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Weight Loss Patent Abstract
A therapy for humans or other animals is described using one or
more antagonists to colipase so as to interfere with macromolecular
nutrient digestion. In one embodiment, the present invention describes
the use of antibodies that can be administered by a variety of routes,
including orally, over a treatment period in a manner that promotes
weight loss or impedes weight gain.
Weight Loss Patent Claims
We claim:
1. A method of treatment, comprising: a) providing i) a subject,
and ii) a preparation comprising an antibody to colipase; and, b)
administering said preparation to said subject.
2. The method of claim 1, wherein said preparation is an oral formulation.
3. The method of claim 2, wherein said oral formulation is a capsule,
microcapsule, tablet, powder, suspension, or solution.
4. The method of claim 2, wherein said oral formulation protects
said antibody from digestive agents.
5. The method of claim 1, wherein said antibody is produced in
an animal.
6. The method of claim 5, wherein said antibody is produced in
a mammal.
7. The method of claim 5, wherein said antibody is produced in
an avian.
8. The method of claim 5, wherein said antibody is produced recombinantly.
9. The method of claim 1, wherein said antibody is unpurified.
10. The method of claims 1, wherein said antibody is purified.
11. The method of claim 1, wherein said antibody is admixed with
a foodstuff intended for consumption.
12. A method of treatment, comprising: a) providing i) a subject
having a starting body weight, and ii) a plurality of preparations,
each of said preparations comprising an antibody to colipase; and,
b) administering said plurality of preparations to said subject
over a treatment period of days.
13. The method of claim 12, wherein said subject, after said treatment
period, has a post-treatment body weight that is lower than said
starting body weight.
14. A method of treatment, comprising: a) providing i) a subject
having a starting body weight, and ii) a plurality of preparations,
each of said preparations comprising an antibody to colipase; and,
b) administering said plurality of preparations to said subject
over a treatment period of days, wherein said subject, during said
treatment period is on a lipid rich diet.
15. The method of claim 14, wherein said subject, after said treatment
period, has a post-treatment body weight approximately equal to
said starting body weight.
16. The method of claim 14, wherein said treatment period is 30
days or more.
Weight Loss Patent Description
[0001] This application for patent under 35 US.C. 111(a) claims
priority to Provisional Application Ser. No. 60/376166 filed Apr.
26, 2002 under 35 US.C. 111(b).
FIELD OF THE INVENTION
[0002] The present invention describes a therapy for bodily weight
loss for humans or other animals using antibodies that interfere
with macromolecular nutrient digestion. Certain enzymes and enzyme
co-factors found in the digestive tracts of animals are essential
for nutrient breakdown prior to absorption and use as an energy
source. The present invention describes the use of antibodies that
interfere with the digestion of dietary lipids to promote weight
loss or impede weight gain.
BACKGROUND OF THE INVENTION
[0003] Obesity is now an epidemic in the United States. Approximately
97 million adults in the United States are either overweight or
obese, with the prevalence increasing markedly over the last decade
(Kucamarski R J et al. Obes. Res. 1997; 5:542-548). Moreover, almost
50% of the population in developed countries such as the United
States are 22% above their recommended weight (Flegal K M et al.
Int. J.Obes Relat. Metab. Disord 1998;2 2:39-47). Obesity is second
only to smoking as a cause of preventable death in the United States
(McGinnis J, et al. JAMA 1993;270:2207-2212). A study in 1991 estimated
the number of overall annual deaths attributed to obesity in the
United States to be 2 80,000 (Allison D S, et al. JAMA 1999;282:1530-1538).
Obesity and hyperlipidemia are conditions associated with a high
rate of morbidity and early mortality (Martin M J, et al. Lancet
1986;2:933-936, Van Itallie T B, Ann. Intern. Med. 1985;103:983-988).
Obesity primarily caused by high fat intakes is associated cardiovascular
disease, type 2 diabetes, osteoarthritis, hypertension and cancer
(National Institutes of Health, National Heart, Lung, and Blood
Institute. US Dept. of Health and Human Services, public Health
Service, NIH, NHLBI; 1998). Dietary fat, primarily as triacylglycerides
contributes a high percentage, from 30-44 of total calories consumed
by people in western society (Kromhout D, Am. J Clin. Nutr. 1983;37:295-299).
[0004] Current dietary guidelines recommend reductions in fat consumption
to 30% or less (Nation Research Council. Diet and Health. Washington
D.C.:Natl Acad. Press, 1989). Because compliance with low-fat diets
has been problematic, pharmacological therapies are often added
to the dietary therapy. About 58 million Americans spend about $30
billion annually for these pharmacological therapies to control
obesity.
