after initiation of
ZAG (.box-solid.) in comparison with PBS (.quadrature.) FIG. 54B is
a graphical diagram showing plasma insulin levels during the
glucose tolerance test described in (FIG. 54A).
[0135] FIG. 54C is a graphical diagram showing glucose uptake into
isolated gastrocnemius muscle of ob/ob mice in the absence or
presence or insulin (100 nM). Ob/ob mice were treated with ZAG with
or without propanolol for 7 days prior to excision of muscle.
[0136] FIG. 54D is a graphical diagram showing glucose uptake into
epididymal adipocytes of ob/ob mice in the absence or presence of
insulin (10 nM). Animals received the treatments indicated for 7
days prior to excision of WAT.
[0137] FIGS. 54E and 54F are graphical diagrams showing levels of
TG (FIG. 54E) and NEFA (FIG. 54F) in ob/ob mice treated with PBS or
ZAG, with or without propranolol for 7 days.
[0138] FIGS. 54G and 54H are pictorial diagrams of Western blots
showing Glut 4 expression in Gastronemius (54G) and WAT (54H) from
ob/ob mice in the presence of ZAG or Insulin or both. Differences
from controls are shown as b, p<0.01 or c, p<0.001, while
differences from ZAG alone are shown as d, p<0.05 or f,
p<0.001.FIG. 55A is a pictorial diagram of a Western blot.
[0139] FIGS. 55A, 55B and 55C are pictorial diagrams showing
expression of .beta.3-AR after treatment of ob/ob mice with ZAG (35
.mu.g; i.v. day.sup.-1) for 5 days. Western blots showing
expression of .beta.3-AR in gastrocnemius muscle (54A), BAT (54B)
and WAT (54C) of ob/ob mice treated with either PBS or ZAG. The
densitometric analysis is the average of three separate Western
blots. Differences from control as shown as c, p<0.001.
[0140] FIGS. 56A, 56B and 56C are pictorial diagrams of expression
of .beta.1- and .beta.2-AR in gastrocnemius muscle (56A), WAT (56B)
and heart (56C) after treatment of ob/ob mice with ZAG (35 .mu.g;
i.v., daily) for 5 days. Differences from PBS treated animals is
shown as a, p<0.05.FIGS. 57A, 57B, 57C and 57D are pictorial
diagrams of the effect of ZAG on expression of uncoupling proteins.
Western blots showing expression of UCP 1 showing expression of
UCP1 in BAT (57A) and WAT (57B), and expression of UCP3 in WAT
(57C) and AMPK in gastrocnemius muscle (57D) in ob/ob mice after
treatment with either PBS or ZAG (35 .mu.g; i.v., daily) for 5
days. The densitometric analysis is the average of three separate
blots. Differences from PBS treated animals are shown as c,
p<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0141] The present invention is based on the observation that
anti-human Zinc-.alpha..sub.2-glycoprotein (ZAG) antibodies reduce
weight loss in models of cachexia. As such, the invention provides
methods for preventing weight loss in cachexia situations in a
subject. Also provided are combinatorial treatments to bring about
a reduction in weight loss in a subject with cachexia.
[0142] Provided herein are formulations and methods for treating
mammals and/or supplementing a human or animal diet. The methods
can include ingesting one or more of the described formulations for
certain time periods and/or in a certain order. Kits comprising one
or more of the formulations are also provided. As such, the present
invention is based on the observation that recombinant
zinc-.alpha.2-glycoprotein (ZAG) produces a decrease in body weight
and increase in insulin responsiveness in subjects with no effect
on food intake.
[0143] Before the present compositions and methods are described,
it is to be understood that this invention is not limited to
particular compositions, methods, and experimental conditions
described, as such compositions, methods, and conditions may vary.
It is also to be understood that the terminology used herein is for
purposes of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only in the appended claims.
[0144] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein which will become apparent to
those persons skilled in the art upon reading this disclosure and
so forth.
[0145] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described.
[0146] The complete amino acid sequence of ZAG has been reported in
a paper entitled "Complete amino acid sequence of human plasma
Zinc-.alpha..sub.2-glycoprotein and its homology to
histocompatibility antigens" by T. Araki et al. (1988) Proc. Natl.
Acad. Sci. USA., 85, 679-683, wherein the glycoprotein was shown as
consisting of a single polypeptide chain of 276 amino acid residues
having three distinct domain structures (A, B and C) and including
two disulfide bonds together with N-linked glycans at three
glycosylation sites. This amino acid sequence of the polypeptide
component is set out in FIG. 10 of the accompanying drawings.
Although some subsequent publications have indicated that the
composition of human ZAG can vary somewhat when isolated from
different body fluids or tissues, all preparations of this material
have substantially the same immunological characteristics. As
reported by H. Ueyama, et al. (1991) "Cloning and nucleotide
sequence of a human Zinc-.alpha..sub.2-glycoprotein cDNA and
chromosomal assignment of its gene", Biochem. Biophys. Res. Commun.
177, 696-703, cDNA of ZAG has been isolated from human liver and
prostate gland libraries, and also the gene has been isolated, as
reported by Ueyama et al., (1993) "Molecular cloning and
chromosomal assignment of the gene for human
Zinc-.alpha..sub.2-glycoprotein", Biochemistry 32, 12968-12976. H.
Ueyama et al. have also described, in J. Biochem. (1994) 116,
677-681, studies on ZAG cDNAs from rat and mouse liver which,
together with the glycoprotein expressed by the corresponding
mRNAs, have been sequenced and compared with the human material.
Although detail differences were found as would be expected from
different species, a high degree of amino acid sequence homology
was found with over 50% identity with the human counterpart (over
70% identity within domain B of the glycoprotein). Again, common
immunological properties between the human, rat and mouse ZAG have
been observed.
[0147] The purified ZAG discussed above was prepared from fresh
human plasma substantially according to the method described by
Ohkubo et al. (Ohkubo et al. (1988) "Purification and
characterisation of human plasma Zn-.alpha..sub.2-glycoprotein"
Prep. Biochem., 18, 413-430). It will be appreciated that in some
cases fragments of the isolated lipid mobilizing factor, of ZAG, or
of anti-ZAG antibodies may be produced without loss of activity,
and various additions, deletions or substitutions may be made which
also will not substantially affect this activity. As such, the
methods of the invention also include use of functional fragments
of anti-ZAG antibodies. The antibody or fragment thereof used in
these therapeutic applications may further be produced by
recombinant DNA techniques such as are well known in the art based
possibly on the known cDNA sequence for
Zn-.alpha..sub.2-glycoprotein which has been published for example
in H. Ueyama et al. (1994) "Structure and Expression of Rat and
Mouse mRNAs for Zn-.alpha..sub.2-glycoprotein" J. Biochem., 116,
677-681. In addition, the antibody or fragment thereof used in
these therapeutic applications may further include post-expression
modifications of the polypeptide, for example, glycosylations,
acetylations, phosphorylations and the like, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring.
[0148] As used herein, ZAG polypeptides or proteins include
variants of wild type proteins which retain their biological
function. As such, one or more of the residues of a ZAG protein can
be altered to yield a variant or truncated protein, so long as the
variant retains it native biological activity. Conservative amino
acid substitutions include, for example, aspartic-glutamic as
acidic amino acids; lysine/arginine/histidine as basic amino acids;
leucine/isoleucine, methionine/valine, alanine/valine as
hydrophobic amino acids; serine/glycine/alanine/threonine as
hydrophilic amino acids. Conservative amino acid substitution also
include groupings based on side chains. For example, a group of
amino acids having aliphatic side chains is glycine, alanine,
valine, leucine, and isoleucine; a group of amino acids having
aliphatic-hydroxyl side chains is serine and threonine; a group of
amino acids having amide-containing side chains is asparagine and
glutamine; a group of amino acids having aromatic side chains is
phenylalanine, tyrosine, and tryptophan; a group of amino acids
having basic side chains is lysine, arginine, and histidine; and a
group of amino acids having sulfur-containing side chains is
cysteine and methionine. For example, it is reasonable to expect
that replacement of a leucine with an isoleucine or valine, an
aspartate with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the properties of the resulting
variant polypeptide.
[0149] Amino acid substitutions falling within the scope of the
invention, are, in general, accomplished by selecting substitutions
that do not differ significantly in their effect on maintaining (a)
the structure of the peptide backbone in the area of the
substitution, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. However, the
invention also envisions variants with non-conservative
substitutions.
[0150] The term "peptide", "polypeptide" and protein" are used
interchangeably herein unless otherwise distinguished to refer to
polymers of amino acids of any length. These terms also include
proteins that are post-translationally modified through reactions
that include glycosylation, acetylation and phosphorylation.
[0151] As discussed above, the present invention includes use of a
function fragment of a
[0152] ZAG polypeptide or protein. A functional fragment, is
characterized, in part, by having or affecting an activity
associated with weight loss, lowering blood glucose level,
increasing body termperature, improving glucose tissue uptake,
increasing expression of Bet3 receptors, increasing expression of
ZAG; increasing expression of Glut 4, and/or increasing expression
of UCP1 and UCP3. Thus, the term "functional fragment," when used
herein refers to a polypeptide that retains one or more biological
functions of ZAG. Methods for identifying such a functional
fragment of a ZAG polypeptide, are generally known in the art.
[0153] As used herein, the term "antibody" includes reference to an
immunoglobulin molecule immunologically reactive with a particular
antigen, and includes both polyclonal and monoclonal antibodies.
The term also includes genetically engineered forms such as
chimeric antibodies (e.g., humanized murine antibodies) and
heteroconjugate antibodies (e.g., bispecific antibodies). The term
"antibody" also includes antigen binding forms of antibodies,
including fragments with antigen-binding capability (e.g., Fab',
F(ab').sub.2, Fab, Fv and rIgG. See also, Pierce Catalog and
Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.). See
also, e.g., Kuby, J., Immunology, 3.sup.rd Ed., W.H. Freeman &
Co., New York (1998). The term also refers to recombinant single
chain Fv fragments (scFv). The term antibody also includes bivalent
or bispecific molecules, diabodies, triabodies, and tetrabodies.
Bivalent and bispecific molecules are described in, e.g., Kostelny
et al. (1992) J Immunol 148:1547, Pack and Pluckthun (1992)
Biochemistry 31:1579, Hollinger et al., 1993, supra, Gruber et al.
(1994) J Immunol: 5368, Zhu et al. (1997) Protein Sci 6:781, Hu et
al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res.
53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.
[0154] An antibody immunologically reactive with a particular
antigen can be generated by recombinant methods such as selection
of libraries of recombinant antibodies in phage or similar vectors,
see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al.,
Nature 341:544-546 (1989); and Vaughan et al., Nature Biotech.
14:309-314 (1996), or by immunizing an animal with the antigen or
with DNA encoding the antigen.
[0155] Typically, an immunoglobulin has a heavy and light chain.
Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). Light
and heavy chain variable regions contain four "framework" regions
interrupted by three hypervariable regions, also called
"complementarity-determining regions" or "CDRs". The extent of the
framework regions and CDRs have been defined. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in three
dimensional space.
[0156] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found.
[0157] References to "V.sub.H" or a "V.sub.H" refer to the variable
region of an immunoglobulin heavy chain of an antibody, including
the heavy chain of an Fv, scFv, or Fab. References to "V.sub.L" or
a "V.sub.L" refer to the variable region of an immunoglobulin light
chain, including the light chain of an Fv, scFv, dsFv or Fab.
[0158] An antibody having a constant region substantially identical
to a naturally occurring class IgG antibody constant region refers
to an antibody in which any constant region present is
substantially identical, i.e., at least about 85-90%, and
preferably at least 95% identical, to the amino acid sequence of
the naturally occurring class IgG antibody's constant region.
[0159] As used herein, the term "monoclonal antibody" is not
limited to antibodies produced through hybridoma technology. The
term "monoclonal antibody" refers to an antibody that is derived
from a single clone, including any eukaryotic, prokaryotic, or
phage clone, and not the method by which it is produced. Monoclonal
antibodies useful with the present invention may be prepared using
a wide variety of techniques known in the art including the use of
hybridoma, recombinant, and phage display technologies, or a
combination thereof. For example, monoclonal antibodies can be
produced using hybridoma techniques including those known in the
art and taught, for example, in Harlow and Lane, "Antibodies: A
Laboratory Manual," Cold Spring Harbor Laboratory Press, New York
(1988); Hammerling et al., in: "Monoclonal Antibodies and T-Cell
Hybridomas," Elsevier, N.Y. (1981), pp. 563-681 (both of which are
incorporated herein by reference in their entireties).
[0160] Thus, in some embodiments, the antibodies of the invention
may be chimeric, primatized, humanized, or human antibodies.
[0161] A "chimeric antibody" is an immunoglobulin molecule in which
(a) the constant region, or a portion thereof, is altered, replaced
or exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202-1207 (1985);
Oi et al., BioTechniques 4:214-221 (1986); Gillies et al., J.
Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;
4,816,567; and 4,816,397, which are incorporated herein by
reference in their entireties.
[0162] The term "humanized antibody" or "humanized immunoglobulin"
refers to an immunoglobulin comprising a human framework, at least
one and preferably all complementarity determining regions (CDRs)
from a non-human antibody, and in which any constant region present
is substantially identical to a human immunoglobulin constant
region, i.e., at least about 85-90%, and preferably at least 95%
identical. Hence, all parts of a humanized immunoglobulin, except
possibly the CDRs, are substantially identical to corresponding
parts of one or more native human immunoglobulin sequences.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. Often, framework
residues in the human framework regions will be substituted with
the corresponding residue from the CDR donor antibody to alter,
preferably improve, antigen binding. These framework substitutions
are identified by methods well known in the art, e.g., by modeling
of the interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. See, e.g., U.S. Pat. Nos. 5,530,101; 5,585,089;
5,693,761; 5,693,762; 6,180,370 (each of which is incorporated by
reference in its entirety). Antibodies can be humanized using a
variety of techniques known in the art including, for example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat.
Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing
(EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28:489-498 (1991);
Studnicka et al., Prot. Eng. 7:805-814 (1994); Roguska et al.,
Proc. Natl. Acad. Sci. 91:969-973 (1994), and chain shuffling (U.S.
Pat. No. 5,565,332), all of which are hereby incorporated by
reference in their entireties.
[0163] In certain embodiments, completely "human" antibodies may be
desirable for therapeutic treatment of human patients. Human
antibodies can be made by a variety of methods known in the art
including phage display methods described above using antibody
libraries derived from human immunoglobulin sequences. See U.S.
Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO
98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO
96/33735; and WO 91/10741, each of which is incorporated herein by
reference in its entirety. Human antibodies can also be produced
using transgenic mice which are incapable of expressing functional
endogenous immunoglobulins, but which can express human
immunoglobulin genes. For an overview of this technology for
producing human antibodies, see Lonberg and Huszar, Int. Rev.
Immunol. 13:65-93 (1995). For a detailed discussion of this
technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; and 5,939,598, which are incorporated by
reference herein in their entireties. In addition, companies such
as Abgenix, Inc. (Fremont, Calif.) and Medarex (Princeton, N.J.)
can be engaged to provide human antibodies directed against a
selected antigen using technology similar to that described
above.
[0164] Completely human antibodies that recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope (Jespers
et al., Biotechnology 12:899-903 (1988).
[0165] The term "primatized antibody" refers to an antibody
comprising monkey variable regions and human constant regions.
Methods for producing primatized antibodies are known in the art.
See e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780, which
are incorporated herein by reference in their entireties.
[0166] As used herein, the terms "epitope" or "antigenic
determinant" refer to a site on an antigen to which an antibody
binds. Epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5 or 8-10 amino acids in a unique spatial
conformation. Methods of determining spatial conformation of
epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed (1996).
[0167] Antibodies of "IgG class" refers to antibodies of IgG1,
IgG2, IgG3, and IgG4. The numbering of the amino acid residues in
the heavy and light chains is that of the EU index (Kabat, et al.,
"Sequences of Proteins of Immunological Interest", 5th ed.,
National Institutes of Health, Bethesda, Md. (1991); the EU
numbering scheme is used herein).
[0168] Methods of preparing polyclonal antibodies are known to the
skilled artisan. Polyclonal antibodies can be raised in a mammal,
e.g., by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include a
protein, such as the polypeptide shown in SEQ ID NO: 1, encoded by
a nucleic acid or a functional fragment thereof. It may be useful
to conjugate the immunizing agent to a protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0169] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler & Milstein, Nature 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The immunizing agent
will typically include a polypeptide as shown in SEQ ID NO: 1 or a
functional fragment thereof.
[0170] Human antibodies can be produced using various techniques
known in the art, including phage display libraries (Hoogenboom
& Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol.
Biol. 222:581 (1991)). The techniques of Cole et al. and Boerner et
al. are also available for the preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy,
p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)).
Similarly, human antibodies can be made by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, e.g., in U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
and in the following scientific publications: Marks et al.,
Bio/Technology 10:779-783 (1992); Lonberg et al., Nature
368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature
Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev.
Immunol. 13:65-93 (1995).
[0171] In some embodiments, the antibody is a single chain Fv
(scFv). The V.sub.H and the V.sub.L regions of a scFv antibody
comprise a single chain which is folded to create an antigen
binding site similar to that found in two chain antibodies. Once
folded, noncovalent interactions stabilize the single chain
antibody. While the V.sub.H and V.sub.L regions of some antibody
embodiments can be directly joined together, one of skill will
appreciate that the regions may be separated by a peptide linker
consisting of one or more amino acids. Peptide linkers and their
use are well-known in the art. See, e.g., Huston et al., Proc.
Nat'l Acad. Sci. USA 8:5879 (1988); Bird et al., Science 242:4236
(1988); Glockshuber et al., Biochemistry 29:1362 (1990); U.S. Pat.
No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et al.,
Biotechniques 14:256-265 (1993). Generally the peptide linker will
have no specific biological activity other than to join the regions
or to preserve some minimum distance or other spatial relationship
between the V.sub.H and V.sub.L. However, the constituent amino
acids of the peptide linker may be selected to influence some
property of the molecule such as the folding, net charge, or
hydrophobicity. Single chain Fv (scFv) antibodies optionally
include a peptide linker of no more than 50 amino acids, generally
no more than 40 amino acids, preferably no more than 30 amino
acids, and more preferably no more than 20 amino acids in length.
