Note: Descriptions are shown in the official language in which they were submitted.
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Colonic Delivery Using Zn/Pectin Beads With a Eudragit Coating
Field of the Invention
The present invention is in the area of oral drug delivery systems to
administer active
agents, such as metallo-specific enzymes, to the colon.
Background of the Invention
Drug delivery systems that specifically deliver active agents to the colon
have been
recognized as having important therapeutic advantages. A large number of
colonic conditions
could effectively be treated more efficaciously if the active ingredient is
released locally.
Examples of such colonic disorders include Crohn's disease, ulcerative
colitis, colorectal
cancer and constipation.
Colonic release can also benefit patients when, from a therapeutic point of
view, a
delay in absorption is necessary. Examples include the treatment of disorders
such as
nocturnal asthma or angor (Kinget R. et al. (1998), Colonic Drug Targeting,
Journal of Drug
Targeting, 6, 129).
Colonic release can also be used to administer therapeutically active
polypeptides.
Polypeptides are typically administered by injection, because they are
degraded in the
stomach. Because injection is painful, research efforts have focused on using
the colon as a
site of absorption for active polypeptides, -including analgesics,
contraceptives, vaccines,
insulin, and the like. The absorption of polypeptides in the colon appears to
be more
effective than in other sites in the digestive tract. This is particularly due
to the relatively
weak proteolytic activity in the small intestine and the absence of peptidase
activity
associated with the membrane of the colonic epithelial cells.
Following their oral administration, antibiotics pass through the stomach and
are then
absorbed in the small intestine to diffuse in the whole organism and treat the
infectious
outbreak site(s) for which they have been administered. All the same, a
fraction of antibiotics
ingested (the importance of this fraction varies with the characteristics of
each antibiotic) is
not absorbed and continues its progress to the colon before being eliminated
in the stool.
These residual antibiotics are combined, in the large intestine, with a
fraction of the
antibiotics absorbed, but which are re-excreted in the digestive tract by
means of biliary
elimination. This fraction is of variable importance as a function of
metabolism and
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elimination pathways for each antibiotic. Finally, for certain antibiotics, a
fraction of the dose
absorbed is directly eliminated from the blood through the intestinal mucosa
back into the
lumen of the digestive tract, a good example is known with ciprofloxacin.
Thus, whether
administered orally or parenterally, a residual fraction of active antibiotics
is generally found
in the colon. This is the case, to varying degrees, for the great majority
antibiotics from the
various families used in therapeutics, with the sole notable exception of
antibiotics from the
amino-glycoside family for which intestinal excretion is negligible. For other
antibiotics,
intestinal excretion of a residual antibiotic activity will have a variety of
consequences, all
harmful. Indeed, the colon harbors a complex and very dense bacterial
ecosystem (several
hundreds of different bacterial species; more than 1011 bacteria per gram of
colonic content)
which will be affected by the arrival of active antibiotic residues. The
following can be
observed:
1. Flora imbalance which is the main cause of banal diarrhea occurring
following
antibiotic treatments (Bartlett J. G. (2002) Clinical practice. Antibiotic
associated diarrhea,
New England Journal of Medicine, 346, 334). Even though this diarrhea is
generally not
serious and ceases rapidly, either spontaneously, or upon completion of the
antibiotic
treatment, it is adversely perceived by patients and adds to the discomfort of
the original
illness for which the antibiotic was prescribed;
2. interference with the resistance to colonization by exogenic bacteria (or
"barrier
effect") with possible risk of infection, such as alimentary salmonella
intoxication (Holmberg
S.D. et al. (1984) Drug resistant Salmonella from animals fed antimicrobials,
New England
Journal of Medicine, 311, 617);
3. selection of microorganisms resistant to the antibiotic. These
microorganisms can
be of various types:
a) first they can be pathogenic bacteria such as for example, Clostridium di,
fficile, a
species capable of secreting toxins causing a form of colitis known as
pseudomembranous
colitis (Bartlett J. G. (1997) Clostridium dicile infection: pathophysiology
and diagnosis,
Seminar in Gastrointestinal Disease, 8, 12);
b) they can also be microorganisms that are relatively weakly pathogenic, but
whose
multiplication can lead to an associated infection (vaginal Candidosis or
Escherichia coli
resistant cystitis).