[0005] Obesity is generally a chronic disease that requires long-term
behavior modification supplemented with drug therapy. Diet-drug
therapies have shown moderate benefits at best though are poorly
tolerated by patients (Jones S P, et al. Int. J Obes. 1995; 19 (suppl
2):41. Abstract 071). Current weight loss therapies primarily function
systemically to alter neurotransmitters to suppress appetite. While
most therapies have been moderately effective, many have systemic
side effects.
[0006] Sibutramine (Meridia), a recently approved drug controls
appetite by modifying levels of the neurotransmitter norepinephrine
producing a chemically induced illusion of satiety. However, this
drug and drugs in this class have a variety of central nervous system
(CNS) aide-effects, is including headache, anxiety, fatigue, insomnia
and hypertension. Some of these appetite-suppressing drugs have
much more serious side-effects. Several drugs such as dexfenfluramine
(Redux) or fenfluramine/phenteramin- e (Fen-phen), which act on
the neurotransmitter serotonin, have been withdrawn from the market
by the United States Food and Drug Administration in 1997. Both
drugs caused life-threatening primary pulmonary hypertension, valvular
heart dysfunction and neurotoxicity. While the safer diet drugs,
such as phenylpropanpamine (Dexatrim) have more moderate side effects,
none of the current therapies work by maximizing the effectiveness
of reducing fat consumption through dieting. Despite recent advances
in treatment, there remains a significant need for a safe, non-systemic
therapy which works in concert with the standard first-line dietary
approach.
[0007] New strategies are being developed to reduce dietary fat
uptake in combination with standard dietary therapy. The use of
fat substitutes such as Olestra, composed of indigestible long-chain
fatty acids, is one approach along these lines. Unfortunately, Olestra
has been met with a less than enthusiastic response by consumers.
In addition to side-effects due to malabsorption, these fat substitutes
can also severely reduce the uptake of fat-soluble vitamins such
as vitamin K.
[0008] Drugs that selectively limit the intestinal absorption of
dietary fat, in addition to that controlled by dietary manipulation
alone, would be a useful therapeutic agent for the treatment of
obesity. Ideally, these drugs would be effective with minimal side-effects
thus promoting wide-spread compliance. A newer approach being explored
is the use of inhibitors to gastrointestinal enzymes involved in
fat metabolism. These enzymes are particularly attractive targets
because they are found within the lumen of the GI tract, and the
inhibitor need not get into circulation to be effective. This luminal
route of action tends to limit the adverse effects associated with
systemic therapies.
[0009] Triacylglyceride lipases are ubiquitous enzymes required
for all aspects of fat metabolism. Found in the gastrointestinal
tract, these enzymes mediate the digestion of triglycerides and
their uptake into tissues. Triacylglycerides contribute to greater
than 95% of the dietary fats in the western diet (Patton J S , et
al. Am. J Physiol. 1981;241:G328-336). Lipases hydrolyze the ester
bonds in the triacylglycerides releasing free fatty acids and mono-acylglycerols.
Dietary triacylglycerides must be cleaved into free fatty acids
and monoacylglycerols before they are absorbed by intestinal enterocytes.
In the absence of lipases, dietary triacylglycerides are not absorbed
and pass into the stool. Consequently, reduced absorption of digested
triacylglycerides should result in weight loss.
[0010] One new drug, Xenical (Orlistat, Hofftnann-La Roche), has
been recently approved by the FDA for treatment of obesity. This
drug is a potent inhibitor of pancreatic lipase; secreted into the
proximal small intestine by acinar cells of the pancreas (Borgstrom
R, Gastroenterol. 1984;86:194-196). Xenical is a synthesized derivative
of tetrahydrolipstatin a naturally occurring lipase inhibitor produced
by Streptomyces toxytricini. It has been shown to be effective at
reducing fat absorption in humans and animals (Hogan S A, et al.
Int J Obes. 1987, 3 (suppl 11):35-42 1987, Hauptman J B, et al.
Am. J Clin. Nutr. 1992;55 (suppl 1): 309S-313S). Xenical is minimally
absorbed and prevents the absorption of about 30% of dietary fat
consumed. In clinical trials of patients on a low-fat diet, those
taking Orlistat lost an average of 3-4% of their body weight in
a year compared with placebo-treated controls. Unfortunately, in
a 1-year trial of maintenance therapy, 76% of patients taking Orlistat
regained weight compared with 84% of the placebo group. Side-effects
were seen in 20-40% of the patients which included flatulence with
discharge, oily spotting, and malabsorption of fat-soluble vitamins
and beta-carotene. While these side effects can be unpleasant, they
do not preclude the use of the drug in most patients. Overall, clinical
trails indicate that Xenical is modestly effective with some unpleasant
side-effects.