In some embodiments, the peptide linker is a concatamer of the
sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO:7), preferably 2, 3, 4, 5,
or 6 such sequences. However, it is to be appreciated that some
amino acid substitutions within the linker can be made. For
example, a valine can be substituted for a glycine.
[0172] Methods of making scFv antibodies have been described. See,
Huse et al., supra; Ward et al. supra; and Vaughan et al., supra.
In brief, mRNA from B-cells from an immunized animal is isolated
and cDNA is prepared. The cDNA is amplified using primers specific
for the variable regions of heavy and light chains of
immunoglobulins. The PCR products are purified and the nucleic acid
sequences are joined. If a linker peptide is desired, nucleic acid
sequences that encode the peptide are inserted between the heavy
and light chain nucleic acid sequences. The nucleic acid which
encodes the scFv is inserted into a vector and expressed in the
appropriate host cell. The scFv that specifically bind to the
desired antigen are typically found by panning of a phage display
library. Panning can be performed by any of several methods.
Panning can conveniently be performed using cells expressing the
desired antigen on their surface or using a solid surface coated
with the desired antigen. Conveniently, the surface can be a
magnetic bead. The unbound phage are washed off the solid surface
and the bound phage are eluted.
[0173] ZAG and/or fragments thereof has been previously shown to
bring about a weight reduction or reduction in obesity in mammals,
as disclosed in U.S. Pat. Nos. 6,890,899 and 7,550,429, and in U.S.
Pub. No. 2010/0173829, the entire contents of each of which is
incorporated herein by reference. In one embodiment, the present
invention demonstrates that anti-ZAG antibodies and/or functional
fragments thereof reduces weight loss in models of cachexia. It is
therefore contemplated that the methods of the instant invention
provide a detectable effect on symptoms associated with cachexia
and/or diseases associated with muscle wasting disease.
[0174] Accordingly, in one aspect, the invention provides a method
of ameliorating the symptoms of cachexia in a subject. The method
includes administering to the subject in need of such treatment a
therapeutically effective dosage of an inhibitor of the biological
activity of a polypeptide having the sequence as shown in SEQ ID
NO: 1. In one embodiment, the treatment regimen may be for months
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months), or years.
In another embodiment, the polypeptide is administered for a period
of up to 21 days or longer. In another embodiment, the amelioration
of symptoms is detectable within days (e.g., 1, 2, 3, 4, 5, 6, or 7
days), weeks (e.g., 1, 2, 3, or 4 weeks), or months (e.g., 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, or 12 months) of initiating treatment. In
another embodiment, the treatment regimen is about 10 days wherein
there is amelioration of symptoms associated with cachexia
following treatment. In another embodiment, the treatment regimen
is about 21 days wherein there is amelioration of symptoms
associated with cachexia following treatment.
[0175] In addition, it has been observed that a lipid mobilizing
agent having similar characteristics of ZAG and/or fragments
thereof has also been used to bring about a weight reduction or
reduction in obesity in mammals, as disclosed in U.S. Published
App. No. 2006/0160723, incorporated by herein by reference in its
entirety. Finally, it has been shown that ZAG and/or functional
fragments thereof increases the insulin responsiveness of
adipocytes and skeletal muscle, and produces an increase in muscle
mass through an increase in protein synthesis coupled with a
decrease in protein degradation regardless of whether a weight
reduction or reduction in obesity is observed during treatment (see
U.S. Ser. No. 12/614,289, incorporated herein by reference).
[0176] Additionally, .beta.3 agonists are reportedly effective
insulin sensitizing agents in rodents and their potential to reduce
blood glucose levels in humans has been a subject of investigation.
Activation of .beta.3 agonists adrenoceptors stimulates fat
oxidation, thereby lowering intracellular concentrations of
metabolites including fatty acyl CoA and diacylglycerol, which
modulate insulin signaling. Furthermore, it is contemplated herein
that certain .beta.3 receptor agonists may not have found success
in clinical trials given that one category of .beta.3 receptors
available to these agents is located in the digestive system and
particularly in the mouth, pharynx, esophagus and stomach,
resulting in minimal, if any, exposure of the agonist to most of
these receptors. This theory is supported by the observation that
several of the .beta.3 agonist therepeutic agents were found to be
efficacious but had limited bioavailability in the plasma
space.
[0177] A number of formulations are provided herein. A formulation
can be in any form, e.g., liquid, solid, gel, emulsion, powder,
tablet, capsule, or gel cap (e.g., soft or hard gel cap). A
formulation typically will include one or more compositions that
have been purified, isolated, or extracted (e.g., from plants) or
synthesized, which are combined to provide a benefit (e.g., a
health benefit in addition to a nutritional benefit) when used to
supplement food in a diet.
[0178] In certain embodiments, recommended amounts per day or per
serving of a formulation or of ingredients provided in a
formulation may be set forth herein. In certain cases, as will be
recognized by one having ordinary skill in the art, one could vary
the form of the formulation, e.g., by substituting a powder for a
capsule, a tablet for a capsule, a gel-cap for a tablet, a gel-cap
for a capsule, a powder for a gel-cap, or any such combination, in
order to provide such recommended amounts per day or per serving of
a formulation.
[0179] Any of the formulations can be prepared using well known
methods by those having ordinary skill in the art, e.g., by mixing
the recited ingredients in the proper amounts. Ingredients for
inclusion in a formulation are generally commercially
available.
[0180] Accordingly, in one aspect, the invention provides a
formulation that includes ZAG or a functional fragment thereof For
example, the ZAG may be mammalian ZAG, such human ZAG as shown in
SEQ ID NO: 1, or fragments thereof. However, it should be
understood that the ZAG may be derived from any source provided
that the ZAG retains the activity of wild-type ZAG. In one
embodiment, the further includes a pharmaceutically acceptable
carrier, which constitutes one or more accessory ingredients.
[0181] The term "subject" as used herein refers to any individual
or patient to which the subject methods are performed. Generally
the subject is human, although as will be appreciated by those in
the art, the subject may be an animal. Thus other animals,
including mammals such as rodents (including mice, rats, hamsters
and guinea pigs), cats, dogs, rabbits, farm animals including cows,
horses, goats, sheep, pigs, etc., and primates (including monkeys,
chimpanzees, orangutans and gorillas) are included within the
definition of subject.
[0182] Cachexia is commonly associated with a number of disease
states, including acute inflammatory processes associated with
critical illness and chronic inflammatory diseases, cancer, AIDS,
sepsis, COPD, renal failure, arthritis, congestive heart failure,
muscular dystrophy, diabetes, sarcopenia of aging, severe trauma
(e.g., orthopaedic immobilization of a limb), metabolic acidosis,
denervation atrophy, and weightlessness.
[0183] The term "therapeutically effective amount" or "effective
amount" means the amount of a compound or pharmaceutical
composition that will elicit the biological or medical response of
a tissue, system, animal or human that is being sought by the
researcher, veterinarian, medical doctor or other clinician.
[0184] In some embodiments, the formulations of the invention are
intended to be orally administered daily. However, other forms of
administration are equally envisioned. As used herein, the terms
"administration" or "administering" are defined to include an act
of providing a compound or pharmaceutical composition of the
invention to a subject in need of treatment. The phrases
"parenteral administration" and "administered parenterally" as used
herein means modes of administration other than enteral and topical
administration, usually orally or by injection, and includes,
without limitation, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticulare, subcapsular, subarachnoid,
intraspinal and intrasternal injection and infusion. The phrases
"systemic administration," "administered systemically," "peripheral
administration" and "administered peripherally" as used herein mean
the administration of a compound, drug or other material other than
directly into the central nervous system, such that it enters the
subject's system and, thus, is subject to metabolism and other like
processes, for example, subcutaneous administration. As such, in
one embodiment, the anti-ZAG antibodies, fragments thereof, and/or
formulations of the invention are administered to a subject via
inhalation, intranasally, buccally, sublingually, intravenously,
intramuscularly, and/or orally. In another embodiment, the
antibodies or compositions thereof are formulated in rapid-melting
compositions, extended release compositions, and the like.
[0185] As used herein, the term "ameliorating" or "treating" means
that the clinical signs and/or the symptoms associated with
cachexia are lessened as a result of the actions performed. The
signs or symptoms to be monitored will be characteristic of
cachexia and will be well known to the skilled clinician, as will
the methods for monitoring the signs and conditions. In addition to
adipose/muscle mass loss, exemplary symptoms associated with
cachexia include, but are not limited to, fever, headache, chronic
pain, body malaise, fainting, seizure associated with the fever,
shock, palpitations, heart murmur, gangrene, epistaxis, hemoptysis,
cough, difficulty in breathing, wheezing, hyperventilation and
hypoventilation, mouth breathing, hiccup and chest pain, abdominal
pain, nausea or vomiting, heartburn, halitosis, and flatulence, as
compared to a normal subject or a subject that does not have
cachexia. As such, an amelioration of the symptoms associate with
cachexia includes but is not limited to, decreasing or reducing
weight loss in the subject and reversing one or more of the
above-listed symptoms.
[0186] As used herein, the terms "reduce" and "inhibit" are used
together because it is recognized that, in some cases, a decrease
can be reduced below the level of detection of a particular assay.
As such, it may not always be clear whether the expression level or
activity is "reduced" below a level of detection of an assay, or is
completely "inhibited." Nevertheless, it will be clearly
determinable, following a treatment according to the present
methods, that amount of weight loss in a subject is at least
reduced from the level prior to treatment.
[0187] ZAG has been attributed a number of biological roles, but
its role as an adipokine regulating lipid mobilization and
utilization is most important in regulating body composition.
Previous studies suggested that the increase in protein synthesis
was due to an increase in cyclic AMP through interaction with the
.beta.-adrenoreceptor, while the decrease in protein degradation
was due to reduced activity of the ubiquitin-proteasome proteolytic
pathway. Studies in db/db mice show that insulin resistance causes
muscle wasting through an increased activity of the
ubiquitin-proteasome pathway. An increased phosphorylation of both
PKR and eIF2.alpha. will reduce protein synthesis by blocking
translation initiation, while activation of PKR will increase
protein degradation through activation of nuclear factor-.kappa.B
(NF-.kappa.B), increasing expression of proteasome subunits. In
vitro studies using myotubes in the presence of high extracellular
glucose showed that activation of PKR led to activation of p38MAPK
and formation of reactive oxygen species (ROS). p38MAPK can
phosphorylate and activate cPLA.sub.2 at Ser-505 causing release of
arachidonic acid, a source of ROS. Hyperactivation of p38MAPK in
skeletal muscle has been observed in models of diet-induced
obesity. In addition caspase-3 activity has been shown to be
increased in skeletal muscle of diabetic animals, which may be part
of the signaling cascade, since it can cleave PKR leading to
activation. Without being bound to theory, the ability of ZAG to
attenuate these signaling pathways provides an explanation
regarding its ability to increase muscle mass. As such, an anti-ZAG
antibody is demonstrated to decrease loss of muscle mass in
cachexia situations.
[0188] Accordingly, in another aspect, the invention provides a
method of supplementing a human or animal diet. The method includes
administering to the subject a ZAG polypeptide or a fragment
thereof. In another embodiment, the method includes ingesting a
formulation that includes a ZAG polypeptide, such as the human ZAG
polypeptide as set forth in SEQ ID NO: 1. A formulation can be
ingested alone or in combination with any other known formulation,
in any order and for varying relative lengths of time. In certain
embodiments, certain formulations are used prior to other
formulations, while other formulations are ingested
concurrently.
[0189] Thus, ZAG is identified as a lipid mobilizing factor capable
of inducing lipolysis in white adipocytes of the mouse in a
GTP-dependent process, similar to that induced by lipolytic
hormones. The data presented herein supports these findings by
showing that ZAG has a similar lipolytic effect in rat adipocytes,
and, moreover, produces a decrease in body weight and carcass fat
in mature male rats, despite the fact that the sequence homology
between rat and human ZAG is only 59.4%.
[0190] ZAG also counters some of the metabolic features of the
diabetic state including a reduction of plasma insulin levels and
improved response in the glucose tolerance test. Thus, in another
aspect, the invention provides a method of decreasing plasma
insulin levels in a subject. The method includes administering to
the subject a therapeutically effective dosage of a polypeptide
having the sequence as shown in SEQ ID NO: 1 or a fragment thereof
In one embodiment, the decrease in plasma insulin occurs within 3
days of initiating treatment. In another embodiment, the treatment
regimen is administered for 10 days or longer. In another
embodiment, the treatment regimen is administered for 21 days or
longer.
[0191] In addition, ZAG has been shown to increase glucose
oxidation and increase the tissue glucose metabolic rate in adult
male mice. This increased utilization of glucose would explain the
fall in both blood glucose and insulin levels in ob/ob mice
administered ZAG. Triglyceride utilization was also increased in
mice administered ZAG, which would explain the fall in plasma
non-esterified fatty acids (NEFA) and triglycerides (TG) despite
the increase in plasma glycerol, indicative of increased lipolysis.
The increased utilization of lipid would be anticipated from the
increased expression of UCP1 and UCP3 in BAT and UCP3 in skeletal
muscle, resulting in an increase in body temperature. Thus, ZAG is
identified as a lipid mobilizing factor capable of inducing
lipolysis in white adipocytes of the mouse in a GTP-dependent
process, similar to that induced by lipolytic hormones. As such, in
one embodiment, amelioration of the symptoms associated with
hyperglycemia also includes an increase in body temperature of
about 0.5.degree. C. to about 1.degree. C. during treatment. In one
embodiment, the increase in body temperature occurs within 4 days
of initiating treatment. In another embodiment, amelioration of the
symptoms associated with hyperglycemia also includes an increase in
pancreatic insulin as compared to pancreatic insulin levels prior
to treatment, since less insulin is needed to control blood glucose
as a result of the presence of ZAG.
[0192] ZAG has also been shown to counter some of the metabolic
features of the diabetic state including a reduction of plasma
insulin levels and improved response in the glucose tolerance test.
In addition ZAG increases the responsiveness of epididymal
adipocytes to the lipolytic effect of a .beta.3-adrenergic
stimulant. ZAG also increases the expression of HSL and ATGL in
epididymal adipose tissue which have been found to be reduced in
the obese insulin-resistant state. Factors regulating the
expression of HSL and ATGL are not known. However, the specific ERK
inhibitor, PD98059 downregulated HSL expression in response to ZAG,
suggesting a role for MAPK in this process. Mice lacking MAPK
phosphatase-1 have increase activities of ERK and p38MAPK in WAT,
and are resistant to diet-induced obesity due to enhanced energy
expenditure. Previous studies have suggested a role for MAPK in the
ZAG-induced expression of UCP3 in skeletal muscle. ERK activation
may regulate lipolysis in adipocytes by phosphorylation of serine
residues of HSL, such as Ser-600, one of the sites phosphorylated
by protein kinase A.
[0193] The results presented herein show that ZAG administration to
the rat also increases the expression of ATGL and HSL in the rat.
ATGL may be important in excess fat storage in obesity, since ATGL
knockout mice have large fat deposits and reduced NEFA release from
WAT in response to isoproterenol, although they did display normal
insulin sensitivity. In contrast HSL null mice, when fed a normal
diet, had body weights similar to wild-type animals. However,
expression of both ATGL and HSL are reduced in human WAT in the
obese insulin-resistant state compared with the insulin sensitive
state, and weight reduction also decreased mRNA and protein
levels.
[0194] Stimulation of lipolysis alone would not deplete body fat
stores, since without an energy sink the liberated NEFA would be
resynthesised back into triglycerides in adipocytes. To reduce body
fat, ZAG not only increases lipolysis, as shown by an increase in
plasma glycerol, but also increases lipid utilization, as shown by
the decrease in plasma levels of triglycerides and NEFA. This
energy is channeled into heat, as evidenced by the 0.4.degree. C.
rise in body temperature in rats treated with ZAG. The increased
energy utilization most likely arises from the increased expression
of UCP1, which has been shown in both BAT and WAT after
administration of ZAG. An increased expression of UCP1 would be
expected to decrease plasma levels of NEFA, since they are the
primary substrates for thermogenesis in BAT. BAT also has a high
capacity for glucose utilization, which could partially explain the
decrease in blood glucose. In addition there was increased
expression of GLUT4 in skeletal muscle and WAT, which helps mediate
the increase in glucose uptake in the presence of insulin. In mice
treated with ZAG there was an increased glucose
utilization/oxidation by brain, heart, BAT and gastrocnemius
muscle, and increased production of .sup.14CO.sub.2 from
D-[U-.sup.14C] glucose, as well as [.sup.14C carboxy] triolein
(FIG. 24). There was also a three-fold increase in oxygen uptake by
BAT of ob/ob mice after ZAG administration.
[0195] While ZAG increased expression of HSL in epididymal
adipocytes there was no increase in either subcutaneous or visceral
adipocytes. A similar situation was observed with expression of
adipose triglyceride lipase (ATGL). Expression of HSL and ATGL
correlated with expression of the active (phospho) form of ERK.
Expression of HSL and ATGL in epididymal adipocytes correlated with
an increased lipolytic response to the .beta.3 agonist, BRL37344.
This result suggests that ZAG may act synergistically with .beta.3
agonists, and suggests that anti-ZAG antibodies may act
synergistically with .beta.3 antagonists.
[0196] As used herein, the term "agonist" refers to an agent or
analog that is capable of inducing a full or partial
pharmacological response. For example, an agonist may bind
productively to a receptor and mimic the physiological reaction
thereto. As used herein, the term "antagonist" refers to an agent
or analog that does not provoke a biological response itself upon
binding to a receptor, but blocks or dampens agonist-mediated
responses. The methods and formulations of the invention may
include administering anti-ZAG antibodies, or a functional fragment
thereof, in combination with a .beta.3 antagonist, such as but not
limited to BRL37344, or a .beta.3 agonist.
