U.S. patent application number 17/530890 was filed with the patent office on 2022-07-07 for targeted oesophageal administration of zn-alpha2-glycoproteins (zag), methods and formulations thereof.
The applicant listed for this patent is Aston University. Invention is credited to Steven Russell, Michael Tisdale.
Application Number | 20220211809 17/530890 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220211809 |
Kind Code |
A1 |
Tisdale; Michael ; et
al. |
July 7, 2022 |
TARGETED OESOPHAGEAL ADMINISTRATION OF ZN-ALPHA2-GLYCOPROTEINS
(ZAG), METHODS AND FORMULATIONS THEREOF
Abstract
The invention provides formulations and methods for ameliorating
symptoms associated with metabolic disorders, such as hypoglycemia,
obesity, diabetes, and the like by targeted administration to the
oesphagus of a subject of 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;
(Warwickshire, GB) ; Russell; Steven; (West
Midlands, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aston University |
Birmingham |
|
GB |
|
|
Appl. No.: |
17/530890 |
Filed: |
November 19, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16192105 |
Nov 15, 2018 |
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17530890 |
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15175579 |
Jun 7, 2016 |
10172917 |
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16192105 |
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14418912 |
Jan 30, 2015 |
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PCT/GB2013/052039 |
Jul 31, 2013 |
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15175579 |
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61677984 |
Jul 31, 2012 |
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International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 47/56 20060101 A61K047/56; A61K 45/06 20060101
A61K045/06; A61K 9/00 20060101 A61K009/00; A61K 31/138 20060101
A61K031/138 |
Claims
1. A method of delivering a therapeutic agent to a mammal in need
thereof, comprising targeting the therapeutic agent to a receptor
in the oesophagus of the subject, wherein the therapeutic agent is
zinc-.alpha..sub.2-glycoprotein (ZAG) or a functional fragment
thereof, and is administered in combination with a .beta.3 agonist
that specifically targets .beta.3-adrenergic receptor (.beta.3-AR)
thereby increasing specific binding of the therapeutic agent to the
receptor, thereby delivering the therapeutic agent to the
subject.
2. The method of claim 1, wherein the therapeutic agent is
administered directly to the oesophagus, or delivered to the
oesophagus via oral, buccal, sublingual, or intranasal delivery
routes.
3. The method of claim 1, wherein the subject has one or more
symptoms associated with muscle wasting, sarcopenia, diabetes,
cachexia, muscle loss, lipidystrophy, obesity or overweight,
including diseases associated with insulin resistance,
hypoglycemia, elevated plasma levels of free fatty acids (NEFA),
triglycerides, or glucose.
4. The method of claim 1, wherein the ZAG is mammalian.
5. The method of claim 2, wherein the ZAG is human.
6. The method of claim 1, wherein the mammal is human.
7. The method of claim 5, wherein the ZAG consists of the amino
acid sequence set forth in SEQ ID NO: 1.
8. The method of claim 4, wherein the ZAG is conjugated to a
non-protein polymer comprising sialylated, pegylated, or modified
to increase solubility or stability.
9. The method of claim 1, wherein the therapeutic agent is
administered in combination with a glycemic reducing agent.
10. The method of claim 1, wherein the therapeutic agent is
formulated with one or more of the following: micronutrients,
dietary supplements, nutrients, edible compounds and flavorings,
excipients selected from the group consisting of phosphate, Tris,
arginine, glycine, Tween 80, sucrose, trehalose, mannitol, casein
proteins, and derivatives thereof.
11. A method for increasing a mammal's endogenous expression of a
zinc-.alpha..sub.2-glycoprotein (ZAG), the method comprising
administering to the oesophagus of the subject a therapeutic agent,
wherein the therapeutic agent is zinc-.alpha..sub.2-glycoprotein
(ZAG) or a functional fragment thereof, in combination with a
.beta.3 agonist that specifically targets .beta.3-adrenergic
receptor (.beta.3-AR) thereby increasing specific binding of the
therapeutic agent to the receptor, thereby increasing the mammal's
endogenous expression of ZAG.
12. The method of claim 11, wherein the therapeutic agent is
administered directly to the oesophagus, or delivered to the
oesophagus via oral, buccal, sublingual, or intranasal delivery
routes.
13. The method of claim 11, wherein the mammal has one or more
symptoms associated with muscle wasting, sarcopenia, diabetes,
cachexia, muscle loss, lipidystrophy, obesity or overweight,
including diseases associated with insulin resistance,
hypoglycemia, elevated plasma levels of free fatty acids (NEFA),
triglycerides, or glucose.
14. The method of claim 11, wherein the ZAG is mammalian.
15. The method of claim 12, wherein the ZAG is human.
16. The method of claim 11, wherein the mammal is human.
17. The method of claim 15, wherein the ZAG consists of the amino
acid sequence set forth in SEQ ID NO: 1.
18. The method of claim 14, wherein the ZAG is conjugated to a
non-protein polymer comprising sialylated, pegylated, or modified
to increase solubility or stability.
19. The method of claim 11, wherein the therapeutic agent is
administered in combination with a glycemic reducing agent.
20. The method of claim 11, wherein the therapeutic agent is
formulated with one or more of the following: micronutrients,
dietary supplements, nutrients, edible compounds and flavorings,
excipients selected from the group consisting of phosphate, Tris,
arginine, glycine, Tween 80, sucrose, trehalose, mannitol, casein
proteins, and derivatives thereof.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application is a continuation of U.S. Ser. No.