[0011] Antibodies to lipase have also been used to impair dietary
lipid metabolism (Pimental, J., International Patent Application,
WO 99/02187). In rodent studies, lipase antibodies were shown to
increase the amount of animal ration needed to gain 1 gram of body
weight. However, the antibodies were not shown to be able to reduce
weight or impede weight gain.
DEFINITIONS
[0012] To facilitate understanding of the invention, a number of
terms are defined below.
[0013] As used herein, the term "neutralizing" is used
in reference to antagonists, which may include antibodies or antibody
preparations, that interfere with enzymes or enzyme co-factors which
are able to reduce(in some cases substantially reduce) a specific
enzymatic activity. Complete elimination of activity is not required.
For certain embodiments, it is sufficient that there is a reduction
in enzymatic activity (for example, in certain embodiments, it is
sufficient that there is approximately a 50% reduction, more preferably
approximately a 75% reduction, still more preferably a reduction
of approximately 90% or more).
[0014] As used herein, the term "purified" or "to
purify" refers to at least the partial removal of one or more
contaminants from a sample. For example, antibodies may be purified
by at least partial removal (for example, approximately a 20% removal,
more preferably approximately a 50% removal, still more preferably
a reduction of approximately 90% or more) of contaminating non-immunoglobulin
proteins. The removal of non-immunoglobulin proteins results in
an increase in the percent of specific target-reactive immunoglobulins
in the antibody preparation. While the present invention is not
limited to the manner of purification, one method for purification
of antibodies from egg yolks is by extraction and precipitation
using polyethylene glycol.
[0015] The term "subject" when used in reference to administration
of compositions comprising antagonists to colipase refers to the
recipient animal to whom said antagonists are administered. The
subject may be an animal, including mammals and more particularly,
humans, in whom it is desirable to reduce bodily weight or impede
bodily weight gain. In the case where the subject is a human, the
present invention is not limited to only humans that are patients;
indeed; in certain embodiments, it is contemplated that treatment
can be accomplished without hospitalization.
[0016] The term "antibody" includes antibody fragments
such as single chain antibodies, Fab fragments, Fab.sub.2 fragments
and other fragments which are capable of binding colipase. It is
not intended that the present invention be limited by the host used
to make antibodies. Moreover, in certain embodiments, the antibody
can be made recombinantly.
[0017] The term "antagonist to colipase" includes antibodies.
However, it is contemplated that antagonists can be fashioned from
colipase as well as the receptor for colipase. For example, small
peptides or mimetics can be made based on sequences of colipase;
such peptides can be variant in amino acid sequence (one or two
different amino acids) or wild type. Such peptides can be protected
from breakdown by protecting groups on the N-terminus and C-terminus.
[0018] In one embodiment, the present invention contemplates formulations
which "protect the antibody from digestive agents." Typically,
such formulations are solid formulations comprising coatings which
shield the antibody from enzymatic breakdown. Complete protection
is not required.
[0019] It is sufficient that some protection is afforded (for example,
approximately a 20% reduction in antibody breakdown, more preferably
approximately a 50% reduction, still more preferably a reduction
of approximately 90% or more).
SUMMARY OF THE INVENTION
[0020] The present invention provides compositions comprising an
antagonist to colipase. Colipase is another key protein involved
in fat metabolism. This protein is an essential enzyme cofactor
of pancreatic lipase. Colipase stabilizes the active conformation
of lipase by binding to the carboxy-terminal, non-catalytic domain
of lipase (Lowe M, Ann. Rev. Nutr. 1997; 17:141-158). This enzyme
complex allows the hydrolysis of the dietary triacylglyceride at
the lipid-water interface.
[0021] Importantly, the interaction of pancreatic lipase and colipase
is needed for activity. Pancreatic lipase is inhibited by physiological
concentrations of bile salts as well as by phospholipids and proteins
present in emulsions of dietary lipids. Colipase restores activity
to pancreatic lipase and permits efficient digestion of dietary
fats under these inhibitory conditions (Lowe M, Ann. Rev. Nutr.
1997;17:141-158). The essential role of colipase in triacylglyceride
digestion makes it an attractive enzyme target for drug therapy
to control fat absorption.
[0022] The present invention relates to a method for decreasing
fat absorption using orally-delivered colipase antagonist therapy.
While it is not intended that the present invention be limited to
any particular mechanism, it is believed that the present invention
describes a treatment to reduce dietary fat metabolism and absorption
in humans as well as other animals through the use of compositions
comprising one or more colipase antagonists. The examples of the
present invention demonstrate the production of neutralizing antibodies
to pancreatic lipase and colipase by hyper-immunizing egg-laying
hens. Both antibodies were tested in rats, showing the relative
ability of these antibodies to effect impedance of body weight gain.