[0197] Examples of .beta.3 agonists that may be used in the present
invention include, but are not limited to: epinephrine
(adrenaline), norepinephrine (noradrenaline), isoprotenerol,
isoprenaline, propranolol, alprenolol, arotinolol, bucindolol,
carazolol, carteolol, clenbuterol, denopamine, fenoterol, nadolol,
octopamine, oxyprenolol, pindolol, [(cyano)pindolol], salbuterol,
salmeterol, teratolol, tecradine, trimetoquinolol,
3'-iodotrimetoquinolol, 3',5'-iodotrimetoquinolol, Amibegron,
Solabegron, Nebivolol, AD-9677, AJ-9677, AZ-002, CGP-12177,
CL-316243, CL-317413, BRL-37344, BRL-35135, BRL-26830, BRL-28410,
BRL-33725, BRL-37344, BRL-35113, BMS-194449, BMS-196085,
BMS-201620, BMS-210285, BMS-187257, BMS-187413, the CONH2
substitution of SO3H of BMS-187413, the racemates of BMS-181413,
CGP-20712A, CGP-12177, CP-114271, CP-331679, CP-331684, CP-209129,
FR-165914, FR-149175, ICI-118551, ICI-201651, ICI-198157,
ICI-D7114, LY-377604, LY-368842, KTO-7924, LY-362884, LY-750355,
LY-749372, LY-79771, LY-104119, L-771047, L-755507, L-749372,
L-750355, L-760087, L-766892, L-746646, L-757793, L-770644,
L-760081, L-796568, L-748328, L-748337, Ro-16-8714, Ro-40-2148,
(-)-RO-363, SB-215691, SB-220648, SB-226552, SB-229432, SB-251023,
SB-236923, SB-246982, SR-58894A, SR-58611, SR-58878, SR-59062,
SM-11044, SM-350300, ZD-7114, ZD-2079, ZD-9969, ZM-215001, and
ZM-215967.
[0198] Examples of .beta.-AR antagonists that may be used in the
present invention include, but are not limited to: propranolol,
(-)-propranolol, (+)-propranolol, practolol, (-)-practolol,
(+)-practolol, CGP-20712A, ICI-118551, (-)-buprranolol, acebutolol,
atenolol, betaxolol, bisoprolol, esmolol, nebivolol, metoprolol,
acebutolol, carteolol, penbutolol, pindolol, carvedilol, labetalol,
levobunolol, metipranolol, nadolol, sotalol, and timolol.
[0199] Induction of lipolysis in rat adipocytes by ZAG is suggested
to be mediated through a .beta.3-AR, and the effect of ZAG on
adipose tissue and lean body mass may also be due to its ability to
stimulate the .beta.3-AR. Induction of UCP1 expression by ZAG has
been shown to be mediated through interaction with a .beta.3-AR.
The increased expression of UCP1 in WAT may also be a .beta.3-AR
effect through remodeling of brown adipocyte precursors, as occurs
with the .beta.3-AR agonist CL3 16,243. Using knock-out mice the
antiobesity effect of .beta.3-AR stimulation has been mainly
attributed to UCP1 in BAT, and less to UCP2 and UCP3 through the
UCP 1-dependent degradation of NEFA released from WAT. Glucose
uptake into peripheral tissues of animals is stimulated by
cold-exposure, an effect also mediated through the .beta.3-AR.
However, targeting the .beta.3-AR has been more difficult in humans
than in rodents, since (.beta.3-AR play a less prominent role than
.beta.1 and .beta.2-AR subtypes in the control of lipolysis and
nutritive blood flow in human subcutaneous abdominal adipose
tissue. However, despite this the .beta.3-AR agonist CL3 16,243 has
been shown to increase fat oxidation in healthy young male
volunteers. This may be due to the ability of .beta.-adrenergic
agonists to increase the number of .beta.3-AR in plasma membranes
from BAT.
[0200] Accordingly, in another aspect, the invention provides a
method of treating a subject to bring about a reduction in weight
loss due to cachexia or a disease associated with muscle wasting.
The method includes administering to the subject in need of such
treatment a therapeutically effective dosage of a .beta.3
antagonist in combination with an antibody, or a fragment thereof,
that binds to the polypeptide having the sequence as shown in SEQ
ID NO: 1. In another embodiment, the method includes administering
to the subject in need of such treatment a therapeutically
effective dosage of a .beta.3-AR antagonist in combination with an
antibody, or a fragment thereof, that binds to the polypeptide
having the sequence as shown in SEQ ID NO: 1.
[0201] Recent results suggest that ZAG expression in adipose tissue
may be more important locally than circulating ZAG, by acting in a
paracrine manner. Thus in humans, while mRNA levels of ZAG in
visceral and subcutaneous fat correlated negatively with BMI, fat
mass and insulin resistance, serum levels, determined by ELISA,
correlated positively with parameters of adiposity (BMI and waist
circumference) and insulin resistance. Thus the ability of ZAG to
induce its own expression in gastrocnemius muscle, WAT and BAT may
be critical for its ability to increase lipolysis and energy
utilization.
[0202] In various embodiments of the invention, the purpose of
combining ZAG, .beta.-3AR agonists and .beta.-AR antagonists varies
depending on the purpose of the treatment and the status of the
subject.
[0203] In one embodiment involving the treatment of obesity or
diabetes in which it is desired to activate the .beta.-3AR
mechanism to achieve the desired lipolysis, glucose consumption,
insulin sensitization, protein synthesis, increased energy
expenditure, and the like. In this circumstance with some subjects
it may be observed that the administered ZAG, or more likely the
.beta.-3AR agonist will exhibit some undesired activity at one or
more of the .beta.-1AR or the .beta.-2AR, causing side effects or
diminishment of desired efficacy. This circumstance would then call
for the additional administration of .beta.-AR antagonists,
sometimes referred to as "classic beta blockers" so as to prevent
the undesired activity at the .beta.-1AR or .beta.-2AR. These
.beta.-AR antagonists would preferably, but not necessarily, be
selected to block the receptor subtype (one of .beta.-1AR,
.beta.-2AR) that is associated with the side effect or mitigation
of efficacy.
[0204] In another embodiment involving the treatment of cachexia.
In cachexia caused by different diseases, and within populations of
subjects with a given diseases, different degrees of cachexia are
observed, and with different proportions of muscle loss and fat
loss.
[0205] In another aspect, a cachectic subject may be suffering from
loss of muscle mass, but with either no loss of fat, or some degree
of fat loss. Because muscle loss is typically a more clinically
undesireable outcome, utilizing ZAG to cause some of the muscle
build-up that occurs in cachetic animals treated with ZAG, while
also causing some degree of fat loss, may be desired. Thus treating
such subjects with ZAG, a .beta.-3AR agonist, and optionally as
described above, .beta.-AR antagonists, could increase muscle
mass.
[0206] In another aspect, a cachectic subject may be suffering from
loss of fat mass, with either no or some degree of loss of muscle
mass. In this case, it may be desirable from a clinical standpoint
to block the loss of fat, and so administration of antibodies
specific to ZAG would be used, in order to block the action caused
by ZAG and therefore decrease the downstream action of
.beta.-3AR.
[0207] In another embodiment, involving treatment of lipidystrophy,
in which fat masses are disproportionate to the normal distribution
within a subject, and in which loss of fat mass is desired. In this
case, the administration of one or more of ZAG, a .beta.-3AR
agonist and a .beta.-AR antagonist would be desired, with reasoning
similar to the first circumstance.
[0208] All methods may further include the step of bringing the
active ingredient(s) into association with a pharmaceutically
acceptable carrier, which constitutes one or more accessory
ingredients. As such, the invention also provides pharmaceutical
compositions for use in treating subjects having symptoms
associated with cachexia. In one embodiment, the composition
includes as the active constituent a therapeutically effective
amount of an anti-ZAG antibody as discussed above, or a functional
fragment thereof, together with a pharmaceutically acceptable
carrier, diluent of excipient.
[0209] Pharmaceutically acceptable carriers useful for formulating
a composition for administration to a subject are well known in the
art and include, for example, aqueous solutions such as water or
physiologically buffered saline or other solvents or vehicles such
as glycols, glycerol, oils such as olive oil or injectable organic
esters. A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize or to increase the absorption of the conjugate. Such
physiologically acceptable compounds include, for example,
carbohydrates, such as glucose, sucrose or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins or other stabilizers or excipients. In
addition, such physiologically acceptable compounds may further be
in salt form (i.e., balanced with a counter-ion such as Ca2+, Mg2+,
Na+, NH4+, etc.), provided that the carrier is compatible with the
desired route of administration (e.g., intravenous, subcutaneous,
oral, etc.). One skilled in the art would know that the choice of a
pharmaceutically acceptable carrier, including a physiologically
acceptable compound, depends, for example, on the physico-chemical
characteristics of the therapeutic agent and on the route of
administration of the composition, which can be, for example,
orally or parenterally such as intravenously, and by injection,
intubation, or other such method known in the art. The
pharmaceutical composition also can contain a second (or more)
compound(s) such as a diagnostic reagent, additional nutritional
substance, toxin, or therapeutic agent, for example, a cancer
chemotherapeutic agent and/or vitamin(s).
[0210] Formulations of the present invention may also include one
or more excipients. Pharmaceutically acceptable excipients which
may be included in the formulation are buffers such as citrate
buffer, phosphate buffer, acetate buffer, and bicarbonate buffer,
amino acids, urea, alcohols, ascorbic acid, phospholipids;
proteins, such as serum albumin, collagen, and gelatin; salts such
as EDTA or EGTA, and sodium chloride; liposomes;
polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol,
and glycerol; propylene glycol and polyethylene glycol (e.g.,
PEG-4000, PEG-6000); glycerol; and glycine or other amino acids.
Buffer systems for use with the formulations include citrate;
acetate; bicarbonate; and phosphate buffers.
[0211] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets, tablets or lozenges, each containing a predetermined
amount of the active compound in the form of a powder or granules;
or as a suspension of the active compound in an aqueous liquid or
non-aqueous liquid such as a syrup, an elixir, an emulsion of a
draught.
[0212] The nutritional supplement formulations can further include
any number of additional ingredients that are known to promote
health and/or weight reduction. Exemplary additional ingredients
include, but are not limited to, low-glycemic ingredients such as
carbohydrate sources, protein sources and sources of dietary fiber.
Such low-glycemic ingredients have been shown to curb appetite and
cause a reduction in daily caloric intake.
[0213] An important macronutrient of the nutritional supplement is
carbohydrate because it has the greatest influence on satiety and
subsequent weight loss. As used herein, satiety, refers to the
sensation of fullness between one meal and the next and satiation
refers to a sensation of fullness that develops during the progress
of a meal and contributes to meal termination. Foods with
low-glycemic-indexes evoke a smaller rise in blood glucose and
insulin and a higher glucagon concentration, which promote satiety
and prevent weight gain better than those carbohydrate-containing
foods with higher ones because they take longer to digest and to be
absorbed than carbohydrates with high- glycemic-indices.
[0214] The "glycemic index" is a system of predicting subsequent
rises in blood glucose after ingestion of carbohydrate-containing
foods (Anderson, J. S. et al., Modern Nutrition in Health and
Disease, ch. 70: 1259-86 (1994); Wolever, T. M. S. et al., Am. J.
Clin. Nutr., 54: 846-54 (1991); Wolever, T. M. S. et al., Diab.
Care, 12: 126-32 (1990)). The glycemic index characterizes the rate
of carbohydrate absorption after a meal. It is defined as the area
under the glycemic response curve during a 2-hour period after
consumption of 50 g of carbohydrate from a test food divided by the
area under the curve of a standard, which is either white bread or
glucose. The glycemic index carbohydrates have the highest peak
circulating glucose in a 2 hour period following ingestion of food.
Conversely, low-glycemic-index carbohydrates cause a lower peak
glucose and smaller area under the curve.
[0215] Many factors determine the glycemic index of foods. These
include carbohydrate type, fiber, protein and fat content and the
method of preparation (overcooked foods evoke a higher response).
Generally high-glycemic-index carbohydrates are highly refined, and
have a relatively high amount of glucose or starch compared to
lactose, sucrose or fructose. Also, they are low in soluble fiber.
The inclusion of fiber is important due to the way fiber
facilitates weight loss by forming a gel with the food in the
stomach. This gelling action reduces the rate of gastric emptying
and hence digestion rates which promote satiety. Other factors
which affect satiety are the amount of carbohydrate, the complexity
of the carbohydrate, and the other foods that are eaten
simultaneously with the carbohydrate (e.g., fiber, protein, fat)
(Ludwig, D. S., J Nutr., 130: 280S-3S (2000); Wolever, T. M. S. et
al., Am. J. Clin. Nutr., 54: 846-54 (1991); Wolever, T. M. S. et
al., Diab. Care, 12: 126-32 (1990)). Bread and potatoes raise blood
glucose more than beans. Other foods containing no or
non-digestible carbohydrate ingested at the same time as
carbohydrates (e.g., fat, fiber and protein) reduces postprandial
blood glucose and insulin levels (Wolever, T. M. S., et al., Am. J.
Clin. Nutr., 54: 846-54 (1991)).
[0216] The low-glycemic-index carbohydrate source can be provided
by a single carbohydrate or a combination. The carbohydrate source
can further provide a source of fiber and may be a natural
sweetener, fructose, barley, konjac mannan, psyllium and
combinations thereof. The protein source is of a high biological
value and is selected from at least one of the following: whey
protein concentrate, casein, soy, milk, egg and combinations of
these. Additionally, the nutritional supplement may contain,
micronutrients, vitamins, minerals, Aietary supplements (e.g.,
herb), nutrients, emulsifiers, flavorings and edible compounds.
[0217] In one embodiment, the nutritional supplement formulation
may further include a carbohydrate for sweetening the nutritional
supplement. Exemplary carbohydrates useful for sweetening the
nutritional supplement include, but are not limited to, fructose,
evaporated cane juice, inulin, agave, honey, maple syrup, brown
rice syrup, malt syrup, date sugar, fruit juice concentrate, and
mixed fruit juice concentrate.
[0218] Dietary fiber that may be suitable for use in the invention
includes but is not limited to cellulose, seeds, hemicellulose
(e.g., bran, whole grains), polyfructose (e.g., inulin and
oligofructans), polysaccharide gums (e.g., Larch Arabinogalactan),
oatmeal, barley, pectins, lignin, resistant starches. Examples of
suitable fiber sources include but are not limited to wheat bran,
cellulose, oat bran, corn bran, guar, pectin, and psyllium.
[0219] Sources of protein can be any suitable protein utilized in
nutritional formulations and can include whey protein, whey protein
concentrate, whey powder, egg, soy protein, soy protein isolate,
caseinate (e.g., sodium caseinate, sodium calcium caseinate,
calcium caseinate, and potassium caseinate), animal and vegetable
protein and mixtures thereof When choosing a protein source, the
biological value of the protein should be considered first, with
the highest biological values being found in caseinate, whey,
lactalbumin, soy, delactosed milk solids, egg albumin and whole egg
proteins. These proteins have high biological value; that is, they
have a high proportion of the essential amino acids.
[0220] The nutritional supplement can also contain other
ingredients such as one or a combination of other vitamins,
minerals, antioxidants, fiber (e.g., ginkgo biloba, ginseng) and
other nutritional supplements. Selection of one or several of these
ingredients is a matter of formulation design, consumer and
end-user preference. The amount of these ingredients added to the
nutritional supplements of this invention are readily known to the
skilled artisan and guidance to such amounts can be provided by the
RDA and DRI (Dietary Reference Intake) doses for children and
adults. Vitamins and minerals that can be added include, but are
not limited to, calcium phosphate or acetate, tribasic; potassium
phosphate, dibasic; magnesium sulfate or oxide; salt (sodium
chloride); potassium chloride or acetate; ascorbic acid; ferric
orthophosphate; niacin amide; zinc sulfate or oxide; calcium
pantothenate; copper gluconate; riboflavin; beta-carotene;
pyridoxine hydrochloride; thiamin mononitrate; folic acid; biotin;
chromium chloride or picolinate; potassium iodide; selenium; sodium
selenate; sodium molybdate; phylloquinone; Vitamin D3;
cyanocobalamin; sodium selenite; copper sulfate; Vitamin A; Vitamin
E; vitamin B6 and hydrochloride thereof; Vitamin C; inositol;
Vitamin B12; potassium iodide.
[0221] The amount of other ingredients per unit serving is a matter
of design and will depend upon the total number of unit servings of
the nutritional supplement daily administered to the patient. The
total amount of other ingredients will also depend, in part, upon
the condition of the patient. Preferably the amount of other
ingredients will be a fraction or multiplier of the RDA or DRI
amounts. For example, the nutritional supplement will comprise 50%
RDI (Reference Daily Intake) of vitamins and minerals per unit
dosage and the patient will consume two units per day.
[0222] Flavors, coloring agents, spices, nuts and the like can be
incorporated into the product. Flavorings can be in the form of
flavored extracts, volatile oils, chocolate flavorings (e.g.,
non-caffeinated cocoa or chocolate, or chocolate substitutes, such
as carob), peanut butter flavoring, cookie crumbs, crisp rice,
vanilla or any commercially available flavoring. Flavorings can be
protected with mixed tocopherols. Examples of useful flavorings
include but are not limited to pure anise extract, imitation banana
extract, imitation cherry extract, chocolate extract, pure lemon
extract, pure orange extract, pure peppermint extract, imitation
pineapple extract, imitation rum extract, imitation strawberry
extract, or pure vanilla extract; or volatile oils, such as balm
oil, bay oil, bergamot oil, cedarwood oil, cherry oil, walnut oil,
cinnamon oil, clove oil, or peppermint oil; peanut butter,
chocolate flavoring, vanilla cookie crumb, butterscotch or toffee.
In a preferred embodiment, the nutritional supplement contains
berry or other fruit flavors. The food compositions may further be
coated, for example with a yogurt coating, if it is produced as a
bar.
[0223] Emulsifiers may be added for stability of the final product.
Examples of suitable emulsifiers include, but are not limited to,
lecithin (e.g., from egg or soy), and/or mono- and di-glycerides.
Other emulsifiers are readily apparent to the skilled artisan and
selection of suitable emulsifier(s) will depend, in part, upon the
formulation and final product.
[0224] Preservatives may also be added to the nutritional
supplement to extend product shelf life. Preferably, preservatives
such as potassium sorbate, sodium sorbate, potassium benzoate,
sodium benzoate or calcium disodium EDTA are used.