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c) they can finally be non-pathogenic commensal drug-resistant bacteria whose
multiplication and fecal elimination will increase dissemination of antibiotic
resistance in the
environment. It is well documented that antibiotic resistance genes are
carried by mobile or
transposable genetic elements that may contain up to 5 or 6 antibiotic
resistance genes, and
are readily transmitted to other bacteria, even across species. Consequently,
these resistant
commensal bacteria may constitute an important source leading to drug
resistance for
pathogenic species. This risk is currently considered seminal in terms of the
disquieting
character of the evolution towards drug multiresistance by numerous species
pathogenic for
humans.
It would therefore be desirable to have drugs and drug delivery systems that
would act
to reduce the quantity of residual antibiotics reaching the colon following
oral or parenteral
antibiotic therapy.
Numerous strategies exploiting the diverse physiological parameters of the
digestive
tract have been devised with the aim to release active ingredients in the
colon. These
strategies have focused on drug delivery systems based on (1) using polymers
that are
sensitive to variations in pH, (2) time-dependent drug release forms, (3)
prodrugs or polymers
degradable by bacteria of the intestinal flora.
It would be advantageous to have additional drug delivery systems which enable
the
administration of active agents to the colon, including but not limited to
agents that reduce the
quantity of residual antibiotics in the colon. The present invention provides
such drug
delivery systems.
Summary of the Invention
Drug delivery systems that can deliver prophylactic, therapeutic and/or
diagnostic
agents to the colon are disclosed. The systems include pectin beads
crosslinked with a metal
cation for instance zinc or any divalent cation of interest, which beads are
then coated with
Eudragitg-type polymers.
Further embodiments are set forth in the claims which are herein incorporated
in their
entirety by reference.
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The drug delivery systems are orally administrable, but can deliver the active
agents to
the colon. In some embodiments, they can administer the agents to various
positions in the
gastro-intestinal tract, including the colon.
In one embodiment, the therapeutic agent is an agent capable of reducing the
residual
quantity of residual antibiotics reaching the colon following oral or
parenteral antibiotic
therapy, such as a metallo-dependent enzyme. Application is illustrated for (3-
la.ctamase L 1
from Stenotrophomonas maltophilia. However, one can also use P-lactamases
which are not
metallo-enzymes (classes A, C or D). Moreover, one can use enzymes, metallo-
dependent or
otherwise, to inactivate other classes of antibiotics such as macrolides,
quinolones and
fluoriquinolones, glycopeptides, lipopeptides, cyclins, oxazolidinones, and
other classes of
antibiotics. The enzymes can have the full sequence of the native enzyme, or
can be truncated
or otherwise modified so long as they maintain acceptable activity. By
delivering agents
capable of reducing the quantity of residual antibiotics reaching the colon
following oral or
parenteral antibiotic therapy, one can limit the development of bacterial
resistance.