16/192,105, filed Nov. 15, 2018, now pending, which is a
continuation of U.S. Ser. No. 15/175,579, filed Jun. 7, 2016, now
abandoned, which is a continuation of U.S. Ser. No. 14/418,912,
filed Jan. 30, 2015, now abandoned, which is a 35 USC .sctn. 371
National Stage application of International Application No.
PCT/GB2013/052039, filed Jul. 31, 2013, now expired, which claims
the benefit of priority under 35 U.S.C. .sctn. 119(e) of U.S. Ser.
No. 61/677,984, filed Jul. 31, 2012, the entire content of each of
which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates generally to medicinal
formulations, and more particularly, to formulations and methods
for altering the metabolism of a subject, as well as ameliorating
disorders such as obesity, diabetes and insulin resistance.
Background Information
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] ZAG has previously been investigated as a treatment for
obesity and type 2 diabetes. ZAG is a soluble protein of Mr41 kDa,
which resembles a class 1 major histocompatability complex (MHC)
heavy chain, and has a major groove capable of binding hydrophobic
molecules, that could be important in its action. ZAG was first
identified as the lipid mobilising factor in cancer cachexia
following its isolation from the cachexia-inducing MAC16 tumour,
and from the urine of cachectic patients. Treatment of either aged,
or obese mice with ZAG produced a time-dependent decrease in body
weight through specific loss of carcass lipid, while there was an
expansion of the non-fat carcass mass. ZAG is produced by a range
of tissues including white (WAT) and brown (BAT) adipose tissue,
liver, heart, lung and skeletal muscle, as well as certain tumours
that induce cachexia. Expression of ZAG mRNA in adipose tissue is
high in cancer cachexia, where lipid stores are low, and low in
obesity, where lipid stores are high. Thus ZAG expression is
negatively correlated with BMI and fat mass. ZAG expression is
negatively regulated by tumour necrosis factor-.alpha.
(TNF-.alpha.), and positively regulated by the PPAR.gamma. agonist
rosiglitazone, .beta.3-adrenergic receptor ((.beta.3-AR) agonists
and glucocorticoids. ZAG also induces its own expression in adipose
tissue through interaction with a .beta.3-AR. In this way
extracellular ZAG can induce expression of intracellular ZAG in
target tissues, which has been suggested to be more important
locally than circulating ZAG.
[0011] Previously studies have administration ZAG by either the
i.p., or i.v. routes. However, neither route is convenient for
clinical use. There remains a lack of effective and safe
alternatives for altering metabolism and treatment of metabolic
diseases, such as obesity and diabetes. There is therefore a need
for new formulations for such uses which provide specific targeting
of therapeutics.
SUMMARY OF THE INVENTION
[0012] The present invention is based in part on the finding that
ZAG has the ability to induce its own expression through binding
.beta.3-ARs present in the oesophagus thereby enabling activation
of the therapeutic effect of ZAG before being digested in lower
regions of the gastrointestinal tract. Such a finding is useful in
methods of targeting the oesophagus for moderating body weight,
improving insulin responsiveness or ameliorating the symptoms
associated with diabetes.
[0013] In one embodiment the present invention provides a
formulation including zinc-.alpha..sub.2-glycoprotein (ZAG), a ZAG
variant, a modified ZAG, or a functional fragment thereof, wherein
the formulation is formulated to specifically target
.beta.3-adrenergic receptors (.beta.3-ARs) of the oesophagus to
increase specific binding of ZAG, the ZAG variant, the modified
ZAG, or the functional fragment thereof with a .beta.3-adrenergic
receptor (.beta.3-AR) of the oesophagus thereby providing targeted
delivery of the formulation to the oesophagus.
[0014] In another embodiment, the invention provides a method of
delivering a therapeutic agent to a subject. The method includes
targeting the therapeutic agent to a receptor in the oesophagus of
the subject, wherein the therapeutic agent is
zinc-.alpha..sub.2-glycoprotein (ZAG), a ZAG variant, a modified
ZAG, or a functional fragment thereof formulated to specifically
target the receptor to increase specific binding of the therapeutic
agent to the receptor, and wherein the receptor is a
.beta.3-adrenergic receptor (.beta.3-AR), thereby delivering the
therapeutic agent to the subject.
[0015] In another embodiment, the invention provides a method for
delivering a zinc-.alpha..sub.2-glycoprotein (ZAG) to a mammalian
subject, the method including administering to the oesophagus of
the subject the formulation as described herein.
[0016] 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 oesophagus of the subject the formulation as
described herein.
[0017] In a further aspect, the present invention provides a method
of ameliorating symptoms of diabetes or obesity in a mammalian
subject. The method includes administering to the oesophagus of the
subject a therapeutically effective dosage of a formulation as
described herein. In one embodiment, the formulation may be
administered in combination with a glycemic reducing agent selected
from insulin, glucagon-like peptide-1 (GLP-1), or analogs thereof
in any sequence or simultaneously.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1C are graphical diagrams showing the effect of
human ZAG on body weight (A), rectal temperature (B) and urinary
glucose excretion (C) in ob/ob mice. ZAG was dissolved in the
drinking water so that animals consumed 50 .mu.gday.sup.-1
(.box-solid.), while a control group received an equal volume of
PBS (.diamond-solid.) (0.5 ml in 5 ml water). Differences from PBS
controls are shown as ***, p<0.001.