[0023] Such antagonist is capable of reducing lipase enzyme activity
that occurs normally in the lumen of the intestinal tract. In a
preferred embodiment, the antagonist is an antibody that binds to
colipase. In a further embodiment, colipase antibodies are produced
in laying hens. In one embodiment, it is intended that the antagonist
of the present invention be formulated as a medicament.
[0024] The antagonists of the present invention find use in humans
or other animals as a medicament to promote bodily weight to as
or to impede bodily weight gain by interfering with dietary fat
metabolism. In a one embodiment, the medicament would be in the
form of a solid dosage form, such as a tablet or capsule, suitable
for oral administration. In another embodiment, the medicament would
be admixed with a foodstuff intended for consumption. In a further
embodiment, the antibodies would be protected by pharmaceutical
excipients and/or chemical coatings to afford protection from digestive
agents.
[0025] In yet another embodiment, the dosage would be in the form
of a powder that could be mixed with potable liquids and administered
orally. Such potable liquids may contain compounds to aid in survival
of the antagonist while passing through the digestive tract of the
subject.
[0026] In one embodiment, the invention contemplates a method of
treatment comprising: a) providing: i) a subject, ii) a preparation
comprising an antagonist to colipase, iii) administering said preparation
(e.g. orally) to said subject. In another embodiment, the present
invention contemplates a method of treatment, comprising: a) providing
i) a subject having a starting body weight, and ii) a plurality
of preparations, each of said preparations comprising an antibody
to colipase; and, b) administering said plurality of preparations
to said subject over a treatment period (e.g. of days, weeks, months
or years). In a preferred embodiment, said subject, after said treatment
period, has a post-treatment body weight that is lower than said
starting body weight.
[0027] In yet another embodiment, the present invention contemplates
a method of treatment, comprising: a) providing i) a subject having
a starting body weight, and ii) a plurality of preparations, each
of said preparations comprising an antibody to colipase; and, b)
administering said plurality of preparations to said subject over
a treatment period (e.g. of days, weeks, months, or years) wherein
said subject, during said treatment period is on a lipid rich diet
(e.g. as defined by any health agency or medical group). It is preferred
said subject, after said treatment period, has a post-treatment
body weight approximately equal to said starting body weight. In
one embodiment, the treatment period is between approximately 5
and approximately 90 days. In another embodiment, the treatment
period is between approximately three and approximately twelve months.
[0028] The present invention also contemplates compositions. In
one embodiment, the present invention contemplates a preparation
comprising an antagonist to colipase (including but not limited
to a polyclonal antibody).
DESCRIPTION OF THE INVENTION
[0029] The present invention is directed to: i) preparations comprising
one or more antagonists to colipase, and; ii) use of such an antagonist,
as a medicament in a variety of forms, to modulate bodily weight.
Because lipids represent a large portion of the nutritional and
caloric content of a typical diet, interfering with lipid breakdown
and absorption would be of considerable use in impeding weight gain
or promoting weight loss.
[0030] i) Compositions of antagonists to colipase. The present
invention contemplates an antagonist to colipase capable of interfering
with the hydrolytic activity of one or more gastrointestinal lipases.
It is not intended that the present invention be limited to any
particular chemical or molecular form of antagonist. To show that
antagonism of colipase is effective in reducing lipase activity,
antibodies directed to colipase were used. Colipase antibodies were
produced in laying hens following immunization with colipase protein.
In a preferred embodiment, the antagonist is an antibody. In yet
another embodiment, the antibody is harvested from the chicken eggs.
It is also contemplated that antagonists may be in the form of small
organic molecules, peptides, antibody fragments, macromolecular
absorbants, resins, carbohydrates, or lipids.
[0031] Antibodies of the present invention may be produced a variety
of animal species and have a variety of compositions. For example,
antibodies may be produced following immunization of an animal with
the entire colipase protein, a portion of the colipase protein,
peptides representing regions or epitopes of the colipase protein,
or other molecules that are capable of inducing antibodies reactive
to colipase. Antibodies resulting from immunization of an animal
would normally be polyclonal, that is, containing multiple antibody
specificities reacting to colipase with varying affinities. The
present invention contemplates the use of polyclonal antibodies
because of their ease and low cost of production. A variety of animal
species could be used to produce polyclonal antibodies of the present
invention. For example, large mammals such as horses, cows, goats,
sheep, and the like, can produce large amounts of antibodies that
can be harvested from their blood and their milk. Alternatively,
avian species could be used and the antibodies harvested from their
eggs.