In other embodiments, the therapeutic agents include, but are not limited to:
= peptides and proteins (including, but not limited to, enzymes, hormones,
cytokines, lymphokines, growth factors, antibodies, and the like) whether
natural, synthetic or recombinant;
= nucleic acids and compounds including elements from nucleic acids
(including, but not limited to, plasmids, oligonucleotides
(oligoribonucleotides, deoxyribonucleotides, SiRNAs or ShRNAs of various
lengths, and mixed molecules, including natural and/or modified bases, and
optionally containing substitutions and modifications), as well as peptide
nucleic acids;
= complex structures of natural, recombinant or synthetic origin, including,
but
not limited to viruses (including DNA and RNA viruses, viruses targeting
animal cells, viruses targeting vegetal cells, or viruses targeting bacteria
better
known as bacteriophages), bacteria (in whatever form, including spores),
mycoplasms, yeasts and other unicellular eucaryotes (in whatever form,
including spores)
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= natural, synthetic or mixed chemical molecules or mixtures thereof of any
size,
class or structure;
= compounds for use in diagnosis, treatment or investigation of humans and
animals for whatever reason or condition, including infectious diseases
5 (including but not limited to those of bacterial and viral origin),
inflammatory
diseases, cancers;
= compounds for assisting, complementing or modifying a treatment with anti-
infectious agents, anti-inflammatory, anti-cancer agents, immuno-modifying
agents, and the like, particularly where such assistance, complementation, or
modification relates to the ability to block or modulate the activity of
receptors
in the colon, or inactivate other therapeutic agents which might modulate the
activity of receptors in the colon.
Colon-specific delivery is obtained by formulating a prophylactic,
therapeutic, and/or
diagnostic agent, such as a metallo-dependent enzyme or other agent capable of
reducing the
quantity of residual antibiotics reaching the colon following oral or
parenteral antibiotic
therapy, with specific polymers that degrade in the colon, such as pectin. The
pectin is
gelled/crosslinked with a cation such as a zinc cation. The formulation,
typically in the form
of ionically crosslinked pectin beads, is subsequently coated with a specific
polymer such as a
Eudragit polymer.
The delivery can be modulated to occur at various pre-selected sites of
delivery within
the intestinal tract by gelling/crosslinking a mixture of the prophylactic,
therapeutic, and/or
diagnostic agent and pectin, with divalent metallic cations such as Ca2} or
Zn2+.
Previous efforts have focused on coating pectin beads with cationic polymers
such as
polyethylene imine (PEI), chitosan or other cationic polymers, to prevent the
pectin beads
from degrading in the upper gastro-intestinal tract. Such efforts are
described, for example, in
U.S. Patent Application No. 10/524,318, and U.S. Patent Application No.
60/651,352, the
contents of which are hereby incorporated by reference.
The present invention relates to coating the pectin beads with Eudragit
polymers
such as FS30D, L30D (also known as L30D-55), NE30D, mixtures thereof or other
desirable
types of Eudragit(t polymers to achieve the desired release of the
prophylactic, therapeutic
and/or diagnostic agent at predefined levels of the gastro-intestinal tract
(GIT).
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When the Eudragit coating is dissolved, according to certain parameters such
as pH
or time, the beads are preferentially degraded by pectinolytic enzymes found
in the lower part
of the intestinal tract. Degradation of pectin then releases the prophylactic,
therapeutic and/or
diagnostic agent encapsulated within the bead.
One aspect of the invention is to provide a stable metallo-enzyme formulation
for the
lower intestinal or colonic delivery of such an enzyme. The use of zinc
cations to crosslink
the pectin is particularly preferred when specific metallo-dependent enzymes,
which are Zn2+
dependent, could interact with other cationic species if they were used to gel
the pectin beads.
Such interactions could drastically affect the activity of such metallo-
dependent enzymes.
Accordingly, one embodiment of the drug delivery system involves using Zn2+
ions as a
crosslinking agent for the pectin beads and in association with Zn2+-dependent
enzymes
which are very sensitive to the presence of other competitive cations. Of
course, if the
enzymes are dependent on other metal cations, such other metal cations (if
they have a
valence exceeding +1) can be used to crosslink the pectin.
The processes to obtain such beads can involve specific process conditions,
such as
time for gelification, washing, and drying that can be optimized to provide
the highest quality
beads, with optimized efficacy in vitro and in vivo. Therefore, another
embodiment of the
invention relates to processes for preparing zinc-crosslinked and Eudragit -
coated pectin
beads.
Brief Description of the Figures
Figure 1 is a graph showing the efficiency of water rinsing to remove excess
metallic
cations from a formulation of (3-lacatamase L1 in pectin beads erosslinked
with zinc acetate,
measured in terms of conductivity (mS/cm) per sample following various washes.