[0019] FIGS. 2A-2G are graphical diagrams showing the effect of
orally administered human ZAG (.box-solid.) compared with PBS
(.diamond-solid.) on glucose and insulin tolerance of ob/ob mice
after 3 days of treatment. Animals were fasted for 12 h before oral
administration of glucose (1 gkg.sup.-1 in a volume of 100 .mu.l).
Blood samples were removed from the tail vein at the time intervals
shown and used for the measurement of serum glucose (FIG. 2A) and
insulin (FIG. 2B). The inset in (FIG. 2A) shows the total area
under the glucose curves (AUC) in arbitrary units. Differences from
PBS controls are shown as ***, p<0.001. Effect of propranolol
(40 mgkg.sup.-1, po, daily) on ZAG-induced reductions in obesity
and diabetes in ob/ob mice. Animals received ZAG (50 .mu.g daily)
in their drinking water as described in the legend to FIG. 1,
either alone (.box-solid.) or in the presence of propranolol
(.tangle-solidup.), while a control group received PBS
(.diamond-solid.). Changes in body weight (FIG. 2C), rectal
temperature (FIG. 2D), and urinary glucose excretion (FIG. 2E) were
monitored over an 8 day period. A glucose tolerance test (FIG. 2F),
with measurement of serum insulin levels (FIG. 2G) was made 3 days
after starting the oral ZAG. Differences from PBS controls are
shown as ***, p<0.001, while differences from ZAG alone are
shown as #, p<0.001.
[0020] FIG. 3A is a pictorial representation of a SDS/PAGE of
purified biosynthetically labelled [.sup.14C] ZAG (15 .mu.g) and
serum from ob/ob mice administered [.sup.14C] ZAG (50 .mu.g; 212
.mu.Ci.mu.mol.sup.-1) orally for 24 h.
[0021] FIG. 3B is a pictorial representation of a western blot of
serum from ob/ob mice administered non-radioactive ZAG for 8 days
in the absence or presence of propanolol (40 mg kg.sup.-1) using
anti-human ZAG monoclonal antibody.
[0022] FIG. 3C is a graphical representation of the effect of a
tryptic digest of ZAG in comparison with intact ZAG on cyclic AMP
production by CHO cells transfected with human .beta.1-AR
(.box-solid.), .beta.2-AR(.quadrature.) and .beta.3-AR) () after 30
min incubation. ZAG (1 mg) was incubated with trypsin (200 .mu.g)
in 1 ml 10 mM Tris.HCl, pH 8 for 4 h at 37.degree. C. and
proteolysis was terminated by addition of the trypsin inhibitor
(200 .mu.g). High molecular weight material was removed by a
Sephadex G25 column followed by dialysis using an Amicon filtration
cell containing a 10 kDa cut-off membrane filter.
[0023] FIGS. 4A-4C are a series of pictorial representation of a
western blots of ZAG. FIG. 4A shows expression of murine ZAG in
serum of ob/ob mice administered human ZAG or PBS orally for 8 days
as shown in FIG. 1. Each lane is a sample from an individual mouse.
The blot was probed with anti-mouse ZAG antibody. FIG. 4B shows
human ZAG was electrophoretically blotted, and probed with
antibodies specific to human and mouse ZAG. FIG. 4C shows
expression of ZAG in WAT quantitated using an anti-mouse ZAG
antibody after 8 days treatment with human ZAG Differences from PBS
treated animals are shown as ***, p<0.001.
[0024] FIGS. 5A-5D are a series of graphical and pictorial
representations showing the effect of propanolol on the stimulation
of glucose uptake into WAT, BAT and skeletal muscle of ob/ob mice
ex vivo after administration of ZAG.
[0025] FIG. 5A is a graphical representation showing glucose uptake
into epididymal (ep), subcutaneous (sc) and visceral (vis)
adipocytes from animals treated with PBS and ZAG with or without
propanolol (Prop) for 8 days in the absence (closed bars), or
presence (open bars) of insulin (10 nM).
[0026] FIG. 5B is a graphical representation showing glucose uptake
into brown adipocytes from mice treated with PBS, ZAG or
ZAG+propanolol for 8 days with or without insulin (10 nM).
[0027] FIG. 5C is a graphical representation showing glucose uptake
into isolated gastrocnemius muscle of ob/ob mice administered
either PBS or ZAG with or without propanolol for 8 days.
[0028] FIG. 5D is a pictorial representation showing quantitation
of serum ZAG in mice treated with PBS, ZAG or ZAG+propanolol for 3
days by immunoblotting using an anti-mouse ZAG monoclonal antibody.
Each lane represents serum from an individual mouse. Differences
from PBS treated animals are shown as *, p<0.05 or ***,
P<0.001, while differences from ZAG treated animals are shown as
##, p<0.001.
[0029] FIGS. 6A-6C are a series of pictorial representations
showing ZAG gene expression in mouse tissues examined by
RT-PCR.
[0030] FIG. 6A shows tissue specificity of expression from mice
treated with ZAG orally.
[0031] FIG. 6B shows ZAG expression in control mice.
[0032] FIG. 6C shows ZAG mRNA expression in mouse tissue after
either oral administration of ZAG (.box-solid.) or PBS
(.quadrature.). Differences from PBS treated animals are shown as
***, P<0.001.
[0033] FIG. 7 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."