[0032] However, the present invention is not limited to the production
of polyclonal antibodies following immunization of animals. For
example, monoclonal colipase antibodies having a single specificity
(or a mixture of two or more monoclonal antibodies) are also contemplated.
Monoclonal antibodies may be produced from hybridoma cell lines
placed in living animals (production of acites fluid) or in bioreactors
(production of culture supernatants). Monoclonal antibodies, or
fragments of whole antibodies, may also be produced recombinantly
using animal, bacterial, yeast, insect, or plant expression systems.
[0033] Antibodies of the present invention may be used in a plurality
of forms. It is contemplated that useful antibodies of the present
invention may be in a crude (that is, unpurified) form as might
be found in animal serum, eggs, cell culture supernatant, or plant
tissues. In a preferred embodiment, the antibodies would be purified
(through separation, fractionation, precipitation, chromatography,
absorption, filtration, and the like) to enrich the concentration
of the desired antibody protein in relation to non-antibody substances.
In one embodiment, antibodies, whether crude or purified, may be
mixed with excipients that function to enhance chemical stability,
prevent aggregation, allow for compaction, or otherwise enable the
antibody protein to be taken by a subject by mouth and be effective
within the lumen of the gastrointestinal tract. In addition to excipients,
the present invention contemplates the use of enteric coatings to
protect the antibody protein from digestive agents within the gastrointestinal
tract. In preferred embodiments, the antibody would be processed
in the form of a capsule, microcapsule, tablet, suspension, powder,
or solution, each of which would be orally deliverable. In yet another
embodiment, the antibodies could be admixed with a foodstuff intended
for consumption.
[0034] ii) Use of colipase antagonists to modulate bodily weight.
The present invention contemplates the use of colipase antagonists
as a medicament to modulate bodily weight by interference with normal
intestinal lipid metabolism. Modulation of bodily weight could include
reduction of weight or impedance of weight gain. It is contemplated
that the medicament would be useful in humans, or other animals,
where bodily weight modulation is desirable.
[0035] The medicament of the present invention could have various
modes of delivery. In a preferred embodiment, the medicament of
the present invention is in a solid dosage form that can be taken
by mouth. In another embodiment, the medicament is mixed with a
potable liquid suitable for drinking. In yet another embodiment,
the medicament is admixed with a foodstuff suitable for consumption.
[0036] The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to
be construed as limiting the scope thereof.
[0037] In the disclosure that follows, various abbreviations are
used: .degree. C. (degrees Centigrade); g (the force of gravity
on Earth); mg (milligram); ug (microgram); ml (milliliter); ul (microliter)
PBS (phosphate buffered saline); PEG (polyethyleneglycol); IgY (immunoglobulin
of yolk); nm (nanometer); EIA (enzyme immunoassay); U/L (units per
liter, and; SEM (standard error of the mean).
EXAMPLE 1
Production of Antibodies to Pancreatic Lipase and Colipase in the
Hen
[0038] This example involved (a) preparation of the immunogen and
immunization of chickens, (b) purification of pancreatic lipase
antibodies and colipase antibodies (IgY) from egg yolk, and (c)
detection of specific antibodies in the purified IgY preparations.
[0039] (a) Preparation of the immunogen and immunization.
[0040] Human pancreatic lipase (catalog number 437709) was purchased
from Calbiochem Corp, La Jolla, Calif. Porcine colipase (catalog
number 142A4000) was purchased from Calzyme Laboratories, San Luis
Obispo, Cailf. The colipase, which was supplied as a freeze-dried
powder was reconstituted in phosphate-buffered saline (PBS) pH 7.0-7.4
at 10 mg/ml. Egg-laying Leghorn hens were immunized with either
approximately 10 ug of pancreatic lipase or 250 ug of colipase each
mixed with 75 ug Quil A adjuvant (Superfos Biosector, Denmark, distributed
by Accurate Chem., Westbury, N.Y.) in PBS. The immunization volume
was 0.5 ml and delivered via one sub-cutaneous or intramuscular
injection. The hens were immunized at least 3 times every 2-4 weeks.
[0041] (b) Purification of pancreatic lipase IgY and colipase IgY
from egg yolks.