Figure 2 is a graph showing the effect of gelification time, rinsing process,
and drying
time on recovery of (3-lactamase L 1 activity.
Figure 3 is a graph showing the enzymatic activity of P-lactamase L i using
CENTA as
a substrate, measured in terms of response (OD/min) versus LI concentration (
g/m1).
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Figure 4 is a series of scanning electron micrographs showing Eudragit-coated
beads
prepared using the methods described herein, and a cross-section of the beads
showing the
approximate thickness of Eudragit layer.
Figure 5 is a chart showing the release kinetics of (3-lactamase L l from
uncoated
beads, and Eudragit-coated beads with or without hydroxypropyl methyl
cellulose (HPMC)
pre-coating, measured in terms of activity ( g/mg beads) versus time
(minutes). Blue
triangles represent uncoated beads; red circles represent beads coated with
40% Eudragit
L30D-55 without pre-coating; green squares represent beads pre-coated with 5%
HPMC and
coated with 40% Eudragit L30D-55.
Figure 6 is a chart showing the hydrolysis of amoxicillin by uncoated, and
Eudragit-
coated beads with or without a hydroxypropyl methylcellulose (HPMC) pre-
coating,
measured in terms of residual amoxicillin (%) versus time (minutes). Blue
triangles represent
uncoated beads; red circles represent beads coated with 40% Eudragit L30D-55
without pre-
coating; green squares represent beads pre-coated with HPMC and coated with
Eudragit
L30D-55.
Figure 7 is a chart showing the effect of Eudragit-coated pectin beads
containing (3-
lactamase L l on the emergence of antibiotic-resistant bacteria in piglets
treated with
amoxicillin, measured in terms of amoxicillin resistant bacteriacae (%) versus
treatment
duration (days). Blue triangles represent untreated animals (n=12); red
diamonds represent
animals treated with amoxicillin and placebo pectin beads (n=12); green
squares represent
animals treated with amoxicillin together with Eudragit-coated pectin beads
containing
~3-
lactamase L 1 (n=4).
Detailed Description of the Invention
The drug delivery systems described herein will be better understood with
reference to
the following detailed description.
1. Pectin Beads
The pectin beads are formed from pectin, zinc ions, and further coating with
Eudragit
(t polymers and encapsulate one or more active agents.
Stability and protection of the pectin beads in gastric medium and intestinal
medium is
ensured by the Eudragit (t polymer coating. In contrast, uncoated beads of
pectin tend not be
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stable in such an environment and may not adequately protect their contents
against
degradation and/or inactivation. The Eudragit coating ensures that they
resist long enough
so that their contents able to reach the colon intact.
Pectin
Pectin is a polysaccharide isolated from the cellular walls of superior
plants, used
widely in the agricultural food industry (as a coagulant or thickener for
jams, ice creams and
the like) and pharmaeeutics. It is polymolecular and polydisperse. Its
composition varies
depending on the source, extraction conditions and environmental factors.
Pectins are principally composed of linear chains of beta-l,4-(D)-galacturonic
acid, at
times interspersed by units of rhamnose. The carboxylic groups of galacturonic
acid can be
partially esterified to yield methylated pectins. Two types of pectins are
distinguished
according to their degree of methylation (DM: number of methoxy groups per 100
units of
galacturonic acid):
- highly methylated pectin (HM: high methoxy) where the degree of methylation
varies between 50 and 80%. It is slightly soluble in water and forms gels in
acidic medium
(pH<3.6) or in the presence of sugars;
- weakly methylated pectin (LM: low methoxy), with a degree of methylation
varying
from 25 to 50%. More soluble in water than HM pectin, it gives gels in the
presence of
divalent cations such as Ca2+ ions. Indeed, Ca2+ ions form "bridges" between
the free
carboxylated groups of galacturonic acid moities. The network that is formed
has been
described by Grant et al. under the name of egg-box model (Grant G.T. et al.