DETAILED DESCRIPTION OF THE INVENTION
[0034] The present invention is based on the observation that
Zinc-.alpha..sub.2-glycoprotein (ZAG) binds .beta.3-AR in the
oesophagus to induce its own expression, as opposed to being
degraded in the lower regions of the gastrointestinal tract. As
such, the invention provides methods and formulation for targeted
delivery of ZAG to .beta.3-AR receptors of the oesophagus to treat
a variety of disorders.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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 its 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.
[0041] 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.
[0042] 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.
[0043] As discussed above, the present invention includes use of a
function fragment of a 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 temperature, improving glucose tissue uptake,
increasing expression of Bet3 receptors, increasing expression of
ZAG, increasing expression of Glut 4, and/or increasing expression
of UCP 1 and UCP 3. 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.
[0044] 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.
[0045] Accordingly, in one aspect, the invention provides a method
of ameliorating the symptoms of insulin resistance, obesity or
diabetes 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 following treatment. In another
embodiment, the treatment regimen is about 21 days wherein there is
amelioration of symptoms following treatment.
[0046] 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).
[0047] 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 (33 agonist therapeutic agents were found to be
efficacious but had limited bioavailability in the plasma
space.
[0048] A number of formulations are provided herein for
specifically targeting .beta.3-ARs of the oesophagus. A formulation
can be in any form which facilitates increased binding of ZAG with
.beta.3-ARs of the oesophagus, e.g., liquid, gel, suspension, or
emulsion. A formulation typically will include one or more
compositions that have been purified, isolated, or extracted (e.g.,
from plants) or synthesized.
[0049] 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.
[0050] In one embodiment the present invention provides a
formulation including zinc-.alpha..sub.2-glycoprotein (ZAG), a ZAG
variant, a modified ZAG, or a functional fragment thereof, wherein
the formulation is formulated to specifically target
.beta.3-adrenergic receptors (.beta.3-ARs) of the oesophagus to
increase specific binding of ZAG, the ZAG variant, the modified
ZAG, or the functional fragment thereof with a .beta.3-adrenergic
receptor (.beta.3-AR) of the oesophagus thereby providing targeted
delivery of the formulation to the oesophagus. 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.
[0051] 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.
[0052] 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.
[0053] In some embodiments, the formulations of the invention are
intended to be administered to the oesophagus daily. 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] ZAG 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 oesophagus
of a 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.
[0058] 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.
[0059] 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.
[0060] ZAG administration to rats has also been shown to increase
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.
[0061] 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 UCP 1, 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.
There was also a three-fold increase in oxygen uptake by BAT of
ob/ob mice after ZAG administration.
[0062] 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) faun 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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 CL316,243. 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 CL316,243 has
been shown to increase fat oxidation in healthy young male
volunteers. This may be due to the ability of .beta.3-adrenergic
agonists to increase the number of .beta.3-AR in plasma membranes
from BAT.
[0067] 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.
[0068] 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.
[0069] 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. Pharmaceutically acceptable carriers useful for
formulating a composition for administration to the oesophagus of a
subject 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 Ca.sup.2+,
Mg.sup.2+, Na.sup.+, NH.sub.4.sup.+, etc.), provided that the
carrier is compatible with the desired route of administration
(e.g., bucal, oral, sublingual, etc.).
[0070] 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.
[0071] Formulations of the present invention are formulated for
specifically targeting the oesophagus of a subject. Such
formulation may be presented as rapid-melt oral formulations,
lozenges, or suspensions of the active compound in an aqueous
liquid or non-aqueous liquid such as a syrup, an elixir, or an
emulsion.
[0072] 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.
[0073] In one embodiment, the formulation of the present invention
is administered directly to the oesophagus or to the oesophagus via
oral, sublingual, bucal or intranasal routes. In such embodiments,
the formulation is at least 70, 75, 80, 85, 90, 95 or 100% as
effective as any other route of administration.
[0074] 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, for example by bolus or 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.
[0075] 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, 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.
[0076] 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
Targeted Administration of Zinc-.alpha..sub.2-Glycoprotein to the
Oesophagus
[0077] In this example it is shown that targeted administration of
human ZAG which is a 41 kDa protein, specifically to the
oesophagus, as opposed to other regions of the gastrointestinal
tract, of ob/ob mice at 50 .mu.g day.sup.-1po in drinking water
produced a progressive loss of body weight (5 g after 8 days
treatment), together with a 0.5.degree. C. increase in rectal
temperature, and a 40% reduction in urinary excretion of glucose.
There was also a 33% reduction in the area under the curve during
an oral glucose tolerance test and an increased sensitivity to
insulin. These results were similar to those after iv
administration of ZAG. However, tryptic digestion was shown to
inactivate ZAG. There was no evidence of human ZAG in the serum,
but a 2-fold elevation of murine ZAG, which was also observed in
target tissues such as white adipose tissue. To determine whether
the effect was due to interaction of the human ZAG with the
.beta.-adrenoreceptor (.beta.-AR) in the gastrointestinal tract
before digestion, ZAG was co-administered to ob/ob mice together
with propanolol (40 mgkg.sup.-1), a non-specific .beta.-AR
antagonist. The effect of ZAG on body weight, rectal temperature,
urinary glucose excretion, improvement in glucose disposal and
increased insulin sensitivity were attenuated by propanolol, as was
the increase in murine ZAG in the serum. These results suggest that
oral administration of ZAG increases serum levels through
interaction with a .beta.-AR in the upper gastrointestinal tract,
and gene expression studies showed this to be specifically in the
oesophagus.
[0078] The following materials and methods were utilized.