[0225] In addition to the carbohydrates described above, the
nutritional supplement can contain artificial sweeteners, e.g.,
saccharides, cyclamates, aspartamine, aspartame, acesulfame K,
and/or sorbitol. Such artificial sweeteners can be desirable if the
nutritional supplement is intended for an overweight or obese
individual, or an individual with type II diabetes who is prone to
hyperglycemia.
[0226] The nutritional supplements of the present invention may be
formulated using any pharmaceutically acceptable forms of the
vitamins, minerals and other nutrients discussed above, including
their salts. They may be formulated into capsules, tablets,
powders, suspensions, gels or liquids optionally comprising a
physiologically acceptable carrier, such as but not limited to
water, milk, juice, sodas, starch, vegetable oils, salt solutions,
hydroxymethyl cellulose, carbohydrate. In one embodiment, the
nutritional supplements may be formulated as powders, for example,
for mixing with consumable liquids, such as milk, juice, sodas,
water or consumable gels or syrups for mixing into other
nutritional liquids or foods. The powdered form has particular
consumer appeal, is easy to administer and incorporate into one's
daily regimen, thus increasing the chances of patient compliance.
The nutritional supplements of this invention may be formulated
with other foods or liquids to provide premeasured supplemental
foods, such as single serving breakfast bars, energy bars, breads,
cookies, brownies, crackers, cereals, cakes, or beverages, for
example.
[0227] Thus, the nutritional supplement formulation may be
administered as a dietary supplement or as an additive to a
consumable carrier such as a foodstuff. The composition may be
incorporated into a foodstuff that is later cooked or baked. The
components of the composition are structurally stable to remain
un-oxidized and are heat stable at temperatures required for baking
or cooking.
[0228] To manufacture such a beverage, the ingredients are dried
and made readily soluble in water or other consumable liquids as
described above. The beverage is a preferred nutritional supplement
form due to its ability to aid in the sensation of satiety if
consumed at least one half hour prior to meals.
[0229] To manufacture such a food bar, the dry ingredients are
added with the liquid ingredients in a mixer and mixed until the
dough phase is reached; the dough is put into an extruder and
extruded; the extruded dough is cut into appropriate lengths; and
the product is ooled.
[0230] For manufacture of other foods or beverages, the ingredients
comprising the nutritional supplement of this invention can be
added to traditional formulations or they can be used to replace
traditional ingredients. Those skilled in food formulating will be
able to design appropriate foods/beverages with the objective of
this invention in mind.
[0231] The nutritional supplement can be made in a variety of
forms, such as puddings, confections, (i.e., candy), nutritional
beverages, ice cream, frozen confections and novelties, or baked or
non-baked, extruded food products such as bars. In one embodiment,
nutritional supplement is in the form of a powder for a beverage or
a non-baked extruded nutritional bar.
[0232] In one embodiment, the consumable carrier is a meat product,
such as natural or cultured meat. In vitro meat, also known as
cultured meat, is animal flesh that has never been part of a
complete, living animal. The process of developing in vitro meat
involves taking muscle cells and applying a protein that helps the
cells to grow into large portions of meat. Once the initial cells
have been obtained, additional animals would not be needed--akin to
the production of yogurt cultures. In one embodiment, the
production of in vitro meat: loose muscle cells and structured
muscle, the latter one being vastly more challenging than the
former. Muscles consist of muscle fibers, long cells with multiple
nuclei. Such cells do not proliferate by themselves, but arise when
precursor cells fuse. Precursor cells can be embryonic stem cells
or satellite cells, specialized stem cells in muscle tissue.
Theoretically, it is relatively simple to culture them in a
bioreactor and then make them fuse. For the growth of real muscle,
however, the cells should grow "on the spot," which requires a
perfusion system akin to a blood supply to deliver nutrients and
oxygen close to the growing cells, as well as to remove the waste
products. In addition, other cell types, such as adipocytes, need
to be grown, and chemical messengers should provide clues to the
growing tissue about the structure. Lastly, muscle tissue needs to
be physically stretched or "exercised" to properly develop (see,
e.g., U.S. Pat. No. 6,835,390 and published International
application no. WO 99/31222, both of which are incorporated herein
by reference). In yet another embodiment, the invention includes
cultured meat that is engineered to express ZAG in sufficient
quantities such that addition of recombinant ZAG is
unnecessary.
[0233] The ingredients can be administered in a single formulation
or they can be separately administered. For example, it may be
desirable to administer a bitter tasting ingredient in a form that
masks its taste (e.g., capsule or pill form) rather than
incorporating it into the nutritional composition itself (e.g.,
powder or bar). Thus, the invention also provides a pharmaceutical
pack or kit comprising one or more containers filled with one or
more of the ingredients of the nutritional compositions of the
invention. Optionally associated with such container(s) can be a
notice in the form prescribed by a government agency regulating the
manufacture, use or sale of pharmaceutical or dietary supplement
products, which notice reflects approval by the agency of
manufacture, use of sale for human administration. The pack or kit
can be labeled with information regarding mode of administration,
sequence of administration (e.g., separately, sequentially or
concurrently), or the like. The pack or kit may also include means
for reminding the patient to take the therapy. The pack or kit can
be a single unit dosage of the combination therapy or it can be a
plurality of unit dosages. In particular, the agents can be
separated, mixed together in any combination, present in a
formulation or tablet. Agents assembled in a blister pack or other
dispensing means is preferred.
[0234] In one embodiment, the formulation includes about 1.0 mg to
1000 mg ZAG. In another embodiment, the formulation includes about
1.0 mg to about 500 mg ZAG. In another embodiment, the formulation
includes about 1.0 mg to about 100 mg ZAG. In another embodiment,
the formulation includes about 1.0 mg to about 50 mg ZAG. In
another embodiment, the formulation includes about 1.0 mg to about
10 mg ZAG. In another embodiment, the formulation includes about
5.0 mg ZAG.
[0235] Accordingly, in another aspect, the invention provides the
use of anti-ZAG antibodies, or functional fragments thereof, as
herein defined, for the manufacture of a medicament useful in human
medicine for treating symptoms and/or conditions associated with
cachexia or diseases associated with muscle wasting disorders.
[0236] In one embodiment, the formulation of the present invention
is administered orally. In such embodiments, the formulation is at
least 70, 75, 80, 85, 90, 95 or 100% as effective as any other
route of administration.
[0237] The total amount of formulation to be administered in
practicing a method of the invention can be administered to a
subject as a single dose, either as a bolus or by ingestion over a
relatively short period of time, or can be administered using a
fractionated treatment protocol, in which multiple doses are
administered over a prolonged period of time (e.g., once daily,
twice daily, etc.). One skilled in the art would know that the
amount of formulation depends on many factors including the age and
general health of the subject as well as the route of
administration and the number of treatments to be administered. In
view of these factors, the skilled artisan would adjust the
particular dose as necessary. In general, the formulation of the
pharmaceutical composition and the routes and frequency of
administration are determined, initially, using Phase I and Phase
II clinical trials.
[0238] Accordingly, in certain embodiments, the methods of the
invention include an intervalled treatment regimen. It was observed
that long-term daily administration of ZAG in ob/ob mice results in
continuous weight loss. As such, in one embodiment, the treatment
of ZAG or anti-ZAG antibodies, alone or in combination with one or
more .beta.-AR antagonists or .beta.3-AR agonists, is administered
every other day. In another embodiment, the treatment is
administered every two days. In another embodiment, the treatment
is administered every three days. In another embodiment, the
treatment is administered every four days.
[0239] The following examples are provided to further illustrate
the advantages and features of the present invention, but are not
intended to limit the scope of the invention. While they are
typical of those that might be used, other procedures,
methodologies, or techniques known to those skilled in the art may
alternatively be used.
EXAMPLE 1
Zinc-.alpha..sub.2-glycoprotein Attenuates Hyperglycemia
[0240] To evaluate the ability of Zinc-.alpha..sub.2-glycoprotein
(ZAG) to attenuate obesity and hyperglycemia ob/ob mice were
administered ZAG which induced a loss of body weight, and a rise in
body temperature, suggesting an increased energy expenditure.
Expression of uncoupling proteins-1 and -3 in brown adipose tissue
were increased, while there was a decrease in serum levels of
glucose, triglycerides and non-esterified fatty acids, despite an
increase in glycerol, indicative of increased lipolysis. There was
a decrease in plasma insulin and an improved response to
intravenous glucose together with an increased glucose uptake into
adipocytes and skeletal muscle. Expression of hormone-sensitive
lipase in epididymal adipocytes was increased. There was an
increase in skeletal muscle mass due to an increase in protein
synthesis and decrease in degradation. This suggests that ZAG may
be effective in the treatment of hyperglycemia.
[0241] Dulbeccos' Modified Eagle's (DMEM) and Freestyle media were
purchased from ivitrogen (Paisley, UK) while fetal calf serum was
from Biosera (Sussex, UK). 2-[1-.sup.14C] Deoxy-D-glucose
(sp.act.1.85GBq mmol.sup.-1) and L-[2,6-.sup.3H] phenylalanine
(sp.act.37Bq mmol.sup.-1) were from American Radiolabeled Chemicals
(Cardiff, UK). Rabbit polyclonal antibody to phospho (Thr-202) and
total ERK1, total p38MAPK, phospho HSL (Ser-552), glucose
transporter 4 (GLUT4), adipose triglyceride lipase, hormone
sensitive lipase, and phospho PLA.sub.2 (Ser-505) and to human ATGL
were purchased from Abcam (Cambridge, UK). Mouse monoclonal
antibody to full length human ZAG was from Santa Cruz (California,
USA), and mouse monoclonal antibody to myosin heavy chain type II
was from Novacastra (via Leica Biosystems, Newcastle, UK). Mouse
monoclonal antibodies to 20S proteasome .alpha.-subunits and p42
were from Affiniti Research Products (Exeter, UK). Mouse monoclonal
antibody to phospho (Thr-180/Tyr-182) p38MAPK and rabbit polyclonal
antisera to total and phospho (Thr-451) PKR, phospho (Ser-162)
eIF2.alpha. and to total eIF2.alpha. were from New England
Biosciences (Herts, UK). Polyclonal rabbit antibodies to UCP1, UCP3
and total PKR and PHOSPHOSAFE.TM. Extraction Reagent were from
Calbiochem (via Merk Chemicals, Nottingham, UK).
Peroxidase-conjugated goat anti-rabbit and rabbit anti-mouse
antibodies were purchased from Dako (Cambridge, UK). Polyclonal
rabbit antibody to mouse .beta.-actin and the triglyceride assay
kit were purchased from Sigma Aldrich (Dorset, UK). Hybond A
nitrocellulose membranes and enhanced chemiluminescence (ECL)
development kits were from Amersham Pharmacia Biotech (Bucks, UK).
A WAKO colorimetric assay kit for NEFA was purchased from Alpha
Laboratories (Hampshire, UK), and a mouse insulin ELISA kit was
purchased from DRG (Marburg, Germany). Glucose measurements were
made using a Boots (Nottingham, UK) plasma glucose kit.
[0242] Production of recombinant ZAG--HEK293F cells were
transfected with full length human ZAG cDNA in the expression
vector pcDNA 3.1, and maintained in FreeStyle medium under an
atmosphere of 5% CO.sub.2 in air at 37.degree. C. ZAG was secreted
into the medium, which was collected, and maximal protein levels
(16 .mu.gml.sup.-1) were obtained after 14 days of culture. To
purify ZAG, media (200 ml) was centrifuged at 700 g for 15 min to
remove cells, and concentrated into a volume of 1 ml sterile PBS
using an Amicon Ultra-15 centrifugal filter with a 10 kDa cut-off.
The concentrate (about 2 mg protein) was added to 2 g DEAE
cellulose suspended in 20 ml 10 mM Tris, pH 8.8 and stirred for 2 h
at 4.degree. C. The DEAE cellulose bound ZAG and it was sedimented
by centrifugation (1500 g for 15 min) and the ZAG was eluted by
stirring with 20 ml 10 mM Tris, pH8.8 containing 0.3M NaCl for 30
min at 4.degree. C. The eluate was washed and concentrated into a
volume of lml in sterile PBS using an Amicon centrifugal filter.
The purified ZAG was free of endotoxin, as determined with a LAL
Pyrogent single test kit (Lonza, Bucks, UK).
[0243] Cell culture and purification of ZAG. Single-cell
suspensions of white adipocytes were prepared from minced adipose
deposits by incubation at 37.degree. C. for 2 h in Krebs-Ringer
biocarbonate buffer containing 1.5 mgml.sup.-1 collagenase, and 4%
bovine serum albumin under an atmosphere of 95% oxygen: 5% CO.sub.2
as previously described. For time-course studies adipocytes were
suspended in DMEM containing 10% fetal calf serum at a
concentration of 10.sup.5 cells ml.sup.-1 and maintained under an
atmosphere of 10% CO, in air at 37.degree. C. Human 293 cells
transfected with a plasmid containing human ZAG were seeded at a
concentration of 10.sup.5 cells ml.sup.-1 in FreeStyle medium and
maintained under an atmosphere of 5% CO.sub.2 in air at 37.degree.
C. Maximal protein levels (16 .mu.gml.sup.-1) were obtained after
14 days of culture. The media (200 ml) was then centrifuged at 700
g for 15 min to remove cells and concentrated into a volume of 1 ml
of sterile PBS using an Amicon Ultra-15 centrifugal filter with a
10 kDa cut-off. After measurement of the protein concentration of
the sample (about 2 mg) it was added to 2 g DEAE cellulose
suspended in 20 ml of 10 mM Tris, pH8.8 and stirred at 4.degree. C.
for 2 h. ZAG being negatively charged binds to the DEAE cellulose,
which was sedimented by centrifugation (1500 g for 15 min), and
eluted by stirring with 20 ml 10 mM Tris, pH8.8 containing 0.3M
NaCl for 30 min at 4.degree. C. The supernatant was washed and
concentrated to a volume of lml in sterile PBS using the Amicon
centrifugal filter.
[0244] Animals--mice. Homozygous obese (ob/ob) mice from the colony
maintained at Aston University were used in the present study. The
origin and characteristics of Aston ob/ob mice have been previously
described. Male mice (20-21 weeks old, weight 90-100 g) were
grouped into three per cage in an air conditioned room at
22.+-.2.degree. C. with a 12 h-light:12 h-dark cycle and fed a rat
and mouse breeding diet (Special Diet Services, Witham, UK) and tap
water ad libitum. They were administered ZAG (35 .mu.g) in PBS (100
.mu.l) b.d. by i.v. administration and body weight and food and
water intake were monitored daily. Control mice received PBS alone.
Body temperature was measured daily by the use of a rectal
thermometer (RS Components, Northants, UK). All animal experiments
were carried out in accordance with the U.K. Animals (Scientific
Procedures) Act 1986. No adverse effects were observed after
administration of ZAG.
[0245] Animal--Rats. Mature male Wistar rats (one year old from our
own colony) weighing 540.+-.82.5 g were housed individually and
treated once daily i.v., with either ZAG in PBS (100 .mu.l) (50
.mu.g per 100 g body weight), or with PBS (100 .mu.l) as a control.
Both food and water intake and body weight were measured daily.
Animals were given free access to food (Special Diet Services,
Essex, UK) and water ad libitum. The animal experiment was carried
out under the welfare conditions imposed by the British Home
Office. After 10 days treatment the animals were terminated and the
body composition determined. Animals were heated to 80-90.degree.
C. for 7 days until constant weight was achieved. The water content
was then determined from the difference between the wet and dry
weight. Lipids were extracted from the dry carcass using a sequence
of chloroform:methanol (1:1), ethanol/acetone (1:1) and diethyl
ether (120 ml of each) as described by Lundholm et al (14). The
solvents were evaporated and the fat weighed. The non-fat carcass
mass was calculated as the difference between the initial weight of
the carcass and the weight of water and fat.
[0246] Lipolytic assay. Samples to be assayed were incubated with
10.sup.5 to 2.times.10.sup.5 adipocytes for 2 h in 1 ml
Krebs-Ringer bicarbonate buffer, pH 7.2. The concentration of
glycerol released was determined enzymatically by the method of
Wieland (Wieland, O. Glycerol UV method. In Methods of Enzymatic
Analysis (ed. Bergmeyer, H. U.) (Academic Press, London, UK, pp
1404-1409, 1974)). Control samples containing adipocytes alone were
analysed to determine the spontaneous glycerol release. Activity
was expressed as .mu.mol glycerol released/10.sup.5 adipocytes/2
h.
[0247] Serum Metabolite Determinations. Non-esterified fatty acids
(NEFA) were determined using a Wako-ASC-ACOD kit (Wako Chemical
GmbH, Neuss, Germany). Triglycerides were determined using a
Triglyceride kit (Sigma Chemical Co., Poole, United Kingdom) and
3-hydroxybutyrate by a quantitative enzymatic determination kit
(Sigma). Glucose was measured using a glucose analyser (Beckman,
Irvine, Calif) and glycerol was determined enzymatically using the
method of Wieland as described in "Methods of Enzymatic Analysis"
(Ed. Bergmeyer, H. U.) Vol. 3, pp 1404-1409, published by Academic
Press, London (1974).
[0248] Isolation of Mouse Adipocyte Plasma Membranes. In a typical
procedure white adipocytes were isolated from mouse epididymal fat
pads as referred to above except that the cells were washed in 250
mM sucrose, 2 mM ethyleneglycol
bis(.beta.-aminoethylether)-N,N,N',N'(EGTA), 10 mM Tris-HCl (pH
7.4). Adipocytes were resuspended in 20 ml of the above buffer and
homogenised by aspirating through a Swinny filter at least 10
times. The cell homogenate was then centrifuged at 300 g for 5 min,
the fat cake removed from the surface and the remaining pellet and
infranatant transferred to clean tubes. These were centrifuged at
30,000 g for 1 h at 4.degree. C. and the membrane pellet formed was
resuspended in the sucrose buffer (200 to 400 .mu.l). Plasma
membranes were separated from other organelle membranes on a
self-forming gradient of PERCOLL.TM. colloidial silica particles.