(1973)
Biological interactions between polysaccharides and divalent cations: the egg-
box model,
FEBS Letters, 32, 195).
There are also amidated pectins. Treatment of pectin by ammonia transfoms some
methyl carboxylate groups (-COOCH3) into carboxamide groups (-CONH2). This
amidation
confers novel properties to the pectins, in particular better resistance to
variations in pH.
Amidated pectins tend to be more tolerant to the variations in pH, and have
also been studied
for the manufacture of matricial tablets for colonic delivery (Wakerly Z. et
al. (1997) Studies
on amidated pectins as potential carriers in colonic drug delivery, Journal of
Pharmacy and
Pharmacology. 49, 622).
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Pectin is degraded by enzymes originating from higher plants and various
microorganisms (fungi, bacteria, and the like) among which bacteria from the
human colonic
flora. The enzymes produced by the microflora encompass a mixture of
polysaccharidases,
glycosidases and esterases.
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Zinc Cations
Divalent zinc cations from various zinc salts can be used to crosslink pectin.
Examples include zinc sulfate, zinc chloride, and zinc acetate.
5 In the present invention, agents for enteric coatings are preferably
methacrylic acid-
alkyl acrylate copolymers, such as Eudragit(t polymers.
Eudra ig t polymers
The coating of drug-loaded cores such as tablets, capsules, granules, pellets
or crystals
offers many advantages over uncoated counterparts, such as higher
physicochemical stability,
10 better compliance and increased therapeutic efficiency of the active
ingredients. Indeed, the
effectiveness of a medication depends not only on the actives it contains, but
also on
formulation and processing.
Poly(meth)acrylates have proven particularly suitable as coating materials.
These
polymers, typically used in amounts of only a few milligrams, are
pharmacologically inactive,
i.e. are excreted unchanged.
EUDRAGIT is the trade name for copolymers derived from esters of acrylic and
methacrylic acid, whose properties are determined by functional groups. The
individual
EUDRAGIT grades differ in their proportion of neutral, alkaline or acid
groups and thus in
terms of physicochemical properties. The skillful use and combination of
different
EUDRA.GIT9 polymers offers ideal solutions for controlled drug release in
various
pharmaceutical and technical applications. EUDRAGIT provides functional films
for
sustained-release tablet and pellet coatings. The polymers are described in
international
pharmacopeias such as Ph.Eur., USP/NF, DMF and JPE.
EUDRA.GIT polymers can provide the following possibilities for controlled
drug release:
= Gastrointestinal tract targeting (gastroresistance, release in the colon)
= Protective coatings (taste and odor masking, protection against moisture)
= Delayed drug release (sustained-release formulations).
EUDRAGITV polymers are available in a wide range of different concentrations
and physical
forms, including aqueous solutions, aqueous dispersion, organic solutions, and
solid
substances.
The pharmaeeutical properties of EUDRAGIT polymers are determined by the
chemical properties of their functional groups. A distinction is made between:
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= poly(meth)acrylates, soluble in digestive fluids (by salt formation)
EUDRAGIT L (Methacrylic acid copolymer), S (Methacrylic acid copolymer), FS
and E (basic butylated methacrylate copolymer) polymers with acidic or
alkaline groups
enable pH-dependent release of the active ingredient.
Applications: from simple taste masking via resistance solely to gastric
fluid, to
controlled drug release in all sections of the intestine.
= poly(meth)acrylates, insoluble in digestive fluids
EUDRAGIT RL and RS (ammonio methacrylate copolymers) polymers with
alkaline and EUDRAGIT NE polymers with neutral groups enable controlled time
release
of the active by pH-independent swelling.