[0079] Materials--FCS (foetal calf serum) was from Biosera (Sussex,
UK), while DMEM (Dulbecco's modified Eagles Medium) was from PAA
(Somerset, UK) and Feestyle medium was purchased from Invitrogen
(Paisley, UK). Hybond A nitrocellulose membranes and
peroxidase--conjugated rabbit anti-mouse antibody were from GE
Healthcare (Bucks, UK), while enhanced chemiluminescene (ECL)
development kits were purchased from Thermo Scientific
(Northumberland, UK). Mouse monoclonal antibodies to full-length
human and mouse ZAG were from Santa Cruz Biotechnology (Santa Cruz,
Calif. A mouse insulin ELISA kit was purchased from DRG (Marburg,
Germany) and glucose measurements in both urine and plasma were
made using a Boots (Nottingham, UK) glucose kit. L-[U-.sup.14C]
tyrosine (sp.act 16.7 GBqmmol.sup.-1) was purchased from Perkin
Elmer Ltd, (Cambridge, UK), while 2-[1-.sup.14C] deoxy-D-glucose
(sp.act 1.85 G Bqmmol.sup.-1) was from American Radiolabeled
Chemicals (Cardiff, UK).
[0080] Production and purification of ZAG--Recombinant human ZAG
was produced by HEK293F cells transfected with pcDNA3.1 containing
human ZAG (1). Cells were grown for 2 weeks in Freestyle medium
containing neomycin (50 .mu.gml.sup.-1) under an atmosphere of 5%
CO.sub.2 in air. The cells were then removed by centrifugation (700
g for 15 min), 1 litre of medium was concentrated to 1 ml, and the
ZAG was extracted by binding to activated DEAE cellulose, following
by elution with 0.3M NaCl before washing and concentrating with
sterile PBS. The ZAG produced was greater than 95% pure mainly due
to ZAG's negative charge, as determined by sodium dodecylsulphate
polyacrylamide electrophoresis (SDS PAGE), and was free of
endotoxin (1). For [.sup.14C] ZAG L-[U-.sup.14C] tyrosine was added
to the media (1 .mu.Ciml.sup.-1), the cells were allowed to grow
for 2 weeks and ZAG was purified as above. The specific activity of
the ZAG was 221 .mu.Ci.mu.mol.sup.-1, and the purity of the product
is shown in FIG. 3A.
[0081] Cyclic AMP determination--CHOK1 cells transfected with human
.beta.1-, .beta.2- and .beta.3-AR were maintained in DMEM
supplemented with 2 mM glutamine, hygromycin B (50 .mu.gml.sup.-1),
G418 (200 mgml.sup.-1) and 10% FCS, under an atmosphere of 10%
CO.sub.2 in air. For cyclic AMP production cells were grown in
24-well plates in 1 ml nutrient medium, and ZAG, after tryptic
digestion as described in the legend to FIG. 3C, was incubated for
30 min. The medium was then removed and 0.5 ml of 20 mM HEPES, pH
7.5, 5 mM EDTA and 0.1 mM isobutylmethylxanthine was added,
followed by heating on a water bath for 5 min, and cooling on ice
for 10 min. The concentration of cyclic AMP was determined using a
Parameter cyclic AMP assay kit (New England Biolabs, Hitchin,
Herts, UK).
[0082] Animals--Obese (ob/ob) mice (average weight 65 g) were bred
in a colony, and were 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. These
animals exhibit a more severe form of diabetes then C57BL/6J ob/ob
mice, and the origins and characteristics of the Aston ob/ob mouse
has been previously described. Animals were grouped (n=5) to
receive either ZAG/PBS (50 .mu.g day.sup.-1), or PBS in their
drinking water, the experiment was repeated three times after a
power analysis was performed. Each mouse consumed 5 ml day.sup.-1
water, and this did not change on ZAG administration. The ZAG was
replaced every 48 h. One group of mice receiving ZAG were also
administered propanolol (40 mgkg.sup.-1, p.o.) daily. The dose of
ZAG was chosen to be the same as that previously administered i.v.
(1), so that a direct comparison could be made, between the two
routes. Body weight, food and water intake, urinary glucose
excretion, and body temperature, determined by the use of a rectal
thermometer (RS Components, Northants, UK), were measured daily. A
glucose tolerance test was performed on day 3. Animals were fasted
for 12 h, followed by oral administration of glucose (1 gkg.sup.-1
in a volume of 100 .mu.l by gavage). Blood samples were removed
from the tail vein at 15, 30, 60 and 120 min and used for the
measurement of glucose. Urinary glucose was measured by collecting
0.5 ml urine and testing glucose concentration using a Boots
glucose monitor. After 8 days of treatment the animals were
terminated by cervical dislocation, tissues were removed and
rapidly frozen in liquid nitrogen, and maintained at -80.degree. C.
Future work would be to repeat this work in diet-induced animals as
alternative to a model with gene alteration, although previous
studies have shown the ob/ob mouse to be a good indicator of
potential human treatments. Animal studies were conducted under
Home Office License according to the UKCCCR Guidelines for the care
and use of laboratory animals.