[0042] Eggs were collected from hens given either pancreatic lipase
or colipase immunizations at least 3-5 days after receipt of the
last booster immunization. Egg yolk immunoglobulin (IgY) was extracted
using a two-step polyethylene glycol (PEG)8000 method performed
according to a modification of the procedure of Polson (Poulson
et al. Immunol. Comm. 1980;9:495). The yolks were separated from
the whites and the yolks were placed in a graduated cylinder. The
pooled yolks were blended with 4 volumes of PBS and PEG was added
to a concentration of 3.5%. When the PEG was dissolved, the protein
and lipid precipitate that formed was removed by centrifugation
at 9,000.times.g for 15 minutes. The supernatant was decanted and
filtered through 4 layers of gauze to remove the floating particulates
and a second PEG step was performed by adding PEG to a final concentration
of 12% (the supernatant was assumed to contain 3.5% PEG). After
a second centrifugation, the supernatant was discarded and the IgY
pellet resuspended in PBS at approximately 1/6 the original yolk
volume. IgY extracted from the eggs of immunized hens are designated
as "pancreatic lipase" or "colipase IgY" while
IgY extracted from the eggs of unimmunized hens is designated "preimmune
IgY." The concentration of the fractionated IgY was estimated
by measuring absorbance at 280 nm (an optical density at 280 nm
of 1.3 equals 1 mg of IgY/ml). The protein concentration of the
IgY preparations were between 20-35 mg/ml.
[0043] (c) Detection of pancreatic lipase and colipase antibody
activity in the IgY preparations.
[0044] In order to determine if an antibody response was generated
against pancreatic lipase or colipase and to determine relative
levels of the responses, enzyme-linked immunosorbant assays (EIA)
were performed. Briefly, ninety-six well Maxisorb (Nunc) micro-titer
plates were coated overnight at 4.degree. C. with 100 ul/well with
either pancreatic lipase or colipase at 2.5 ug/ml in PBS. The wells
are then blocked with PBS containing 1% BSA and 0.05% Tween 20 and
incubated for about 1 hour at 37.degree. C. The blocking solution
was removed. The immune or preimmune IgY was diluted in PBS containing
BSA and the plates were incubated for 1 hour at 37.degree. C. The
plates were washed 3 times with PBS containing 0.05% Tween 20 and
three times with PBS alone. Alkaline phosphatase-conjugated anti-chicken
IgG was diluted 1:1000 in PBS containing 1% BSA and 0.05% Tween
20, added to the plates and incubated 1 hour at 37.degree. C. The
plates were washed as above and p-nitrophenyl phosphate at 1 mg/ml
in 0.05 M Na.sub.2CO.sub.3, pH 9.5, 10 mM MgCl.sub.2 was added.
The absorbance at 410 nm of the solution in each well was determined
using a Dynatech plate reader about 30 minutes after substrate addition.
[0045] The EIA results indicated that antibody activity could be
readily measured in both the pancreatic lipase IqY and colipase
IgY preparations. EIA titer is a convenient measure of antibody
activity. EIA titer is defined as the reciprocal of the highest
immune IgY dilution generating an absorbance signal that is about
3-fold higher than that of preimmune IgY control sample. EIA titers
to both pancreatic lipase and colipase were at least 10,000. These
results indicated hens readily respond to pancreatic lipase and
colipase by producing antibodies. None of the hens receiving an
immunization exhibited an adverse reaction.
EXAMPLE 2
Demonstration of Pancreatic Lipase IgY Neutralizing Ability Using
an in vitro Enzyme Assay.
[0046] In this example, the pancreatic lipase IgY was evaluated
in an in vitro assay to determine if pancreatic lipase enzyme activity
could be neutralized by antibodies in the IgY preparation. The effectiveness
of pancreatic lipase IgY at neutralizing the enzymatic activity
of pancreatic lipase was determined using a commercially-available
in vitro assay (Lipase-PS assay catalog number 805-A, Sigma Chemical
Co., St. Louis, Mo.). This assay was designed to measure serum lipase
activity which is widely used for the diagnosis of acute pancreatitis.
The assay is a colorimetric method in-which 1,2-diglyceride is hydrolyzed
to 2-monoglyceride and fatty acid. This assay was modified for our
purposes to determine if pancreatic lipase IgY could inhibit the
pancreatic lipase activity in the presence of substrate. The Lipase
PS assay was also modified to conform to a microtiter plate assay
format.
[0047] Different concentrations of pancreatic lipase IgY or preimmune
IgY as a control were diluted in phosphate-buffered saline (PBS)
and 100 ul was added in duplicate to a 95 well-microtiter plate
(Nunc, Maxisorb). Another set of controls consisted of two sets
of wells containing 100 ul of only PBS to serve as a blank and PBS
plus the lipase standard to serve as a positive control. To each
well, 100 ul of Lipase-PS assay kit substrate reagent diluted in
substrate diluent buffer was added. The reconstituted substrate
reagent contains lipase substrate (1,2 diglyceride), cofactors (porcine
colipase, 1340 U/L) and substrates for the chromogenic reaction.