Informations on Eudragit polyemers are found at:
http://www.pharma-polymers.com/pharmapolymerslenleudragit/entericcoatings/
and at
http://www.pharma-polymers.
com/pharmapolymers/enleudragit/regulatorytoxicology/
Enteric Coatings: Gastoresistance and Release in the Colon
Enteric EUDRAGIT coatings provide protection against drug release in the
stomach
and enable controlled release in the intestine. Targeted drug release in the
gastrointestinal
tract is recommended for particular applications or therapeutic strategies,
for example when
the drug is sparingly soluble in the upper digestive tract, or when the drug
may be degraded
by gastric fluid. Secondly, this dosage form is very patient-friendly as it
does not stress the
stomach and the number of doses of the therapeutic drug can be considerably
reduced, thanks
to prolonged delivery. The dominant criterion for release is the pH-dependent
dissolution of
the coating, which takes place in a certain section of the intestine (pH 5 to
over 7) rather than
in the stomach (pH 1-5). For these applications, anionic EUDRAGITO grades
containing
carboxyl groups can be mixed with each other. This makes it possible to finely
adjust the
dissolution pH, and thus to define the drug release site in the intestine.
EUDRAGIT L and S
grades are suitable for enteric coatings. EUDRA.GIT FS 30 D (aqueous
dispersion of an
anionic copolymer based on methyl acrylate, methyl methacrylate and
methacrylic acid) is
specifically used for controlled release in the colon.
Application benefits of enteric EUDRAGIT(t coatings include:
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= pH-dependent drug release
= protection of actives sensitive to gastric fluid
= protection of the gastric mucosa from aggressive actives
= increase in drug effectiveness
= good storage stability
= controlled release in the colon/Gl targeting
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Active Agents
The active agent can be an anti-infectious, for example antibiotics, anti-
inflammatory
compounds, anti-histamines, anti-cholinergics, antivirals, antimitotics,
peptides, proteins,
enzymes, nucleic acids (RNA or DNA), peptide nucleic acids, plasmids, genes,
anti-sense
oligonucleotides, interfering RNAs, ribozymes, small molecules with specific
binding
capacities or activities (such as targeted chemotherapeutics), diagnostic
agents,
immunosuppressive agents, viruses, bacteria, other micro-organisms or
eukaryotic cells.
The active agent can be introduced into the drug delivery system as a powder,
a
solution, a suspension, or complexed with a solubilizing agent, such as a
cyclodextrin or any
other suitable compound.
Some of the active agents described herein can be administered in the form of
prodrugs. Prodrugs have been widely studied for the colonic targeting of
various active
ingredients (such as steroid and non-steroid anti-inflammatory drugs, and
spasmolytics).
These systems are based on the capacity of the enzymes produced by the colonic
flora to act
on the prodrugs to release the active form of the active ingredient.
The prodrugs can be based on the action of bacterial azoreductases, so that
the active
agents are targeted to the colon with the drug delivery systems described
herein, and the
active agents are formed by reaction of the prodrug with a bacterial
azoreductase, which
provides a dual mechanism for ensuring that the drugs are administered to the
colon.
Representative chemistry for forming such prodrugs is described, for example,
in Peppercorn
M.A. et al. (1972) The role of intestinal bacteria in the metabolism of
salicylazosulfapyridin,
The Journal of Pharmacology and Experimental Therapeutics, 181, 555 and 64,
240.
Another approach consists in using bacterial hydrolases such as glycosidases
and
polysaccharidases (Friend D.R. (1995) Glycoside prodrugs: novel
pharmacotherapy for
colonic diseases, S.T.P.Pharma Sciences, 5, 70; Friend D.R. et al. (1984) A
colon-specific
drug-delivery system based on drug glycosides and the glycosidases of colonic
bacteria,
Journal of Medicinal Chemistry, 27, 261; Friend D.R. et al. (1985) Drug
glycosides: potential
prodrugs for colon-specific drug delivery, Journal of Medicinal Chemistry, 28,
51; and Friend
D.R. et al. (1992) Drug glycosides in oral colon-specific drug delivery,
Journal of Controlled
Release, 19, 109). Prodrugs have thus been developed by coupling, for example,
sugar with
steroids (glucose, galactose, cellobiose, dextrane (international application
WO 90/09168)),
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cyclodextrins Hirayama F. et al. (1996) In vitro evaluation of Biphenylyl
Acetic Acid-beta -
Cyclodextrin conjugates as colon-targeting prodrugs: drug release behavior in
rat biological
media, Journal of Pharmacy and Pharmacology, 48, 27).