[0083] Glucose uptake into adipocytes--Single cell suspensions of
white and brown adipocytes were obtained by incubation of minced
epididymal subcutaneous and visceral WAT and BAT for 2 and 2.5 h,
respectively, with Krebs-Ringer bicarbonate (KRBB) containing 1.5
mgml.sup.-1 collagenase and 4% BSA under 95% oxygen-5% CO.sub.2 at
37.degree. C. Adipocytes were washed twice in 1 ml KRBB, pH7.2, and
then incubated for 10 min at room temperature in 0.5 ml KRBB,
containing 18.5 MBq 2-[1-.sup.14C] deoxy-D-glucose (2-DG), together
with non-radioactive 2-DG, to give a final concentration of 0.1 mM,
in the absence or presence of insulin (10 nM). Uptake was
terminated by addition of 1 ml ice-cold KRBB without glucose.
Adipocytes were washed three times with 1 ml KRBB and lysed by the
addition of 0.5 ml 1M NaOH. The uptake of 2-[1-.sup.14C] DG was
determined by liquid scintillation counting.
[0084] Glucose uptake into soleus muscle--The uptake of
2-[1-.sup.14C] DG into freshly isolated soleus muscles in the
absence and presence of insulin (10 nM) was determined as
previously described.
[0085] Western blotting analysis--Tissues were thawed, washed in
PBS and lysed in Phosphosafe.TM. Extraction reagent for 5 min at
room temperature, followed by sonication at 4.degree. C. Cytosolic
protein (5-20 ug) formed by centrifugation at 18,000 g for 5 min at
4.degree. C., was resolved on 12% SDS PAGE by electrophoresis at
180V for about 1 h. To determine ZAG in serum 30 .mu.l samples
containing 20 .mu.g total protein were electrophoresed as above.
Protein was transferred 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. Membranes
were washed for 15 min in 0.1% Tween 20 buffered saline prior to
adding the primary antibodies. Both primary and secondary
antibodies were used at a dilution of 1:1000. Incubation was for 1
h at room temperature, and development was by ECL. Blots were
scanned by a densitometer to quantify differences.
[0086] PCR--Total RNA was extracted from tissues (50-120 mg) and
adipocytes with Trizol. RNA samples used for real-time PCR were
treated with a DNA-free kit (Ambion) to remove any genomic DNA. The
RNA concentration was determined from the absorbance at 260 nM. 1
.mu.g of total RNA of each sample was reverse transcribed to cDNA
in a final volume of 20 .mu.l by using a Reverse-iT first strand
kit (ABgene). 1 .mu.l of each cDNA sample was then amplified in a
PCR mixture containing 0.02 mM of each primer and 1.1 Reddy Mix PCR
Master Mix (ABgene) in a final volume of 25 .mu.l. Human b-actin
was used as a house keeping gene. The PCR products were sequenced
commercially to confirm their identity (MWG Biotech). (7)
[0087] RT-PCR--Relative ZAG mRNA levels were quantified using
real-time PCR with an ABI Prism 7700 Sequence Detector (Applied
Biosystems). Mouse .beta.-actin mRNA levels were similarly measured
and served as the reference gene. Primers and Taqman probes were
designed using PrimerExpress software (Applied Biosystems).
[0088] Statistical analysis--Results are shown as mean.+-.SEM for
at least three replicate experiments. Differences in means between
groups were determined by one-way analysis of variance (ANOVA)
followed by Tukey-Kramer multiple comparison tests, p values
<0.05 were considered significant.
[0089] Previous studies have shown that animals treated with iv ZAG
consume the same amount of food and water as PBS controls. It was
therefore convenient for oral administration to dissolve the ZAG in
drinking water, since this would avoid the stress associated with
dosing by gavage. The concentration of ZAG in the drinking water
was such that the animals would consume 50 .mu.g per day, so that a
direct comparison could be made with the iv route. The effect of
oral ZAG on the body weight of ob/ob mice is shown in FIG. 1A.
After 5 days of treatment the difference in body weight between the
ZAG and PBS groups was 3.5 g, which was the same as that found
after i.v. administration, while after 8 days of treatment there
was 5 g weight difference between the groups. As with i.v.
administration of ZAG there was an increase in rectal temperature,
which became significant after 4 days of treatment (FIG. 1B), while
there was a 40% reduction in urinary glucose excretion, which
became significant after 1 day of treatment (FIG. 1C). This
suggests that the oral ZAG also reduced the severity of diabetes in
the ob/ob mouse. A glucose tolerance test, performed on animals
after 3 days of oral ZAG, showed a reduced peak blood glucose
concentration, and a 33% reduction in the total area under the
curve (AUC) during the entire glucose tolerance test in ZAG treated
animals (FIG. 2A). ZAG also decreased the insulin response to the
glucose challenge (FIG. 2B) although a direct comparison has not
been made. These results suggest that oral administration of ZAG is
as effective in inducing weight loss, and reducing the severity of
diabetes in ob/ob mice as when given by the i.v. route. To
determine whether this effect was due to interaction with a
.beta.-AR, ZAG was administered orally to ob/ob mice that were
co-administered the non-specific .beta.-AR antagonist propanolol
(40 mg/kg). As shown in FIG. 2C while mice administered ZAG orally
lost weight this was blocked with propanolol, which had no effect
on weight gain of mice, administered PBS. Initially propanolol was
administered at 20 mgkg.sup.-1, but this did not prevent the weight
loss with ZAG so the dose was increased to 40 mgkg.sup.-1.
Antagonists of .beta.1- and .beta.2-AR are known to be less
effective against .beta.3-AR responses. Propanolol also completely
attenuated the ZAG induced increase in rectal temperature (FIG. 2D)
and the reduction in urinary excretion of glucose (FIG. 2E).