The constituents were 250 mM sucrose, 2 mM EGTA, 10 mM Tris-HCl, pH
7.4; PERCOLL.TM.; and 2M sucrose, 8 mM EGTA, 80 mM Tris-HCl, pH
7.4, mixed in a ratio of 32:7:1 together with the membrane
suspension (in a total volume of 8 ml). This mixture was
centrifuged at 10,000 g for 30 mM at 4.degree. C. The gradient was
fractionated into 0.75 ml portions and each portion was assayed for
the presence of succinate dehydrogenase, NADH-cytochrome c
reductase, lactate dehydrogenase and 5'-nucleotidase to locate the
plasma membrane fraction. The membrane fractions were resuspended
in 150 mM NaCl, 1 mM EGTA, 10 mM Tris-HCl, pH 7.4 and centrifuged
at 10,000 g at 4.degree. C. for 2 min. The process was repeated
twice. The washed plasma membranes were then diluted in 10 mM
Tris-HCl, pH 7.4, 250 mM sucrose, 2 mM EGTA and 4 .mu.M
phenylmethylsulfonyl fluoride (PMSF) at 1-2 mg/ml, snap frozen in
liquid nitrogen and stored at -70.degree. C. until use.
[0249] Lipolytic activity in rat adipocytes--White adipocytes were
prepared from finely minced epididymal adipose tissue of male
Wistar rats (400g) using collagenase digestion, as described (Beck
S A, et al. Production of lipolytic and proteolytic factors by a
murine tumor-producing cachexia in the host. Cancer Res
47:5919-5923, 1987). Lipolytic activity was determined by
incubating 10.sup.5-2.times.10.sup.5 adipocytes for 2 h in 1 ml
Krebs-Ringer bicarbonate buffer, pH 7.2, and the extent of
lipolysis was determined by measuring the glycerol released
(Wieland O. Glycerol UV method. Methods of Enzymatic Analysis,
edited by Bergmeyer H U. Academic Press, London, pp 1404-1409,
1974). Spontaneous glycerol release was measured by incubating
adipocytes alone. Lipolytic activity was expressed as .mu.mol
glycerol released/10.sup.5 adipocytes/2 h.
[0250] Gel Electrophoresis. Gels were prepared according to the
method of Laemmli and generally consisted of a 5% stacking gel and
a 15% SDS-PAGE resolving gel (denaturing or reducing conditions) or
a 10% SDS-PAGE resolving gel (non-denaturing or non-reducing
conditions). Samples were loaded at 1-5.mu.g/lane. Bands were
visualized by staining either with Coomassie brilliant blue R-250
or by silver. Samples were prepared for reducing conditions by
heating for 5 min at 100.degree. C. in 0.0625M Tris-HCl, pH 6.8,
10% glycerol, 1% SDS, 0.01% bromophenol blue and 5%
2-mercaptoethanol.
[0251] Glucose uptake into adipocytyes. Isolated adipocytes
(5.times.10.sup.4) were washed twice in lml Krebs-Ringer
bicarbonate buffer, pH 7.2 (KRBS) and further incubated for 10 min
at room temperature in 0.5 ml KRBS containing 18.5MBq
2-[1-.sup.14C] deoxy-D-glucose and non-radioactive
2-deoxy-D-glucose to a final concentration of 0.1 mM. Uptake was
terminated by the addition of lml ice-cold glucose-free KRBS, and
the cells were washed three times with 1 ml KRBS, lysed by addition
of 0.5 ml 1M NaOH and left for at least 1 h at room temperature
before the radioactivity was determined by liquid scintillation
counting.
[0252] Glucose uptake into gastrocnemius muscle--Gastrocnemius
muscles were incubated in Krebs-Henseleit bicarbonate buffer for 45
min at 37.degree. C. and then incubated for a further 10 min in 5
ml Krebs-Henseleit buffer containing 185M Bq 2-[1-.sup.14C]
deoxy-D-glucose and non-radioactive 2-deoxy-D-glucose to a final
concentration of 0.1 mM. The muscles were then removed and washed
in 0.9% NaCl for 5 min followed by dissolution in 0.5 ml 1M NaOH
and the radioactivity was determined by liquid scintillation
counting.
[0253] Glucose uptake into soleus muscle. Soleus muscles were
incubated in Krebs-Henseleit bicarbonate buffer for 45min at
37.degree. C. and then incubated for a further 10 min in 5 ml
Krebs-Henseleit buffer containing 185 MBq 2-[1-.sup.14C]
deoxy-D-glucose and non-radioactive 2-deoxy-D-glucose to a final
concentration of 0.1 mM. The muscles were then removed and washed
in 0.9% NaCl for 5 min, followed by dissolution in 0.5 ml 1M NaOH
and the radioactivity was determined by liquid scintillation
counting.
[0254] Protein synthesis and degradation in muscle. The method for
the determination of protein synthesis and degradation in muscle
has been previously described (Smith, K. L. & Tisdale, M. J.
Increased protein degradation and decreased protein synthesis in
skeletal muscle during cancer cachexia. Br. J. Cancer 67, 680-685
(1993)). Gastrocnemius muscles were excised using ligatures and
incubated for 30 min at 37.degree. C. in RPMI 1640 medium lacking
phenol red and saturated with O.sub.2:CO.sub.2 (19:1) and then
washed with PBS. Protein synthesis was measured by the
incorporation of L-[2,6-.sup.3H] phenylalanine (640 MBq) into
acid-insoluble material using a 2 h period in which the muscles
were incubated at 37.degree. C. in RPMI/640 without phenol red and
saturated with O.sub.2:CO.sub.2 (19:1). Muscles were then rinsed in
non-radioactive medium, blotted and homogenised in 2% perchloric
acid. The rate of protein synthesis was calculated by dividing the
amount of protein-bound radioactivity by the amount of acid soluble
radioactivity. Protein degradation was determined by the release of
tyrosine from gastrocnemius muscle over a 2 h period in 3 ml of
oxygenated Krebs-Henseleit buffer, pH7.4, containing 5 mM glucose
and 0.5 mM cycloheximide.
[0255] Measurement of proteasome and caspase activity. The
`chymotrypsin-like` activity of the proteasome was determined
fluorometrically by measuring the release of
7-amido-4-methylcoumarin (AMC) at an excitation wavelength of 360
nm and an emission wavelength of 460 nm from the fluorogenic
substrate N-succinyl Lys Lys Val Tyr.AMC (SEQ ID NO: 2) as
previously described for myotubes (Whitehouse, A. S. & Tisdale,
M. J. Increased expression of the ubiquitin-proteasome pathway in
murine myotubes by proteolysis-inducing factor (PIF) is associated
with activation of the transcription factor NF-.kappa.B. Br. J.
Cancer 89, 1116-1122 (2003)). Gastrocnemius muscle was homogenised
in 20 mM Tris, pH7.5, 2 mM ATP, 5 mM MgCl.sub.2 and 50 mM DTT at
4.degree. C., sonicated and centrifuged at 18,000 g for 10 min at
4.degree. C. to pellet insoluble material, and the resulting
supernatant was used to measure `chymotrypsin-like` enzyme activity
in the presence or absence of the proteasome inhibitor lactacystin
(10 .mu.M). Only lactacystin suppressible activity was considered
as true proteasome activity. The activity of caspase-3 was
determined by the release of AMC from AcAsp.Gly.Val.Asp.AMC (SEQ ID
NO: 3), and the activity of caspase-8 was determined by the release
of 7-amino-4-trifluromethylcoumarin (AFC) from the specific
substrate Z-Ile Glu Phe Thr Asp-AFC (SEQ ID NO: 4), using the
supernatant from above (50 .mu.g protein), and either the caspase-3
or -8 substrate (10 .mu.M) for 1 h at 37.degree. C., in the
presence or absence of the caspase-3 (AcAspGluValAsp-CHO) (SEQ ID
NO: 5) or caspase-8 (Ile Glu Phe Thr Asp-CHO) (SEQ ID NO: 6)
inhibitors (100 .mu.M). The increase in fluorescence due to AFC was
determined as above, while the increase in fluorescence due to AFC
was measured with an excitation wavelength of 400 nm and an
emission wavelength of 505 nm. The difference in values in the
absence and presence of the caspase inhibitors was a measure of
activity.
[0256] Western blot analysis. Freshly excised gastrocnemius muscles
were washed in PBS and lysed in PHOSPHOSAFETM Extraction Reagent
for 5min at room temperature followed by sonication at 4.degree. C.
The lysate was cleared by centrifugation at 18,000 g for 5 min at
4.degree. C. and samples of cytosolic protein (5 .mu.g) were
resolved on 12% sodium dodecyl suflate-polyacrylamide gel
electrophoresis at 180V for approximately 1 h. This was followed by
transference to 0.45 .mu.m nitrocellulose membranes, which were
then blocked with 5% Marvel in Tris-buffered saline, pH 7.5, at
4.degree. C. overnight. Both primary and secondary antibodies were
used at a dilution of 1:1000 except anti-myosin (1:250). Incubation
was for lh at room temperature, and development was by ECL. Blots
were scanned by a densitometer to quantify differences.
[0257] Samples of epididymal WAT, BAT and gastrocnemius muscle
excised from rats treated with ZAG or PBS for 5 days were
homogenized in 0.25M sucrose, 1 mM HEPES, pH 7.0 and 0.2M EDTA, and
then centrifuged for 10 min at 4,500 rpm. Samples of cytosolic
protein (10 .mu.g) were resolved on 12% sodium dodecylsulphate
polyacrylamide gel electrophoresis and the proteins were then
transferred onto 0.45 .mu.m nitrocellulose membranes, which had
been blocked with 5% Marvel in Tris-buffered saline, pH 7.5, at
4.degree. C. overnight, and following four 15 min washes with 0.1%
Tween in PBS, incubation with the secondary antibody was performed
for 1 h at room temperature. Development was by ECL.
[0258] Statistical analysis. The results are shown as means .+-.SEM
for at least three replicate experiments. Difference in means
between groups was determined by one-way analysis of variance
(ANOVA) followed by the Tukey-Kramer multiple comparison test. P
values less than 0.05 were considered significant.
[0259] Results--mice. Purification of ZAG resulted in a product
that was greater than 95% pure (FIG. 1A), confirmed as ZAG by
immunoblotting (FIG. 1B). ZAG stimulated lipolysis in epididymal
adipocytes (FIG. 1D) but the lipolytic effect was considerably
reduced in adipocytes from both subcutaneous and visceral deposits,
although it was significantly elevated over basal levels (FIG. 1E).
There was no significant difference in the extent of stimulation of
lipolysis between isoprenaline and ZAG in any adipocyte group,
although ZAG was more potent at inducing lipolysis than
isoprenaline on a molar basis. The effect of ZAG on the body weight
of ob/ob mice over a 5 day period is shown in FIG. 1F. While
control animals remained weight stable, animals treated with ZAG
showed a progressive weight loss, such that after 5 days there was
a 3.5 g weight difference between the groups, despite equal food
(PBS 32.+-.3.1 g; ZAG 30.+-.2.5 g) and water (PBS 140.+-.8.2 ml;
ZAG 135.+-.3.2 ml) intake over the course of the experiment. There
was a significant rise of body temperature of 0.4.degree. C. after
4 days of ZAG administration (FIG. 1G), indicative of an increase
in basal metabolic rate. Measurement of plasma metabolite levels
suggest an increase in metabolic substrate utilization in ZAG
treated animals (Table 1). Thus there was a significant decrease in
plasma glucose, triglycerides (TG) and non-esterified fatty acids
(NEFA) in ZAG-treated animals, despite an increased glycerol
concentration indicative of an increased lipolysis. There was a 36%
decrease in plasma insulin levels suggesting that ZAG is effective
in reducing the diabetic state. ZAG mRNA levels in various tissues
are shown in FIG. 1C.
TABLE-US-00001 TABLE 1 Plasma metabolite and insulin levels in
ob/ob mice treated with ZAG for 120 h PBS ZAG Glucose (mmol/L) 24.5
+ 0.4 20.3 + 0.8 p < 0.01 TG (mmol/L) 1.2 + 0.3 0.9 + 0.1 p <
0.05 Glycerol (.mu.mol/L) 359 + 23 429 + 36 p < 0.001 Insulin
(ng/mL) 41.2 + 0.6 26.3 + 0.52 p < 0.001 BAT (g) 0.35 .+-. 0.09
0.73 .+-. 0.12 p < 0.01 NEFA (mEq/L) 0.6 + 0.12 0.23 + 0.05 p
< 0.001 Soleus (g) 0.52 .+-. 0.13 0.80 .+-. 0.09 p < 0.01
Gastrocnemius (g) 0.85 .+-. 0.12 1.12 .+-. 0.14 p < 0.01 Insulin
Pancreas 4.52 .+-. 2.91 16.3 .+-. 3.1 p = 0.0042 (pg/g
pancerase)
[0260] To investigate this, a glucose tolerance test was performed,
on fed animals, after 3 days of ZAG administration (FIG. 2A). While
blood glucose levels were significantly elevated in PBS controls,
there was only a small rise in ZAG treated animals, which remained
significantly below the control group throughout the course of the
study. In addition plasma insulin levels were significantly lower
in ZAG treated animals at the onset of the study and remained so
during the 60 min of observation (FIG. 2B). ZAG administration
increased glucose uptake into epididymal, visceral and subcutaneous
adipocytes in the absence of insulin and also increased glucose
uptake into epididymal and visceral adipocytes in the presence of
low (1 nM) insulin (FIG. 2C). Glucose uptake into gastrocnemius
muscle was also significantly enhanced in ZAG treated animals both
in the absence and presence of insulin (100 nM) (FIG. 2D). The
glucose uptake in gastrocnemius muscle of ZAG treated mice was
greater than the response to insulin in non-treated animals.
[0261] ZAG administration also attenuated the effect of
hyperglycemia on skeletal muscle atrophy. Thus ob/ob mice treated
with ZAG showed a significant increase in the wet weight of both
gastrocnemius and soleus muscles (Table 1). This was associated
with over a two-fold increase in protein synthesis in soles muscle
(FIG. 3A), and a 60% decrease in protein degradation (FIG. 3B).
Gastrocnemius muscles from mice treated with ZAG showed a decreased
activity of the proteasome `chymotrypsin-like` enzyme activity
(FIG. 3C), which was not significantly different from that found in
non-obese mice, and a decreased expression of both the 20S
proteasome a-subunits (FIG. 3D), and p42, an ATPase subunit of the
19S regulator (FIG. 3E), suggesting a reduced activity of the
ubiquitin-proteasome pathway. Myosin levels were increased in
ZAG-treated mice (FIG. 3F), while actin levels did not change (FIG.
3G). In addition there was a reduction in the level of
phosphorylated forms of the dsRNA-dependent protein kinase (PKR)
(FIG. 4A) and eukaryotic initiation factor 2.alpha. (eIF2.alpha.)
(FIG. 4B), which have been shown to be responsible for muscle
atrophy induced by tumor catabolic factors, and high levels of
extracellular glucose. Other enzymes in this pathway including
phospholipase A.sub.2 (PLA.sub.2) (FIG. 4C), p38 mitogen activated
protein kinase (FIG. 4D) and caspases-3 and -8 (FIG. 4E) were also
attenuated in gastrocnemius muscles of ob/ob mice treated with ZAG.
These changes were commensurate with a decrease in catabolic
signaling in muscle in response to ZAG.
[0262] ZAG, but not isoprenaline increased expression of phospho
HSL in adipocytes which was completely attenuated by the
extracellular signal-regulated kinase (ERK) inhibitor
PD98059.sup.14. While ZAG increased expression of HSL in epididymal
adipocytes there was no increase in either subcutaneous or visceral
adipocytes (FIGS. 5B-5D). A similar situation was observed with
expression of adipose triglyceride lipase (ATGL) (FIGS. 5E-5G).
[0263] Expression of HSL and ATGL correlated with expression of the
active (phospho) form of ERK (FIGS. 5H-5J). Expression of HSL and
ATGL in epididymal adipocytes correlated with an increased
lipolytic response to the .beta.3 agonist, BRL37344 (FIG. 5K). This
result suggests that ZAG may act synergistically with .beta.3
agonists.
[0264] As previously reported, ZAG administration increased its
expression in adipose tissue (FIG. 6A). ZAG expression remained
elevated, for a further 3 days in tissue culture in the absence of
ZAG (FIG. 6B). Expression of HSL was also elevated in adipocytes
for 3 days in tissue culture in the absence of ZAG (FIG. 6C).
Administration of ZAG increased the expression of UCP1 (FIG. 6D)
and UCP3 (FIG. 6E) in BAT (FIG. 6D) and UCP3 in skeletal muscle
(FIG. 6F). An increased expression of uncoupling proteins would be
expected to channel metabolic substrates into heat as observed
(FIG. 1G).
[0265] After 21 days, the plasma metabolite levels in the ob/ob
mice were observed (Table 2), with monitored parameters shown in
Table 3. A further drop in blood glucose (from 2.03 to 15.2 mM) and
a rise in glycerol were observed, which seems greater since the
control is lower than before. No change in NEFA, TG or insulin was
observed at Day 21, as compared to Day 5 (Table 1). It was noted
that there is much more insulin in the pancreas in ZAG treated
animals showing the drop in plasma insulin, which is not due to
lower insulin production (e.g., as would happen with a toxin to
pancreatic beta cells), but rather due to the fact that less
insulin is needed to control blood glucose in the ZAG treated
animals.