a) Agents that Inactivate Antibiotics
In one embodiment, the active agent is an enzyme capable of inactivating
antibiotics
in the colon. Any agent that inactivates an antibiotic can be administered.
When the antibiotic is a beta-lactam antibiotic, (3-lactamases can be used.
The selected
enzyme, i.e. (3-lactamase Ll, a Zn2"-dependent (3-lactamase from
Stenotrophomonas
maltophilia, was chosen from a series of P-lactamases because its
characteristics showed the
best profile for the targeted application. Also, it has been demonstrated to
have an excellent
stability profile. The characteristics of various P-lactamases evaluated are
described hereafter.
There are a variety of enzymes that are known to be metallo-dependent, in
addition to
the (3-lactamase L1 enzyme discussed above. When it is desired to administer
such enzymes
to a patient via oral administration, care must be taken to avoid having the
enzyme digested in
the stomach or upper intestine. Accordingly, the drug delivery system
described herein can
advantageously be used to deliver such metallo-dependent enzymes. The cation
used to
crosslink the pectin comprises the cation on which the enzyme depends.
One representative enzyme is (3-lactamase L1, a Zn2+-dependent (3-lactamase
from
Stenotrophomonas maltophilia, which was chosen from a series of (3-lactamases
because its
characteristics showed the best profile for the targeted application. Also, it
has demonstrated
to have an excellent stability profile. The characteristics of various (3-
lactamases evaluated are
described hereafter.
When the antibiotic is from another class of antibiotics, enzymes or other
molecules
that inactivate such antibiotics can be used. One such example would be to use
an
erythromycin esterase to inactivate macrolide antibiotics.
One representative erythromycin esterase is that disclosed by Andremont A. et
al.
((1985) "Plasmid mediated susceptibility to intestinal microbial antagonisms
in Escherichia
coli," Infect. Immun. 49(3):751), the contents of which are hereby
incorporated by reference.
When the antibiotic is a quinolone, the active agent can be one capable of
inactivating
quinolones. Representative agents include those disclosed by Chen, Y et al.
((1997)
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"Microbicidal models of soil metabolisms biotransformations of danofloxacin,"
Journal of
Industrial Microbiology and Biotechnology 19:378).
Patients can be treated with combinations of these agents.
Representative P-lactamase enzymes, and their efficacy on various antibiotics,
is
5 shown in Table 1.