Propanolol also blocked the reduced peak blood glucose
concentration in the glucose tolerance test (FIG. 2F) and the
increase in insulin sensitivity (FIG. 2G). Propanolol also
completely attenuated the decrease in serum glucose and insulin
levels after ZAG administration to ob/ob mice. The elevation of
serum glycerol level, suggesting that it blocked lipolysis induced
by ZAG, along with the decrease in serum triglycerides and
non-esterified fatty acids (Table 1).
[0090] One possibility by which this could occur is that ZAG
escapes digestion by proteolytic enzymes, and is absorbed directly
into the blood stream. To investigate this ZAG was biosynthetically
labelled with L-[U-'.sup.4C] tyrosine. SDS/PAGE showed that the
purified product contained a single band of radioactivity of Mr43
kDa (FIG. 3A). The [.sup.14C]ZAG was then administered to ob/ob
mice by the oral route. SDS PAGE of serum proteins provided no
evidence for intact ZAG (FIG. 3A). Western blotting of serum using
mouse monoclonal antibody to full-length human ZAG, confirmed the
absence of human ZAG (FIG. 3B). Another possibility is that a
tryptic digest of ZAG could mediate the effect, but there is no
evidence for absorption of peptides into the blood stream (FIG.
3A). Alternatively a peptide could act within the gastrointestinal
tract. The effect of ZAG has been shown to be manifested through
interaction with a .beta.3-AR. However, treatment of CHO cells
transfected with human .beta.1-, .beta.2 or .beta.3-AR with a
tryptic digest of ZAG had no effect on cyclic AMP production (FIG.
3C), while intact ZAG stimulated cyclic AMP production in cells
with .beta.2- and .beta.3-AR. This suggests that interaction with
trypsin in the stomach would inactivate ZAG. Therefore ZAG must act
before it reaches the stomach.
[0091] Previous studies have shown that ZAG can induce its own
expression through interaction with a .beta.3-AR. and may be able
to induce its own expression through interaction with .beta.3-AR in
the oesophagus before being digested in the stomach and other parts
of the gastrointestinal tract. Since there was an absence of human
ZAG in the serum of orally dosed mice (FIG. 3B). Western blotting
of serum from mice dosed orally with ZAG for 8 days showed a
two-fold (P<0.001) increased level of murine ZAG (FIG. 4A). The
specificity of the antibodies against human ZAG is shown in FIG.
4B. Thus the anti-mouse ZAG antibody did not detect human ZAG.
Therefore the human ZAG administered orally has resulted in an
increase in mouse ZAG in the serum, and this has also caused a two
fold rise of mouse ZAG in WAT (P<0.001) (FIG. 4C).
Administration of propanolol also attenuated the oral route
ZAG-induced stimulation of glucose uptake ex vivo into epididymal,
subcutaneous and visceral adipocytes in the absence and presence of
insulin (FIG. 5A). It also attenuated glucose uptake into BAT in
the absence and presence of insulin (FIG. 5B), and glucose uptake
ex vivo into gastrocnemius muscle in the presence of insulin (FIG.
5C). In addition there was no increase in murine ZAG in the serum
(FIG. 5D), and no evidence of human ZAG (FIG. 3B) in animals
co-administered propanolol. These results suggest that oral
administration of ZAG increases circulatory levels by interaction
with a .beta.-AR, probably in the oesophagus, since ZAG mRNA
appears to be dramatically increased in oesophageal tissue compared
to that of the stomach, small intestine or the colon and is on par
with that seen in the liver in mice treated with ZAG orally (FIGS.
6A and B). Gene expression for ZAG in the various sections of the
GI Track are shown in (FIG. 6C).
[0092] Previous studies have shown ZAG to bind to a high affinity
binding site on the .beta.3-AR, with a Kd value of 78.+-.45 nM and
Bmax of 282.+-.1 fmole mg protein.sup.-1. Many of the effects of
ZAG are also found with .beta.3-AR agonists, including an increased
lipid mobilization and reduction of body fat, an increase of rectal
temperature and induction of UCP1 in BAT, normalization of
hyperglycaemia and hyper insulinaemia, improvement in glucose
tolerance and reduction of the insulin response during a glucose
tolerance test, and also attenuation of muscle wasting. The
.beta.3-AR is found predominantly on adipocytes, but has also been
reported on BAT and prostate as well as in the smooth muscle of the
gastrointestinal tract in a variety of species, and mediates
relaxation in the ileum, gastric fundus, jejunum, colon and
oesophagus. This study has shown that the previously described
presence of a .beta.-AR in the gastrointestinal tract, coupled with
the ability of ZAG to induce its own expression through a .beta.-AR
enables ZAG to be administered orally and this stimulus to be
converted into circulating ZAG.
[0093] The .beta.-AR responsive to oral ZAG must be in the mouth or
oesophagus, since tryptic digestion of ZAG produced a product with
no stimulation of the .beta.-AR. Using RT-PCR analysis of ZAG mRNA
this study shows a large increase in the oesophagus of animals
receiving ZAG orally. The lack of expression of ZAG in the lower
part of the gastrointestinal tract, despite the reported presence
of .beta.-AR would support the contention that ZAG is digested in
the stomach. Previous studies have suggested that a tryptic digest
of a cancer lipolytic factor called toxohormone L still retains
biological activity. The mechanism by which the ZAG signal is
transmitted from the gastrointestinal tract to the general
circulation has been elucidated by administration of human ZAG to a
mouse, and depends on the specificities of the antibodies to human
and murine ZAG. As expected human ZAG is digested, but murine ZAG
appears in the serum and responsive tissue such as WAT. This effect
is mediated through a .beta.-AR, since mice treated with the
non-specific .beta.-AR antagonist, propanolol, showed no murine ZAG
in their serum, and the effects of ZAG on body weight, lipolysis
and glucose disposal were completely attenuated. Previous studies
have shown that the lipolytic effect of ZAG in vitro was also
completely attenuated by propanolol. Agents that have been reported
to be specific for .beta.3-AR, such as SR59230A, were not used
since previous studies have indicated that this antagonist also
attenuates activation through both the .beta.1 and .beta.2-AR while
other investigators have shown it to be an antagonist of the al-AR.