TABLE-US-00002 TABLE 2 Plasma metabolite and insulin levels in
ob/ob mice treated with ZAG at Day 21. PBS ZAG Glucose (mmol/l)
24.1 .+-. 2.3 15.2 .+-. 2.1 p = 0.0085 NEFA(mEq/l) 0.62 .+-. 0.008
0.22 .+-. 0.06 p = 0.0025 Glycerol 290 .+-. 25.2 450 .+-. 36.2 p =
0.0058 Triglycerides 1.72 .+-. 0.05 0.89 .+-. 0.08 p = 0.0072
(mmol/l) Insulin (ng/ml) 39.5 .+-. 0.96 28.5 .+-. 0.34 p = 0.0056
Insulin Pancreas 6.2 .+-. 3.2 14.5 .+-. 2.5 p = 0.0035 (pg/g
pancerase)
TABLE-US-00003 TABLE 3 Parameters monitored in ob/ob mice treated
with ZAG at Day 21. Parameter PBS ZAG p Start weight 92.5 .+-. 3.1
93.1 .+-. 1.9 Finish weight 89.9 .+-. 1.3 83.95 .+-. 2.2 Food (g)
135 .+-. 6 145 .+-. 4 Water (ml) 268 .+-. 15 259 .+-. 20 BAT (g)
0.36 .+-. 0.21 0.41 .+-. 0.35 Gastrocnemius (g) 0.26 .+-. 0.15 0.39
.+-. 0.12 0.01 Soleus (g) 0.15 .+-. 0.06 0.18 .+-. 0.07
[0266] In addition, body temperature of the ob/ob mice increased
0.5.degree. to 1.degree. C. (FIG. 1G) within four days and peaked
at 38.1.degree. C. (FIG. 7) just before they lost the maximum
amount of weight. This would correlate with the weight of brown
adipose tissue which increases from 0.33.+-.0.12 g in the control
to 0.52.+-.0.08 g in the ZAG treated animals (FIG. 7). The weight
of the gastrocnemius muscles was also increased from 0.2.+-.0.05 g
to 0.7.+-.0.1 g, while there was a progressive decrease in urinary
glucose excretion (FIGS. 8A and 8B).
[0267] Results--rats. The lipolytic effect of human ZAG towards rat
epididymal adipocytes in comparison with isoprenaline is shown in
FIG. 11. At concentrations between 233 and 700 nM ZAG produced a
dose-related increase in glycerol release, which was attenuated by
anti-ZAG monoclonal antibody, showing the specificity of the
action. The extent of lipolysis in rat adipocytes was similar to
that previously reported in the mouse. As in the mouse, the
lipolytic effect of ZAG was completely attenuated by the
.beta.3-adrenergic receptor (.beta.3-AR) antagonist SR59230A,
suggesting that the action of ZAG was mediated through .beta.3-AR.
These results suggest that ZAG may be effective in inducing fat
loss in rats.
[0268] The effect of single daily i.v. injection of ZAG (50
.mu.g/100 g b.w.) on the body weight of mature male Wistar rats
(540.+-.83g) is shown in FIG. 12A. Compared with control rats
administered the same volume of solvent (PBS), rats administered
ZAG showed a progressive decrease in body weight, such that after
10 days, while rats treated with PBS showed a 13 g increase in body
weight, animals treated with ZAG showed a 5 g decrease in body
weight (Table 4). There was no difference in food (ZAG: 102.+-.32
g; PBS:98.+-.25 g) or water (ZAG: 135.+-.35 ml; PBS: 125.+-.25 ml)
intake between the two groups during the course of the study, but
ZAG-treated animals showed a consistent 0.4.degree. C. elevation in
body temperature, which was significant within 24 h of the first
administration of ZAG (FIG. 12B), indicating an elevated energy
expenditure. Body composition analysis (Table 4) showed that the
loss of body weight induced by ZAG was due to a loss of carcass
fat, which was partially offset by a significant increase in lean
body mass. There was a 50% increase in plasma glycerol
concentration in rats treated with ZAG (Table 5), indicative of an
increased lipolysis, but a 55% decrease in plasma levels of
non-esterified fatty acids (NEFA), suggesting an increased
utilisation. Plasma levels of glucose and triglycerides were also
reduced by 36-37% (Table 5), also suggesting an increased
utilization. There was a significant increase in the uptake of
2-deoxygluocse into epididymal adipocytes of rats treated with ZAG
for 10 days, which was increased in the presence of insulin (FIG.
12C). However, there was no significant difference in glucose
uptake into adipocytes from ZAG or PBS treated animals in the
presence of insulin (FIG. 12C). There was a small, non-significant
increase in glucose uptake into gastrocnemius muscle and BAT of
rats treated with ZAG in comparison with PBS controls, but a
significant increase in uptake in the presence of insulin (FIG.
12D). These results suggest that the decrease in blood glucose is
due to increased utilization by BAT, WAT and skeletal muscle, and
this is supported by an increased expression of glucose transporter
4 (GLUT4) in all three tissues (FIG. 13).
TABLE-US-00004 TABLE 4 Body composition of male rats after
treatment with either PBS or ZAG Starting Final Weight weight
weight change Water Fat Non fat Treatment (g) (g) (g) (g) (%) (g)
(%) (g) (%) PBS 510 .+-. 30 523 .+-. 2 +13 .+-. 3 326 .+-. 32 62
.+-. 2 105 .+-. 14 20 .+-. 3 90 .+-. 6 17 .+-. 3 ZAG 530 .+-. 45
525 .+-. 1 -5 .+-. 1 331 .+-. 5 63 .+-. 3 .sup. 92 .+-. 5.sup.b 18
.+-. 1 .sup. 96 .+-. 2.sup.a 18 .+-. 2 Differences from animals
treated with PBS are shown as .sup.ap < 0.05 or .sup.bp <
0.01
TABLE-US-00005 TABLE 5 Plasma metabolite and insulin levels in rats
treated with either PBS or ZAG for 10 days Metabolite PBS ZAG
Glucose (mmol/l) 25.5 .+-. 2.3 16.2 .+-. 2.1.sup.c Trigylcerides
(mmol/l) 1.75 .+-. 0.01 1.1 .+-. 0.09.sup.a Glycerol (umol/l) 300
.+-. 52 450 .+-. 51.sup.c NEFA (mEq/l) 0.58 .+-. 0.008 0.26 .+-.
0.06.sup.b Differences from animals treated with PBS are shown as
either .sup.ap < 0.05; .sup.bp < 0.01 or .sup.cp <
0.001
[0269] ZAG administration increased expression of the uncoupling
proteins (UCP)-1 and -3 in both BAT and WAT by almost two-fold
(FIGS. 13A and 13B), which would contribute to increased substrate
utilization. In rats treated with ZAG there was also an increased
expression of the lipolytic enzymes adipose triglyceride lipase
(ATGL) and hormone sensitive lipase (HSL) in epididymal adipose
tissue (FIG. 15), again with a two-fold increase. ATGL is mainly
responsible for the hydrolysis of the first ester bond in a
triacylglycerol molecule forming diacylgylcerol, while its
conversion to monacylglycerol is carried out by HSL. Expression of
ZAG was also significantly increased in skeletal muscle, (FIG.
16A), WAT (FIG. 16B) and BAT (FIG. 16C) of rats treated with ZAG
for 10 days, showing that exogenous ZAG boosts its own production
in peripheral tissues.
[0270] There was a significant reduction in the expression of the
phosphorylated forms of both dsRNA-dependent protein kinase (PKR)
and eukaryotic initiation factor 2 (eIF2) on the .alpha.-subunit in
gastrocnemius muscle of rats administered ZAG, while the total
amount did not change (FIGS. 17A and 17B). Similar changes have
been observed in ob/ob mice administered ZAG (unpublished results)
and were consistent with a depression of protein degradation and
increase in protein synthesis in skeletal muscle.
EXAMPLE 2
Interval Administration of Zinc-.alpha..sub.2-glycoprotein
[0271] It was observed that long-term daily administration of ZAG
in ob/ob mice results in a cessation of weight loss. As such, it
was determined that a break of 3-4 days followed by re-infusion ZAG
resulted in continued weight loss and amelioration of the symptoms
associated with hyperglycemia.
[0272] While not wanting to be limited by theory, it may be that
the subjects are receiving too much ZAG or that there is receptor
desensitization as is seen with TNF. A pilot study was performed
with 2 mice in each group to determine optimal scheduling of ZAG
delivery. An 8-10g weight loss from a 90g mouse was observed in
about 3 weeks.
[0273] Adipocytes were removed from mice after 5 days of ZAG and
their responsiveness to isoprenaline (iso) was measured after
culture in the absence of ZAG (FIG. 9). The responsiveness to iso
is higher in ZAG treated mice and this continues for a further 4
days (which was when expression of ZAG and HSL were increased) and
then falls on day 5 (when expression was not increased) down to
values of PBS control.
EXAMPLE 3
Zinc-.alpha..sub.2-glycoprotein Attenuates Muscle Atrophy in ob/ob
Mouse
[0274] This example demonstrates the mechanism by which ZAG
attenuates muscle atrophy in the ob/ob mouse using a newly
developed in vitro model (Russell et al, Exp. Cell Res. 315, 16-25,
2009). This utilizes murine myotubes subjected to high
concentrations of glucose (10 or 25 mM). As shown in FIG. 18 high
glucose stimulates an increase in protein degradation (FIG. 18A),
and depresses protein synthesis (FIG. 18B), and both of these
effects were completely attenuated by ZAG (25 .mu.g/ml). It was
therefore determined if the effect of ZAG was mediated through a
.beta.3-AR using the antagonist SR59230A. However the SR compound
(i.e., SR59230A) can also act as a .beta.-agonist, which it seemed
to do in these experiments. Thus protein degradation induced by
both 10 and 25 mM glucose was attenuated by both ZAG and the SR
compound, and the combination was additive rather than antagonistic
(FIG. 19). For protein synthesis (FIG. 20) the SR compound seems to
be similar to ZAG with no evidence of reversal, while with 10 mM
glucose the SR compound causes an increase in the depression of
protein synthesis.
EXAMPLE 4
Zinc-.alpha..sub.2-glycoprotein Attenuates ROS Formation
[0275] It has been shown that formation of reactive oxygen species
(ROS) is important in protein degradation induced by high glucose
load. The data in FIG. 21 shows that ZAG completely attenuates the
increase in ROS produced by glucose, corresponding with the
decrease in protein degradation (FIG. 18A). High glucose also
induces activation (phosphorylation) of PKR (FIG. 22A) and the
subsequent phosphorylation of eIF2.alpha. (FIG. 22B) as is seen in
skeletal muscle of ob/ob mice, which was also attenuated by ZAG.
These results suggest that this in vitro model will be useful to
study how ZAG affects muscle mass at the molecular level.
EXAMPLE 5
Zinc-.alpha..sub.2-glycoprotein Increases Insulin Tolerance
[0276] An insulin tolerance test was also carried out in ob/ob mice
administered ZAG for 3 days (FIG. 23). Animals were administered
two doses of insulin (10 and 20 U/kg) by i.p. injection and blood
glucose was measured over the next 60 min. As can be seen (FIG.
23A) animals treated with ZAG showed an increased sensitivity to
insulin (10 U/kg) than those given PBS. At the higher concentration
of insulin (20 U/kg) this difference disappeared (FIG. 23B). The
glucose disappearance curve for 20 U/kg+PBS was almost identical to
10 U/kg+ZAG, so at this dose level ZAG is reducing the requirement
for insulin by 50%, but this can be overcome by giving more
insulin.
EXAMPLE 6
Anti-Zinc-.alpha..sub.2-glycoprotein Antibodies Reduce Weight
Loss
[0277] The data shown in FIGS. 26-29 is from a study where the
.beta.3 agonist, BRL37344 was administered alone and in combination
with an anti-ZAG antibody at 50 .mu.g per day on a daily basis.
Within 24 hours of administration, mice that were administered the
antibody showed significant reduction in weight loss, as compared
to mice administered BRL37344.
EXAMPLE 7
5-day Administration of Zinc-.alpha..sub.2-glycoprotein
[0278] A 5 day study was performed where ZAG was administered at 35
.mu.g per day i.v. on a daily basis for 5 days. At the end of the
experiment tissues were removed and blotted, or functional assays
were carried out with isolated adipocytes. As can be seen in FIG.
6A, ZAG administration increased its expression in epididymal (ep),
subcutaneous (sc) and visceral (vis) fat about two-fold. When ep
adipocytes were prepared and maintained in tissue culture (RPMI
1640+10% FCS) ZAG expression was maintained for a further 3 days,
even though no ZAG was added to the culture medium (FIG. 6B). In
addition adipocytes from ZAG treated mice showed an increased
response to isoprenaline (10 .mu.M), and this was also maintained
for 4 days in tissue culture in the absence of ZAG (FIG. 9). The
increased response to isoprenaline is due to an increased
expression of HSL by ZAG, and this was also maintained in tissue
culture for 4 days in the absence of ZAG (FIG. 6C). These results
show that the effects of ZAG are maintained for a further 3 days
when ZAG is withdrawn and therefore it need not be administered on
a daily basis. In fact, as discussed above, too much ZAG is more
likely to lead to resistance rather than an increased response.
[0279] An increased expression of HSL was only seen in ep
adipocytes after 5 days ZAG (FIGS. 5B-5D), as was ATGL (FIGS.
5E-5G). There was an increase in expression of pERK only in ep
adipose tissue (FIGS. 5H-5J), and an inhibitor of pERK (PD98059 10
.mu.M) attenuated the increase in expression of HSL in ep
adipocytes incubated with ZAG for 3 h (FIG. 5A). ZAG increased
expression of UCP1 and UCP3 in BAT (FIGS. 6D and 6E) and muscle
(FIG. 6F) which would account for the increase in body temperature
and fall in TG and NEFA in serum despite the increase in
lipolysis.
EXAMPLE 8
Role of .beta.-Adrenergic Receptors in the Anti-obesity and
Anti-diabetic Effects of Zinc-.alpha..sub.2-glycoprotein
[0280] The goal of the study was to determine whether the
.beta.-adrenoreceptor (.beta.-AR) plays a role in the anti-obesity
and anti-diabetic effects of zinc-.alpha.2-glycoprotein (ZAG). This
has been investigated in CHO-K1 cells transfected with the human
.beta.1-, .beta.2-, .beta.3-AR and in ob/ob mice. In CHO-K1 cells
transfected with the .beta.3-AR the lowest concentration of ZAG to
stimulate cyclic AMP production was 350 nM, while higher
concentrations (580 nM) were required for cells transfected with
the .beta.2-AR, and there was no increase in cyclic AMP in cells
transfected with the .beta.1-AR. This correlated with the Kd values
for binding to the .beta.3-AR (46.+-.4 nM) and .beta.2-AR (71.+-.2
nM), while there was no binding to the .beta.1-AR. Freeze-thawing
of ZAG, which destroyed its biological activity eliminated binding
to .beta.2- and .beta.3-AR. Treatment of ob/ob mice with ZAG
increased protein expression of .beta.3-AR in gastrocnemius muscle,
and in white and brown adipose tissue, but had no effect on
expression of .beta.1- and .beta.2-AR. The effect of ZAG on
reduction of body weight and urinary glucose excretion, increase in
body temperature, reduction in maximal plasma glucose and insulin
levels in the oral glucose tolerance test, and stimulation of
glucose transport into skeletal muscle and adipose tissue, was
completely attenuated by the non-specific .beta.-AR antagonist
propanolol. These results evidence that the effect of ZAG on body
weight and insulin sensitivity in ob/ob mice are manifested through
a .beta.-3AR, or possibly a .beta.2-AR.
[0281] Zinc-.alpha..sub.2-glycoprotein (ZAG) was first recognised
to play a role in lipid metabolism when tryptic fragments of a
lipid mobilizing factor (LMF), thought to be responsible for loss
of adipose tissue in cancer cachexia, were shown to be identical in
amino acid sequence to ZAG. Both ZAG and LMF were shown to be
immunologically identical, and both stimulated lipolysis in murine
adipocytes by the same amount, at the same concentration, by
activation of adenylyl cyclase in a GTP-dependent process. Initial
studies suggested that ZAG originated from the tumour, since
tumours initiating cachexia showed high levels of expression, while
other tumours which did not induce cachexia showed no expression.
Later studies showed that ZAG was also produced in normal tissues
including liver, brown adipose tissue (BAT) and white adipose
tissue (WAT), so that ZAG can be classified as an adipokine.
Moreover, in both cachectic mice and humans expression of ZAG mRNA
in WAT was found to be increased 10-fold and 2.7-fold respectively.
In cachectic cancer patients ZAG mRNA showed negative correlation
with body mass index (BMI), but a positive correlation with weight
loss and serum glycerol levels. In contrast ZAG mRNA levels in WAT
have been shown to be downregulated in obesity and correlated
negatively with fat mass, BMI, plasma insulin and leptin. Treatment
of ob/ob mice with ZAG decreased body weight and fat mass and
improved the parameters of insulin resistance including decreasing
plasma levels of glucose, insulin and non-esterified fatty acids
(NEFA), improving insulin sensitivity, and increasing muscle
mass.
[0282] Serum ZAG levels have been found to be significantly lower
in mice fed a high fat diet than those fed a normal diet, as well
as in obese humans and mice. While ZAG overexpression in mice
reduced both the body weight and weight of epididymal fat, ZAG
knock-out animals showed an increased body weight, especially when
fed a high fat diet. These results suggest that ZAG, like leptin,
is closely associated with fat mass. However, while leptin is
positively correlated with fat mass, ZAG is negatively
correlated.
[0283] The lipolytic effect of ZAG was shown to be attenuated by
the .beta.3-adrenoreceptor (.beta.3-AR) antagonist, SR59230A, while
LMF has been shown to bind to the .beta.3-AR through a high
affinity binding site (Kd 78.+-.4.5 nM). These results suggest that
lipolysis mediated by ZAG is manifested through a .beta.3-AR. This
study examined the role of the .beta.3-AR in the action of ZAG, as
well as determine the binding to .beta..sub.1- and .beta..sub.2-AR,
and the role of the .beta.-AR in the anti-obesity and anti-diabetic
effects of ZAG.