The chromogenic reaction which occurs when active lipase is present
in the sample is directly proportional to pancreatic lipase activity.
Human pancreatic lipase PS enzyme standard (1.5 ul/well)(supplied
in the assay kit) at 258 U/L was added to each well except the blank
PBS wells, followed by incubation for 5 minutes at 37.degree. C.
Lastly, the Lipase-PS activator reagent (30 ul) containing bile
salts, was added to each well to initiate the enzymatic reaction.
The microtiter plate was incubated at 37.degree. C. in a ELx808
plate reader, (Bio-TeK Instruments Inc, Winooski, Vt.). The absorbance
of each well at 550 nm was read after 3 minutes of incubation at
37.degree. C. (initial A) and then again 2 minutes later (final
A). Lipase activity in U/L was determined by calculating the change
in absorbance of the test sample minus the blank divided the change
in the absorbance of standard control minus the blank multiplied
by the labeled activity (258 U/L) of the standard.
[0048] The assay results showed that anti-pancreatic lipase IgY
neutralized the activity of pancreatic lipase. Concentrations of
anti-pancreatic lipase at 0.04 mg/ml to 0.66 mg/ml inhibited 80-90%
of the standard lipase activity. In contrast, equivalent concentrations
preimmune IgY failed to neutralize any of the standard enzyme activity.
Higher IgY concentrations of both the preimmune or the pancreatic
lipase IgY nonspecifically inhibited lipase activity in this assay.
The results demonstrate that the use chicken antibodies against
pancreatic lipase is an effective method to inhibit pancreatic lipase
activity.
EXAMPLE 3
Demonstration of Colipase IgY Neutralizing Ability Using an in
vitro Enzyme Assay
[0049] This example involved the testing of the colipase IgY neutralizing
ability in an in vitro enzyme assay. The ability of colipase IgY
to neutralize the bioactivity of colipase was determined by using
the Lipase-PS assay for lipase activity described in Example 2.
Since colipase is an obligatory enzyme cofactor of pancreatic lipase
in the presence of bile salts, the inhibition of colipase should
prevent the enzymatic reactions involved in the Lipase-PS assay.
Therefore, the inhibition of colipase was measured indirectly by
determining the effect of colipase IgY on the enzymatic activity
in the lipase assay.
[0050] The Lipase-PS assay using colipase IgY was performed essentially
as described in Example 2. Different concentrations of colipase
or preimmune IgY diluted in PBS were preincubated at 37.degree.
C. with the lipase substrate reagent. The lipase substrate reagent
contained a fixed amount of porcine colipase at 1340 U/L. The human
lipase enzyme standard and activator were then added as the assay
proceeded as described above. The wells are read at 550 nm after
3 and minutes of incubation at 37.degree. C. Pancreatic lipase activity
was calculated as described in Example 2.
[0051] The assay results indicated that colipase IgY was effective
at inhibiting the hydrolysis of diglyceride in the lipase assay.
Concentrations of colipase IgY at 0.17 mg/ml and 0.04 mg/ml inhibited
49% and 29% of the standard lipase activity. Since colipase was
fixed component within Lipase-PS substrate buffer, improving the
inhibitory effect by colipase IgY by modifying the amount of colipase
within the reaction was not possible. Importantly however, preimmune
IgY at equivalent concentrations was unable to inhibit any lipase
activity. At higher concentrations of IgY, lipase activity was nonspecifically
inhibited in this assay. The results indicate that the colipase
IgY could inhibit colipase activity and that this antibody is an
effective way to inhibit this enzymatic pathway.
EXAMPLE 4
The Reduction of Body Weight Gain in Sprague-Dawley Rats Using
Oral Pancreatic Lipase IgY or Colipase IgY
[0052] This example involved the testing the effect of pancreatic
lipase IgY and colipase IgY in an animal model of diet-induced obesity.
This example illustrates that the inhibition of colipase in vivo
using orally administered colipase IgY can prevent weight gain in
rats fed a high fat diet. Pancreatic lipase IgY treatment also had
a modest effect on weight gain in this model.
[0053] Diet-induced obesity was studied in rats using a model substantially
as described by Ackroff, K. using Sprague-Dawley rats (Ackroff K,
et al. Am. J Physiol. 1996; 271: R48-R54). Rats that overeat and
become obese when given palatable food serve as a good model for
dietary obesity in man (Schemmel R O, et al J Nutr. 1970;100: 1041-1048).