SR59230A has also been seen to bind to albumin when used in vivo.
The specific .beta.-AR involved can only be determined using
specific .beta.-AR "knock-out" animals. The ability of propanolol
to attenuate the reduction in body weight, increase in temperature,
reduction in blood glucose, insulin, NEFA, and triglycerides,
increase in serum glucose, disposal of glucose and increased
insulin sensitivity induced by ZAG in ob/ob mice suggests that
these effects are mediated through a .beta.-AR.
[0094] The effects of orally administered human ZAG at a dose of 50
.mu.g day.sup.-1 are almost identical to those found when human ZAG
was administered by the i.v. route, suggesting a quantitative
transfer of the message from human ZAG into the serum as mouse ZAG.
ZAG is unusual in inducing its own expression, and the mechanism is
unknown apart from a requirement of the .beta.3-AR. The .beta.3-AR
agonist BRL37344 has also been shown to increase levels of ZAG mRNA
in 3T3 L1 adipocytes, suggesting a common mechanism. The cyclic AMP
faulted from interaction with a .beta.3-AR would lead to activation
of protein kinase A (PKA), the C-subunits of which are capable of
passively diffusing into nucleus, where they can regulate gene
expression through direct phosphorylation of cyclic AMP response
element binding protein (CREB).
[0095] Plasma ZAG protein has been shown to be decreased in ob/ob
mice and a similar decrease has been reported in high fat diet-fed
mice. Serum ZAG levels have also been found to be low in obese
human subjects. Most of the serum ZAG is thought to come from
adipose tissue and liver, and expression levels of ZAG mRNA in
these tissues in ob/ob mice have been shown to be significantly
reduced. This is at least partly due to the pro-inflammatory
cytokine tumour necrosis factor-.alpha. (TNF-.alpha.), which is
elevated in adipose tissue of obese subjects. Many of the effects
of obesity may be due to this low expression of ZAG, because of its
function in regulating lipid metabolism, and ZAG's ability to
increase expression of .beta.3-AR in gastrocnemius muscle, BAT and
WAT (unpublished results), which are low in obesity. The ability of
ZAG to increase serum levels when administered by the oral route
provides a mechanism for countering some of the effects of obesity.
It also raises the possibility of some uncooked foods such as
broccoli, rich in ZAG functioning to control obesity and type 2
diabetes, through conversion of vegetable ZAG to human ZAG.
[0096] 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
11276PRTHomo sapiens 1Gln Glu Asn Gln Asp Gly Arg Tyr Ser Leu Thr
Tyr Ile Tyr Thr Gly1 5 10 15Leu Ser Lys His Val Glu Asp Val Pro Ala
Phe Gln Ala Leu Gly Ser 20 25 30Leu Asn Asp Leu Gln Phe Phe Arg Tyr
Asn Ser Lys Asp Arg Lys Ser 35 40 45Gln Pro Met Gly Leu Trp Arg Gln
Val Glu Gly Met Glu Asp Trp Lys 50 55 60Glu Asp Ser Gln Leu Gln Lys
Ala Arg Glu Asp Met Glu Thr Leu Lys65 70 75 80Asp Ile Val Glu Tyr
Tyr Asn Asp Ser Asn Gly Ser His Val Leu Gln 85 90 95Gly Arg Phe Gly
Cys Glu Ile Glu Asn Asn Arg Ser Ser Gly Ala Phe 100 105 110Trp Lys
Tyr Tyr Tyr Asp Gly Lys Asp Tyr Ile Glu Phe Asn Lys Glu 115 120
125Ile Pro Ala Trp Val Pro Phe Asp Pro Ala Ala Gln Ile Thr Lys Gln
130 135 140Lys Trp Glu Ala Glu Pro Val Tyr Val Gln Arg Ala Lys Ala
Tyr Leu145 150 155 160Glu Glu Glu Cys Pro Ala Thr Leu Arg Lys Tyr
Leu Lys Tyr Ser Lys 165 170 175Asn Ile Leu Asp Arg Gln Asp Pro Pro
Ser Val Val Val Thr Ser His 180 185 190Gln Ala Pro Gly Glu Lys Lys
Lys Leu Lys Cys Leu Ala Tyr Asp Phe 195 200 205Tyr Pro Gly Lys Ile
Asp Val His Trp Thr Arg Ala Gly Gln Val Gln 210 215 220Glu Pro Glu
Leu Arg Gly Asp Val Leu His Asn Gly Asn Gly Thr Tyr225 230 235
240Gln Ser Trp Val Val Val Ala Val Pro Pro Gln Asp Thr Ala Pro Tyr
245 250 255Ser Cys His Val Gln His Ser Ser Leu Ala Gln Pro Leu Val
Val Pro 260 265 270Trp Glu Ala Ser 275
* * * * *