[0284] FCS (foetal calf serum) was from Biosera (Sussex, UK), while
DMEM (Dulbecco's modified Eagle's medium) was from PAA (Somerset,
UK) and Freestyle media and Superscript II reverse transcriptase
were purchased from Invitrogen (Paisley, UK). 2-[1-.sup.14C]
deoxy-D-glucose sp.act. 1.85 GBq mmol.sup.-1) was purchased from
American Radiolabeled Chemicals (Cardiff, UK). Na [.sup.125I]
(specific radioactivity >17 Ci mg.sup.-1) was purchased from
Perkin Elmer Limited. Chicken polyclonal antibody to .beta.3-AR and
rabbit polyclonal antibodies to .beta.1-AR and .beta.2-AR were
purchased from Abcam (Cambridge, UK) and peroxidise-conjugated goat
anti-chicken antibody was from Santa Cruz (USA). Polyclonal rabbit
antibodies to UCP1 and UCP3 were from Calbiochem (via Merck
Chemicals, Nottingham, UK). Peroxidase-conjugated goat anti-rabbit
antibody was from Dako (Cambridge, UK). Polyclonal rabbit antibody
to mouse .beta.-actin, Tri-Reagent and propanolol were from Sigma
Aldrich (Dorset, UK). Hybond A nitrocellulose membranes were from
GE Healthcare (Bucks, UK). The Parameter cyclic AMP assay kit was
purchased from New England Biolabs (Hitchin, UK). The iodo beads
and enhanced chemiluminescence (ECL) development kits were
purchased from Thermo Scientific (Northumberland, UK). A mouse
insulin ELISA kit was purchased from DRG (Marburg, Germany) and
glucose measurements were made using a Boots (Nottingham, UK)
plasma glucose kit. Primers for reverse transcription and Easy-A
one tube RT.PCR system were from Agilent Technologies (Cheshire,
UK).
[0285] Animals. Obese (ob/ob) hyperglycaemic mice having an average
weight of 71 g were bred. The background of these animals has been
previously described (Bailey C J, et al. Influence of genetic
background and age on the expression of the obese hyperglycaemic
syndrome in Aston ob/ob mice. Int J Obes 6: 11-21, 1982), and they
exhibit a more severe form of diabetes than C57BL/6J ob/ob mice.
Male mice (about 20 weeks of age) were grouped into three per cage
and kept in an air conditioned room at 22.+-.2.degree. C., with ad
libitum feeding of a rat and mouse breeding diet (Special Diet
Services, Witham, UK) and tap water. Mice were administered ZAG (50
.mu.g, i.v. in 100 .mu.l PBS) or PBS daily with or without
propanolol (40 mgkg.sup.-1, po, daily) and body weight and food and
water intake were determined, as well as urinary glucose excretion
and body temperature, determined by the use of a rectal thermometer
(RS Components, Northants, UK). A glucose tolerance test was
performed on day 3. Glucose (1 gkg.sup.-1 in a volume of 100 .mu.l)
was administered orally to animals which had been fasted for 12 h.
Blood samples were removed from the tail vein at 15, 30, 60 and 120
min after glucose administration and used for the measurements of
glucose and insulin. At the end of the experiment the animals were
terminated by cervical dislocation, tissues removed and rapidly
frozen in liquid nitrogen, and maintained at -80.degree. C.
[0286] Production of recombinant human ZAG. Human HEK293F cells,
which had been transfected with pcDNA3.1 containing human ZAG were
maintained in Freestyle medium, containing neomycin (50
.mu.gml.sup.-1), under an atmosphere of 5% CO.sub.2 in air. After 2
weeks of growth cells were removed by centrifugation (700 g for 15
min) and the medium was concentrated into a volume of lml sterile
PBS using an Amicon Ultra-15 centrifugal filter with a M.W. cut-off
of 10 kDa. The ZAG was purified as described (Russell S T and
Tisdale M J, Antidiabetic properties of zinc-.alpha.2-glycoprotein
in ob/ob mice. Endocrinol 151: 948-957, 2010), by binding to DEAE
cellulose, since ZAG has a high electronegativity, and was eluted
with 0.3 MNaCl. The ZAG produced by this method was greater than
95% pure and was free of endotoxin, as determined by the LAL
Pyrogent single test kit (Lonza). The purified ZAG ras stored at
4.degree. C. in PBS.
[0287] [.sup.125I] labelling of ZAG. One iodo bead that had been
washed and dried was incubated with Na [.sup.125I] (1 mCi per 100
.mu.g protein) for 5 min in PBS, then ZAG (100 .mu.g protein) was
added and left for a further 15 min. The reaction was terminated by
removal of the iodo bead, while free Na [.sup.125I] was removed
using a Sephadex G25 column eluted with 0.1MNal. The [.sup.125I]
ZAG was concentrated against PBS using a Microcon microconcentrator
with a filter cut-off of Mr 10,000. The specific activity of the
[.sup.125I] ZAG was 8 Cimg protein.sup.1.
[0288] Binding studies and cyclic AMP determination. CHO-K1 cells
transfected with the human .beta.1- and .beta.2-AR obtained from
University of Nottingham, UK, while CHOK1 cells transfected with
the .beta.3-AR were obtained from Astra Zeneca, Macclesfield,
Cheshire, UK. Gene expression was under the control of hygromycin,
together with a .beta.-gal reporter construct, selected for
resistance to G418. They were maintained in DMEM supplemented with
2 mM glutamine, hygromycin B (50 mgml.sup.-1), G418 (200
mgml.sup.-1), and 10% FCS, under an atmosphere of 10% CO.sub.2 in
air. To determine the effect of agonists on cyclic AMP production,
cells were grown in 24-well plates containing lml of nutrient
medium. ZAG or isoproterenol at the concentrations shown in FIG. 51
were added to the cells and incubation was continued for 30min. The
medium was removed and replaced with 0.5 ml of 20 mM HEPES, pH 7.5,
5 mM EDTA and 0.1 mM isobutylmethylxanthine, and the plates were
heated on a boiling water bath for 5 min and cooled on ice for 10
min. The concentration of cyclic AMP was determined with an ELISA
assay.
[0289] For binding studies cells were sonicated in 2 mM Tris HCl,
pH7.5, containing 0.5M MgCl.sub.2 and crude total membranes were
pelleted by centrifugation (13,000 g; 15 min) at 4.degree. C.
Binding studies were carried out at 37.degree. C. by incubating
membranes (500 .mu.g protein) in 0.4 ml 50 mM Tris HCl, pH 7.5,
containing 0.5 mM MgCl.sub.2 for 60 min with various concentrations
of ZAG (3,000 to 15,000 cpm) in the absence or presence of 100
.mu.M non-labelled ZAG. The membranes were then precipitated by
centrifugation at 13000 g for 20 min, the supernatant was removed
and the [.sup.125I] bound to the pellet was quantitated using a
Packard Corbra Model 5005 Auto-gamma counter. Binding was analysed
using non-linear regression analysis (GraphPad Prism, Version
5.04). Specific binding was regarded as the amount of labelled ZAG
displaced by non-radioactive ZAG.
[0290] RNA isolation and RT-PCR._Quantitation of the mRNA
transcripts for .beta.1-, .beta.2- and .beta.3-AR in the three
CHO-K1 cells was based on the methodology already described
(Moniotte S, et al. Real-time RT-PCR for the detection of
beta-adrenoceptor messanger RNAs in small human endomyocardial
biopsies. J Mol Cell Cardiol 33: 2121-2133, 2001). Total RNA was
extracted with Tri Reagent and quantitated by spectrophotometry,
800.+-.34 ng total RNA was reverse transcribed, together with
2000pmol random hexamers as primers using Superscript II reverse
transcriptase at 43.degree. C. for 50 min. The probe sequences were
selected to obtain T.sub.mS approximately 10.degree. C. lower than
the matching primer pair. PCR was carried out using Easy-A one tube
RT.PCR system according to the manufacturer's instructions. The PCR
conditions included a denaturing at 95.degree. C. for 10 min, an
annealing step at 42-65.degree. C., and an extension step at
68.degree. C. for 2 min, and with a final extension at 68.degree.
C. for 10 min. There were 40 cycles of amplification. Expression of
.beta.-AR mRNA was determined by the .DELTA.-CT method using
Stratagenes MxPro, QPCR software v3.00.
[0291] Western blot analysis. WAT, BAT, heart and gastrocnemius
muscle were thawed, washed in PBS, and lysed in Phosphsafe.TM.
Extraction reagent for 5 min at room temperature, followed by
sonication at 4.degree. C. The supernatant formed by centrifugation
at 18,000 g for 5 min at 4.degree. C. was used for Western
blotting. Cytosolic protein (5 .mu.g for UCP's and 20 .mu.g for
.beta.-AR) was resolved on 12% sodium dodecylsulphate
polyacrylamide gels by electrophoresis at 180V for about 1 h and
transferred on to 0.45 .mu.m nitrocellulose membranes, which had
been blocked with 5% (w/v) non-fat dried milk (Marvel) in
Tris-buffered saline, pH 7.5, at 4.degree. C. overnight. Prior to
adding the primary antibodies membranes were washed for 15min in
0.1% Tween 20-buffered saline. Both primary and secondary
antibodies were used at a dilution of 1:1000. Incubation was for lh
at room temperature and development was by ECL. Blots were scanned
by a densitometer to quantify differences.
[0292] Glucose uptake into adipose tissue and skeletal muscle.
Uptake of 2-[1-.sup.14C] deoxy-D-glucose (2-DG) into freshly
isolated epididymal adipocytes and gastrocnemius muscle was
determined as previously described (Russell S T and Tisdale M J,
Antidiabetic properties of zinc-.alpha.2-glycoprotein in ob/ob
mice. Endocrinol 151: 948-957, 2010).
[0293] Statistical analyses. Results are shown as mean.+-.SEM for
at least three replicate experiments. Differences in means between
groups was determined by one-way analysis of variance (ANOVA)
followed by Tukey-Kramer multiple comparison test. p values
<0.05 were considered significant.
[0294] The effect of human ZAG on cyclic AMP production in CHO
cells transfected with human .beta.1, .beta.2 and .beta.3-AR is
shown in FIG. 51. At low concentrations (up to 460 nM) there was
specific stimulation of cyclic AMP production only in cells
transfected with the .beta.3-AR (FIG. 51A). However, at 580 nM
there was also a significant increase in cyclic AMP level in CHO
cells transfected with the .beta.2-AR, although the magnitude of
the change was less than in cells transfected with the .beta.3-AR.
There was no increase in cyclic AMP in CHO cells transfected with
the .beta.1-AR at any concentration of ZAG (FIG. 51A). In contrast
isoprenaline (10 .mu.M) showed significant increases in cyclic AMP
level in CHO cells transfected with .beta.1-, .beta.2- and
.beta.3-AR, showing the lack of specificity to the three isoforms
of the .beta.-AR (FIG. 51B). The increase in cyclic AMP by
isoprenaline through .beta.1-, .beta.2- and .beta.3-AR was
attenuated by SR59230A, showing a lack of specificity of this agent
to the .beta.3-AR.
[0295] To determine whether expression of the .beta.-AR was the
same in the three cell lines mRNA levels of .beta.1-AR, .beta.2-AR
and .beta.3-AR was determines by RT-PCR as described (Moniotte S,
et al. Real-time RT-PCR for the detection of beta-adrenoceptor
messanger RNAs in small human endomyocardial biopsies. J Mol Cell
Cardiol 33: 2121-2133, 2001). The data in FIG. 51C show that the
level of expression of each .beta.-AR is the same in relation to
the housekeeping gene GAPDH. Moreover, the level of adenylate
cyclase, as determined by cyclic AMP production in the presence of
forskolin, was also similar in the three cell lines (FIG. 51D).
These results suggest that a comparison between the .beta.-AR in
the three cell lines is valid.
[0296] The affinity of binding of ZAG to the three .beta.-AR was
determined using .sup.125I labelled ZAG and crude membranes from
CHO-K1, .beta.1, .beta.2 and .beta.3 cells (FIGS. 51E, F and G).
The data was evaluated using non-linear regression analysis and the
Kd and Bmax values are shown. The binding data reflect the
stimulation of cyclic AMP production by ZAG as shown in FIG. 51A.
Thus ZAG bound predominantly to .beta.3-AR (high Bmax and lowest
Kd), less so to .beta.2-AR (Bmax 20% of .beta.3-AR and Kd twice
.beta.3-AR), and not at all to .beta.1-AR (no Bmax and high Kd).
Non-specific binding was determined by the binding of [.sup.125I]
ZAG in the presence of 100 .mu.M non-labelled ZAG, and these values
were subtracted from the total binding to give the specific binding
values. The lipolytic activity of ZAG was shown to be destroyed by
a single freezing and thawing cycle (FIG. 51H), probably due to a
change in onformation of the protein. To determine whether this
disrupted binding to .beta.-AR two experiments were performed: (i)
Freeze-thaw [.sup.125I] ZAG was used in the binding studies, which
completely attenuated binding to the .beta.2- and .beta.3-AR (FIGS.
51F and G). (ii) Freeze/thawed non-labelled ZAG was used in a
competition assay with [.sup.125I]ZAG, as in the determination of
non-specific binding above. In contrast with fresh non-labelled ZAG
this had no effect on either the Kd or Bmax for binding to
.beta.2-AR or .beta.3-AR. These results suggest that freeze/thawing
ZAG destroys biological activity by preventing binding to
.beta.-AR.
[0297] To determine whether the effects of ZAG on body weight and
insulin sensitivity were due to interaction with a .beta.-AR, ob/ob
mice were treated with ZAG (50 .mu.g, iv, daily), as previously
reported (Russell S T and Tisdale M J, Antidiabetic properties of
zinc-.alpha.2-glycoprotein in ob/ob mice. Endocrinol 151: 948-957,
2010), in the absence and presence of the non-specific .beta.-AR
antagonist propanolol (40 mg kg.sup.-1, po, daily). This dose level
is higher than that commonly employed with .beta.2-AR agonists,
since higher levels are required to counteract the effect of
.beta.3-AR agonists (Liu Y L and Stock M J, Acute effects of the
beta 3-adrenoreceptor agonist, BRL 35135, on tissue glucose
utilisation. Br J Pharmacol. 114: 888-894, 1995). Propanolol
completely attenuated the decrease in body weight produced by ZAG
(FIG. 52A), although animals treated with propanolol alone did not
show such a large weight gain as did PBS controls. As previously
reported (Russell ST and Tisdale MJ, Antidiabetic properties of
zinc-.alpha.2-glycoprotein in ob/ob mice. Endocrinol 151: 948-957,
2010) mice treated with ZAG showed an increased body temperature
(FIG. 52B), and this was completely attenuated by propanolol, as
was the reduction in the urinary excretion of glucose (FIG. 52C).
ZAG alone had no effect on liver lipids, although there was some
increase in glycogen (FIG. 52D). Propanolol also blocked the
reduction in peak plasma glucose levels, and the area under the
glucose curve (AUC) induced by ZAG in the oral glucose tolerance
test (FIG. 53A), as well as the corresponding reduction in peak
plasma insulin levels (FIG. 53B). Animals treated with ZAG showed
an increased glucose uptake into gastrocnemius muscle in the
presence of insulin (10 nM) (FIG. 53C), and this was completely
attenuated in gastrocnemius muscle from mice receiving propanolol.
Epididymal adipocytes from mice treated with ZAG also showed an
enhanced glucose uptake in the absence and presence of insulin
(FIG. 53D), and this was also completely attenuated in animals
treated with propanolol. The decrease in serum levels of
triglycerides (TG) and non-esterified fatty acids (NEFA) produced
by ZAG were also attenuated by propranolol (FIGS. 53E and F).These
results suggest that the biological effects of ZAG are mediated
through a .beta.-AR. To determine whether ZAG can increase insulin
signalling the effect on Glut4 expression was determined. Both
insulin and ZAG increased expression of Glut4 in gastrocnemius
muscle (FIG. 53G) and WAT (FIG. 53H), but the combination did not
produce an increase over that of insulin alone. These results
suggest that ZAG influences the same signalling pathways as
insulin, but does not increase insulin signalling.
[0298] A number of .beta.3-agonists are known to increase
expression of the .beta.3-AR. To determine whether ZAG had the same
effect, tissue .beta.3-AR expression was quantified by Western
blotting after 5 days of treatment of ob/ob mice with ZAG. The
results in FIG. 54 show a two-fold increase in .beta.3-AR
expression in gastrocnemius muscle (FIG. 54A) an 89% increase in
BAT (FIG. 54B) and a 85% increase in WAT (FIG. 54C). In contrast
there was no change in expression of .beta.1-AR or .beta.2-AR in
either gastrocnemius muscle (FIG. 55A) or WAT (FIG. 55B) and no
change in expression of .beta.1-AR in heart (FIG. 55C), but a small
increase in .beta.2-AR which just reached significance (FIG.
55C).
[0299] The increased expression of the .beta.3-AR in BAT and WAT
(FIG. 54) would be expected to lead to an increased expression of
UCP1, which is observed in both BAT (FIG. 56A) and WAT (FIG. 56B)
after ZAG administration. In vitro experiments have shown that
induction of expression of UCP3 by ZAG was attenuated by the
mitogen activated protein kinase kinase (MAPKK) inhibitor PD98059,
suggesting the involvement of MAPK in this process. Previous
studies have shown an increase in expression of ERK in WAT of
ZAG-treated mice. This would be expected to lead to an increase in
expression of UCP3 in WAT, as was observed (FIG. 56C). It was also
previously reported that an increase in UCP3 in skeletal muscle of
ob/ob mice after administration of ZAG. The increased expression of
UCP's would provide a sink for the NEFA released from adipose
tissue, generating heat as previously reported.
[0300] In addition treatment with ZAG produced an increase in
expression of AMPK in skeletal muscle (FIG. 56D), which would lead
to an increase oxidation of long-chain fatty acids, decreasing the
availability for the synthesis of triglycerides, as well as
stimulating glucose uptake through increased expression of
GLUT4.
[0301] This study has found that ZAG binds predominantly to the
.beta.3-AR, with intermediate binding to the .beta.2-AR, and no
binding to the .beta.1-AR. The human .beta.3-AR is 51% homologous
in amino acid sequence to the .beta..sub.1-AR and 46% homologous to
the .beta.2-AR. The Bmax for ZAG binding to the .beta.3-AR is about
three times that for the .beta.2-AR, while the Kd is about half.