Four groups ("Group" 1-4) containing 6-7, 6 week-old male
rats (Charles River, Wilmington, Mass.) were all given a standard
balanced low-fat rodent chow (Formulab 5008, PMI Feeds, Inc, Richmond,
Ind.) ad libitum. Rats in Group 1 were fed only the rodent chow
and served as an untreated control. In addition to rodent chow,
the diets of the rats in Groups 2, 3, and 4 were supplemented with
egg yolks as a high-fat, dietary component. Approximately 35% of
egg yolk by weight consists of fat. Rats in Groups 2, 3, and 4 were
given a choice between a fat (egg yolk) and moderately low fat (rat
chow) diet. Rats in Group 2 were fed egg yolks from eggs harvested
from preimmune hens. Rats in Groups 3 and 4 were fed egg yolk from
eggs of the hens immunized with either pancreatic lipase IgY or
colipase IgY. Egg yolks were harvested and pooled in a clear plastic
animal water bottles with volume graduations. The yolks were then
mixed as a suspension consisting of 2 parts yolk to 1 part 0.3%
sodium benzoate in water (final sodium benzoate concentration of
0.1%). The dilution of the egg yolks with a solution of sodium benzoate
minimized microbial contamination and permitted the flow of yolks
through the water bottle sipper.
[0054] During the study, the rats consumed the egg yolks mixture
preferentially over the balanced rat chow. Daily egg yolk consumption
in each group was very comparable and consisted of about 200 ml
of yolk or about 12 eggs/day. Rats from each group were weighed
approximately once per week to compare the rate of weight gain between
the 4 groups. The average weight with standard error (SEM) for each
group during treatment is shown in Table 1.
1TABLE 1 Mean Body Weight* of Rats Group Day 0 Day 16 Day 27 Day
35 Day 50 Untreated Control 165 (2) 313 (6) 365 (11) 409 (17) 458
(19) Preimmune IgY 167 (2) 337 (7) 418 (7) 466 (8) 533 (6) Pancreatic
lipase 168 (1) 344 (7) 404 (11) 452 (13) 504 (16) IgY Colipase IgY
167 (1) 326 (9) 363 (14) 404 (15) 450 (16) *Mean weights are shown
in grams and the standard errors of the mean are shown in grams
(in parenthesis).
[0055] The experimental results indicated that colipase IgY could
significantly impede weight gain in the rat obesity model compared
to the preimmune IgY treated rats. Using the Mann Whitney U-test,
p values of .ltoreq.0.05 are considered significant. Mean rat weights
in the colipase IgY treated group began to differ significantly
from the preimmune IgY treated rats at day 27 (p=0.0025). The mean
weight differences between these two groups remained significant
throughout the treatment period. Remarkably, the mean weight gain
of the colipase IgY treated rats was not significantly different
at any time in the treatment period from the weight gain of the
untreated control animals that were not given supplemental dietary
fat.
[0056] After 50 days of treatment, the statistical differences
in mean weight between the preimmune IgY and colipase IgY treated
rats increased. The mean weight of the colipase IgY treated rats
were 16%, or 83 grams, less than the preimmune IgY treated rats
(p=0.001). Moreover, at that time, every member treated with colipase
IgY weighed less than any rat in the preimmune IgY treated group.
For example, the lightest member in the preimmune IgY treated group
(510 g) still weighed 11 grams more than the heaviest colipase IgY
treated member (499 g).
[0057] The experimental results also indicated that colipase IgY
was more effective than pancreatic lipase IgY at impeding weight
gain in the rat obesity model. After 35 days of treatment, the rats
in the colipase IgY treated groups weighed significantly less than
the pancreatic lipase IgY treated group (p=0.025). After 50 days
of colipase IgY treatment, the weights of the rats in that group
remained significantly lower than the pancreatic lipase IgY treated
rats (p<0.05).
[0058] Rats treated with pancreatic lipase IgY did not show a statistically
significant difference in weight when compared to the preimmune
IgY treated controls at any of the measurement points. For example,
at day 50 the calculated p value between those treatment groups
was <0.1.
[0059] The overall results in this study indicated that oral colipase
IgY treatment could significantly reduce weight gain in rats fed
a high-fat diet. These results indicate that oral colipase IgY therapy
is an effective method to impede weight gain in animals consuming
a fat-rich diet. These results also show that antibodies directed
to colipase are more effective than similar antibodies to pancreatic
lipase and that the effectiveness increases over the treatment period
(thus, treatment periods for humans of 30 days or more are preferred).
While not significant at the p.ltoreq.0.05 level, the pancreatic
lipase IgY treated animals did show reduced mean weight as compared
to the preimmune IgY treated animals.
[0060] From the forgoing is should be clear that the present invention
provides compositions, methods and uses for an effective medicament
comprising an antagonist to colipase.
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