The Kd for ZAG for binding to the .beta.3-AR is about 100-fold
lower than that of CGP12177, a partial agonist, while the Bmax is
only slightly lower. These results were obtained using [.sup.125I]
ZAG which may have non-equivalent binding activity to native ZAG,
which could lead to over or under estimation of the Kd. While most
of the studies with ZAG have been carried out in rodents there is a
difference between human and rodent .beta.3-AR. Thus BRL37344 is
less effective at stimulating adenylyl cyclase via human than
rodent .beta.3-AR, while CGP 12177 is an effective agonist at human
.beta.3-AR, but a poor partial agonist at the rat .beta.3-AR. How
ZAG binds to the .beta.3-AR and .beta.2-AR, while not binding to
the .beta.1-AR, is not known, but the conformation of the protein
is very important, since binding is destroyed by a single
freeze/thaw cycle, as is also the ability of ZAG to stimulate
lipolysis in murine adipocytes. The Kd values are in the range
expected, both from studies on the stimulation of lipolysis, and
cyclic AMP production by ZAG. However, they are more than 10-times
lower than the human plasma concentration reported using an ELISA
(600 nM), but are comparable with that reported using mass
spectrometry (85 nM). If the former value was true ZAG would be
maximally stimulating the .beta.2- and .beta.3-AR at normal plasma
concentrations, which is clearly not correct. Care must be taken in
interpreting plasma concentrations of ZAG using an ELISA, since
there may be other components which bind to the anti-ZAG antibody,
giving apparently higher concentrations. Thus ZAG has been shown to
non-specifically bind to a monoclonal antihuman erythropoietin
antibody giving apparently higher values in samples containing
increased amounts of urinary ZAG.
[0302] The effect of ZAG on obesity and diabetes in the ob/ob mouse
model may be due to its ability to bind to .beta.3-AR. Thus
.beta.3-AR agonists show anti-obesity effects in rodent models
similar to ZAG, which induced an increased mobilisation of
triglycerides from WAT depots, increased fat oxidation, and
increased BAT-mediated thermogenesis, resulting in a selective
reduction in body fat and preservation of fat-free mass. As with
ZAG the anti-diabetic effects of .beta.3-AR agonists are
independent of the anti-obesity effects, and occur at dose levels
which do not induce weight loss. Treatment of ob/ob mice with the
.beta.3-AR agonist BRL 35135 normalised plasma glucose levels and
significantly decreased plasma insulin and non esterified fatty
acid (NEFA) levels. As with ZAG BRL 35135 stimulated Glucose uptake
into three types of skeletal muscle, BAT, WAT, heart and diaphragm,
which was independent of the action of insulin. Another
.beta.3-agonist L-796568 increased lipolysis and energy expenditure
in obese men when administered as a single dose. However, treatment
for 28 days had no major lipolytic or thermogenic effect, although
it lowered triacylglycerol concentration. This may be due to
insufficient recruitment of .beta.3-AR responsive tissues in
humans, or down-regulation of .beta.3-AR with chronic dosing.
Studies in human subcutaneous abdominal adipose tissue show that
.beta.3-AR play a weaker role in the control of lipolysis than
found in rodents, and that mobilisation of lipids is mainly through
.beta.1 and .beta.2-AR subtypes. Thus ZAG may exert its effect in
humans via a .beta.2-AR rather than .beta.3-AR.
[0303] This study has shown that propanolol, a non-specific
.beta.3-AR antagonist attenuates the effect of ZAG in reducing body
weight and urinary glucose excretion, increasing body temperature,
improving the response to glucose in the oral glucose tolerance
test and increasing glucose uptake into skeletal muscle and WAT of
ob/ob mice, when administered at high dose levels. In addition
freeze-thawing, which destroyed the ability of ZAG to induce
lipolysis in WAT, and reduce body fat in aged obese mice also
completely attenuates its ability to bind to human .beta.2- and
.beta.3-AR. These results confirm that the anti-obesity and
anti-diabetic effects of ZAG are mediated through a .beta.-AR.
[0304] This study has also shown that administration of ZAG to
ob/ob mice increases the expression of .beta.3-AR protein in BAT,
WAT and skeletal muscle. This effect is also seen with other
.beta.3-AR agonists. Thus chronic treatment of ob/ob mice with the
.beta.3-AR agonist BRL35135 resulted in a two-fold increase in
.beta.3-AR mRNA in BAT. Similar effects were reported with another
.beta.3-AR agonist CL 316,243 in Zucker fa/fa rats, and in
adipocytes of adult humans. Thus the ability of ZAG to induce
expression of the .beta.3-AR would enhance its effect on obesity
and diabetes. The reduced .beta.3-AR mediated lipolysis and fat
oxidation seen in obese subjects may be due to low levels of ZAG,
and that administration of ZAG could improve sensitivity. Certainly
ZAG administration to ob/ob mice increased sensitivity of
epididymal adipocytes to the lipolytic effect of the .beta.3-AR
agonist, BRL 37344. The ability of ZAG to induce expression of
.beta.3-AR would explain the lack of response of adipose tissue
from ZAG `knock-out mice` to the lipolytic effect of the .beta.3-AR
agonist CL316243.
[0305] Using knock-out mice the antiobesity effect of .beta.3-AR
stimulation has been shown to be through the UCP-1 dependent
degradation of fatty acids released from WAT. Until recently BAT
was considered to be restricted to rodents and neonatal humans.
However three independent studies conclusively identified BAT in
adult humans primarily behind the muscles of the lower neck and
collar bone, as well as along the spine of the chest and the
abdomen. .beta.3-AR agonists have been shown to stimulate
remodelling of WAT into BAT, determined histologically, or by the
appearance of UCP1. The appearance of UCP1 in WAT in response to
ZAG would suggest that it initiates a similar process. Previous
studies have suggested a role for the .beta.3-AR in the induction
of UCP1 by ZAG. .beta.3-AR agonists have been shown to induce
upregulation of UCP1 in BAT through stimulation of p38 mitogen
activated protein kinase (p38 MAPK) downstream of cyclic
AMP/protein kinase A, leading to activation (phosphorylation) of
peroxisome proliferator-activated receptor (PPAR) .gamma.
coactivator 1 (PCG-1.alpha.), as well as ATF-2, allowing the CRE
and PPAR elements of the UCP1 enhancer to be occupied.
[0306] ZAG is a naturally occurring ligand with selective agonist
activity towards the .beta.3-AR. Very few proteins display such
activity, although the hypotensive peptide adrenomedullin may also
activate .beta.3-AR leading to relaxation of ileal muscle. Since
ZAG is much larger than the normal catecholamine agonists it is
possible that activation occurs through allosteric modulation.
However, previous studies using LMF have shown binding to be
completely attenuated by propranolol, suggesting direct interaction
with a .beta.3-AR. It is likely that only part of the ZAG molecule
is required for binding, since evidence suggested that tryptic
fragments of a lipolytic factor (Mr about 5 kDa) were still
biologically active. It is possible that certain groups in amino
acids, such as serine hydroxyl, can mimic the hydrogen bonding
interactions seen between catecholamines and the .beta.3-AR.
Molecular modelling studies may provide further information on the
interactions involved. .beta.3-AR agonists such as BRL37344 have
been shown to increase ZAG expression in adipocytes, and induction
of ZAG expression by ZAG has also been suggested to occur through a
.beta.3-AR. Thus the .beta.3-AR is important in both the production
and biological effects of ZAG, and ZAG is a natural agonist of
.beta.2- and .beta.3-AR.
EXAMPLE 9
[0307] Use of Zinc-.alpha..sub.2-glycoprotein in Skeletal Muscle
Synthesis for Treatment of Cachexia
[0308] The goal of the study was to [explore the mechanism of net
protein gain in ob/ob mice when treated with ZAG. In some ZAG
treatment experiments it is observed that ob/ob mice lose
significant body fat but simultaneously gain a (countervailing)
amount of muscle mass as protein.
[0309] Protein synthesis was measured by the incorporation of
L-[2,6-3H] phenylalanine into acid-insoluble material with 2 h
incubation at 37.degree. C. without phenol red and saturated with
O2CO2 (95:5). The rate of protein synthesis was calculated by
dividing the amount of protein-bound radioactivity by the amount of
acid soluble radioactivity.
[0310] Protein degradation was determined by the release of
tyrosine (21) from gastrocnemius muscle over 2 h in oxygenated
Krebs-Henselit buffer containing 5 mM glucose and 0.5 mM
cycloheximide.
[0311] The results shown in FIG. 30 indicate indicate that the net
protein gain in skeletal muscle is a consequence of both a slowing
of protein degradation and an increase in protein synthesis.
EXAMPLE 10
Oral Administration of Zinc-.alpha..sub.2-glycoprotein for Weight
Loss and Reduction in Glucose
[0312] The goal of the study was to explore the ability of ZAG to
generate weight loss through fat loss and lowering of plasma and
urinary glucose levels over an extended period of time and by means
of oral administration of ZAG. Surprisingly, recombinant human ZAG
administered orally was able to generate the same set of responses
as intravenous administration of recombinant human ZAG, and was
able to do so without entering the plasma space from the digestive
space of the body. A novel mechanism of action is at work to
transduce the signal of recombinant human ZAG present in the
digestive space, causing generation of endogenous murine ZAG in the
plasma space and WAT and other tissues, as seen in FIGS. 34 and
35.
[0313] 50 ug per day of rhZAG was administered p.o. to Aston ob/ob
mice. Oral dosing was achieved by assuming 5 mL/day consumption of
water, and adding ZAG to achieve 50 ug per day dose based on that
assumption. No attempt was made to correct for variations of
consumption on a given day.
[0314] ZAG administration p.o generally duplicates the results
obtained by i.v. administration. This wide range of effects
includes significant weight loss (FIGS. 31, 36, 41, 47), a slight
increase in body temperature emblematic of increased energy
expenditure (FIGS. 32, 37, 43), a lowering of urinary and plasma
glucose (FIGS. 33, 42), and a significant improvement in response
to the oral glucose tolerance test (FIGS. 40, 48).
[0315] The mechanism of action mirrors that of intraveneous
injection, with a critical difference. The mechanisms are similar
in that there is a wide-ranging set of responses in WAT, BAT,
plasma, liver and skeletal muscle that are identical. The critical
difference is that the orally-administered rhZAG never enters the
space occupied by blood and the body's organs. Instead the
administered rhZAG remains in the digestive system space,
persisting 24 hours or longer in the stomach. The surprising and
critical difference in mechanism is that the animal responds to
oral dosing of rhZAG by creating its own endogenous ZAG, which
mediates the subsequent set of responses named above.
[0316] Three experiments are described in detail below.
[0317] Experiment One (8 day oral ZAG study): 50 ug of ZAG was
administered p.o. daily in drinking water. It was observed that
weight loss, increase in body temperature and a lowering of urinary
glucose occurred. An increase in murine ZAG in serum and WAT, but
an absence of rhZAG in serum demonstrates that rhZAG administered
orally is upregulating expression of mouse ZAG.
[0318] Experiment Two (8 day oral ZAG plus propranolol study): 50
ug of ZAG administered p.o. daily, with and without propranolol.
Propanolol blocks all of the effects of ZAG including decrease in
body weight and blood glucose in the tolerance test, also blocking
the rise in body temperature and the rise in plasma mouse ZAG,
confirming that this occurs through a beta adrenergic receptor.
Propranolol totally attenuates weight loss by ZAG as well as the
increase in body temperature. It appears to do this by preventing
the rise in mouse ZAG in the serum after oral administration of the
rhZAG. The second blot shows there is no rhZAG in the serum, as
would be expected if rhZAG remains sequestered in the GI tract
without transfer to the bloodstream.
[0319] Thus oral ZAG works by binding to GI tract beta adrenergic
receptors, leading to a rise in serum ZAG and the consequent
effects on body weight and blood glucose. FIG. 38 shows that
propranolol blocks the increase in murine serum ZAG due to
treatment with rhZAG p.o. Additionally, FIG. 39 shows that human
ZAG is not detected in mouse serum.
[0320] Experiment Three (Oral Study): ZAG administered p.o. daily
over an extended time frame, with a recovery group split from the
treated group beginning at 30 days (data not shown).
[0321] Weight loss, body temperature and decrease in urinary
glucose mirror and extend results achieved by intravenous
injection. Animals lost as much as 13.5% body weight at half way
through the study. After half study duration of treatment, treated
animals showed the following. In urinary glucose, 12 days passed
before 50% reversion to control occurred, and complete reversion to
control levels of urinary glucose occured by the end of the study
duration (data not shown). Body weight loss reached 13.5%, and at
day study end the animals had reverted only 46% towards the control
weights (data not shown). Like the action of ZAG when administered
intravenously, orally-administered ZAG caused weight loss but not
changes in activity (not shown), consumption of food (data not
shown) or consumption of water (data not shown).
EXAMPLE 11
[0322] Administration of Zinc-.alpha..sub.2-glycoprotein Achieves
Loss of Body Fat and a Simultaneous Gain in Muscle Mass in Skeletal
Muscles
[0323] In some experiments it has been observed that the ob/ob mice
will lose significant body fat but simultaneously gain a
(countervailing) amount of muscle mass as protein. This has been
explored and the net protein gain is due to a slowing of protein
degradation and concomitant increase in protein synthesis (FIG.
30).
EXAMPLE 12
Oral Administration of Zinc-.alpha..sub.2-glycoprotein Compared to
I.V. Administration
[0324] The goal of the study was to compare the efficacy of ZAG via
various routes of administration. Mice were orally administered 50
.mu.g ZAG or PBS (control). The results of are shown in FIGS. 31,
32, 33 (8 day oral ZAG study); 36, 37, 40 (8 day oral ZAG plus
propranolol study); and FIGS. 47, 48 (oral ZAG gavage study). As
shown in these repeated studies, ZAG was unexpectedly shown to be
effective in bringing about weight change when administered orally
by simply mixing low doses of ZAG in the drinking water of mice
without requiring systemic absorption of administered ZAG Typical
oral dosing of proteins, such as insulin, can require up to
10.times. (or mega dosing) the intravenous dose to achieve the same
level of efficacy and such limited efficacy requires systemic
absorption of such proteins.
[0325] Additional data was generated (Table 6) showing that,
surprisingly, oral dosing of ZAG achieved as much as 75% of the
weight-loss efficacy of intravenous administration with exactly the
same dose. Also, after 5 days of dosing, the efficacy of lowering
of urinary glucose is equally as good when dosed orally or i.v.
[0326] Oral dosing with rhZAG causes the animals to generate
endogenous ZAG in response, as shown in FIGS. 34, 35 and 38.
Propranolol blocks the increase in murine serum ZAG due to
treatment with rhZAG p.o. (FIG. 38), but administered human ZAG is
not found in plasma (FIG. 39).
[0327] FIG. 38 is a Western blot of ZAG using Anti-mouse ZAG in
mouse serum from Mice treated with and without ZAG in the absence
or presence of propranonol. Human ZAG is not detected in mouse
serum. FIG. 39 is a Western blot of ZAG using Anti-human ZAG in
mouse serum from Mice treated with and without ZAG in the absence
or presence of propranonol (FIG. 39).
TABLE-US-00006 TABLE 6 Body weight loss (and % of i.v. loss over
the same time) due to daily dosing of ZAG at 50 ug/day in 70 g
ob/ob mice by ROA for 8 or 20 days: ROA 8 Days 20 Days intraveneous
-6.0% (100%) -9.0% (100%) oral-water(1) -4.3% (72%) -6.1% (68%)
oral-gavage(2) -2.3% (38%) N/A oral-casein(3) -3.4% (57%) -6.8%
(76%) (1)Literally in the drinking water (2)Gavage places the ZAG
dose directly into the stomach, bypassing the digestive system path
preceding the stomach (mouth, pharynx, esophagus) (3)Casein was
included with the ZAG in the drinking water.
[0328] Although the invention has been described with reference to
the above example, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
71276PRTHomo sapiens 1Gln Glu Asn Gln Asp Gly Arg Tyr Ser Leu Thr
Tyr Ile Tyr Thr Gly 1 5 10 15 Leu Ser Lys His Val Glu Asp Val Pro
Ala Phe Gln Ala Leu Gly Ser 20 25 30 Leu Asn Asp Leu Gln Phe Phe
Arg Tyr Asn Ser Lys Asp Arg Lys Ser 35 40 45 Gln Pro Met Gly Leu
Trp Arg Gln Val Glu Gly Met Glu Asp Trp Lys 50 55 60 Glu Asp Ser
Gln Leu Gln Lys Ala Arg Glu Asp Met Glu Thr Leu Lys 65 70 75 80 Asp
Ile Val Glu Tyr Tyr Asn Asp Ser Asn Gly Ser His Val Leu Gln 85 90
95 Gly Arg Phe Gly Cys Glu Ile Glu Asn Asn Arg Ser Ser Gly Ala Phe
100 105 110 Trp Lys Tyr Tyr Tyr Asp Gly Lys Asp Tyr Ile Glu Phe Asn
Lys Glu 115 120 125 Ile Pro Ala Trp Val Pro Phe Asp Pro Ala Ala Gln
Ile Thr Lys Gln 130 135 140 Lys Trp Glu Ala Glu Pro Val Tyr Val Gln
Arg Ala Lys Ala Tyr Leu 145 150 155 160 Glu Glu Glu Cys Pro Ala Thr
Leu Arg Lys Tyr Leu Lys Tyr Ser Lys 165 170 175 Asn Ile Leu Asp Arg
Gln Asp Pro Pro Ser Val Val Val Thr Ser His 180 185 190 Gln Ala Pro
Gly Glu Lys Lys Lys Leu Lys Cys Leu Ala Tyr Asp Phe 195 200 205 Tyr
Pro Gly Lys Ile Asp Val His Trp Thr Arg Ala Gly Gln Val Gln 210 215
220 Glu Pro Glu Leu Arg Gly Asp Val Leu His Asn Gly Asn Gly Thr Tyr
225 230 235 240 Gln Ser Trp Val Val Val Ala Val Pro Pro Gln Asp Thr
Ala Pro Tyr 245 250 255 Ser Cys His Val Gln His Ser Ser Leu Ala Gln
Pro Leu Val Val Pro 260 265 270 Trp Glu Ala Ser 275 24PRTArtificial
sequenceSynthetic construct 2Lys Lys Val Tyr 1 34PRTArtificial
sequenceSynthetic construct 3Asp Gly Val Asp 1 45PRTArtificial
sequenceSynthetic construct 4Ile Glu Phe Thr Asp 1 5
54PRTArtificial sequenceSynthetic construct 5Asp Glu Val Asp 1
65PRTArtificial sequenceSynthetic construct 6Ile Glu Phe Thr Asp 1
5 75PRTArtificial SequenceSynthetic construct peptide linker 7Gly
Gly Gly Gly Ser 1 5
* * * * *