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Table 1
Antibiotics FEZ-1 * L-1(wt) **
Kcat (s t) Km (gM) Kcat/Km Kcat (s ') Km (!tM) Kcat/Km
( M/ s'1 (M/ s"i)
Penicillin
Benzylpenicillin 70 590 0.11 600 38 16
Ampicillin >5.5 >5000 0.011 520 55 9.5
Cabenicillin 35 1600 0.023
Pipericillin 50 4200 0.012
Azlocillin Mezlocillin
Ticarcillin >65 >5000 0.013
Timocillin
Ce halos orin
Cephaloridin 16 1000 0.016
Cephalothin 300 120 2.5 82 8.9 9.2
Cefoxitin 1 3 0.33
Cefuroxin
Cefotaxim 165 70 2.4 270 10 27
Ceftazidin
Cefepim >6 >1000 0.006
Cefpirom
Nitrocefin 90 100 0.9 41 4 10
Moxalactam 3 18 0.17
Carbapenem
Imipenem >200 >1000 0.2 370 57 6.5
Meropenem 45 85 0.5 157 15 10
Biapenem 70 >1000 0.07 134 32 4.2
Monobactams
Aztreonam
Carumonam
Mechanism-
Based
Inactivators
Sulbactam
Tazobactam 40 700 1.06
Clavulanic Acid <0.01 >1000 <0.00001 11 22 0.5
*(Mercuri et al., Antimicrob. Agents. Chemother. 2001 Apr; 45(4):1254-1262)
** (Carenbauer et al., B1VC Biochem. 2002; 3:4. Epub 2002 Feb. 13; Frere,
2005,
unpublished data)
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Table 1 cont'd
Antibiotics IMP-1 *** VIM-2
Kcat ($ t) Km (t-tM) Koat/Km **** K. (}iM) Koat/Km
( M/ s` K,.r (s 1 ( Ml s"r)
Penicillin
Benzylpenicillin 320 520 0.62 56 49 1.114
Ampicillin 950 200 4.8 125 90 1.4
Cabenicillin 0.02 185 205 0.9
Pipericillin 0.72 300 125 2.4
Azlocillin 200 200 1
Mezlocillin 200 125 1.4
Ticarcillin 1.1 740 0.0015 180 125 1.6
Timocillin >2000 <0.0001 7.7 390 0.002
Ce halos porin
Cephaloridin 53 22 2.4 140 50 2.8
Ce halothin 48 21 2.4 130 11 12
Cefoxitin 16 8 2 15 13 1.2
Cefuroxin 8 37 0.22 8 20 0.4
Cefotaxim 1.3 4 0.45 70 12 5.8
Ceftazidin 8 44 0.18 3.6 72 0.05
Cefepim 7 11 0.66 >40 >400 0.1
Cefpirom 9 14 0.64 180 180 1
Nitrocefin 63 27 2.3 770 18 43
Moxalactam 90 55 1.6
Carbapenem
Imipenem 46 39 1.2 34 9 3.8
Meropenem 50 10 5 2 2.5
Biapenem 160 28 6 8.5 15 0.55
Monobactams
Aztreonam >0.01 >1000 <0.0001 <0.01 >1000 <0.0001
Carumonam >0.01 >1000 <0.0001
Mechanism-
Based
Inactivators
Sulbactam 23 320 0.072
Tazobactam 28 875 0.032
Clavulanic Acid
*** (Murphy et al., 2003, Antimicrob. Ag. Chemother.2003 Feb, 47(2):582-7;
Laraki et al.,
Antimicrob. Ag. Chemother. 1999 Apr., 43(4):902-6)
****(Docquier et al., J. Antimicrob. Chemother. 2003 Feb., 51(2):257-266)
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b) Agents that Treat Colon Cancer
When the drug delivery systems are used to treat colon cancer, any type of
antitumor agent can be used. The anti-tumor agents can be, for example, anti-
proliferative agents, agents for DNA modification or repair, DNA synthesis
inhibitors,
DNA/RNA transcription regulators, RNA processing inhibitors, agents that
affect
protein expression, synthesis and stability, agents that affect protein
localization or
their ability to exert their physiological action, agents that interfere with
protein-
protein or protein-nucleic acid interactions, agents that act by RNA
interference,
receptor binding molecules of any chemical nature (including small molecules
and
antibodies), targeted toxins, enzyme activators, enzyme inhibitors, gene
regulators,
HSP-90 inhibitors, molecules interfering with microtubules or other
cytoskeletal
components or cell adhesion and motility , agents for phototherapy, and
therapy
adjuncts.
Representative antiproliferative agents include N-acetyl-D-sphingosine (C2
ceramide), apigenin, berberine chloride, dichloromethylenediphosphonic acid
disodium salt, loe-emodine, emodin, HA 14-1, N-hexanoyl-D-sphingosine (C6
ceramide), 7b-hydroxycholesterol, 25-hydroxycholesterol, hyperforin,
parthenolide,
and rapamycin.
Representative agents for DNA modification and repair include aphidicolin,
bleomycin sulfate, carboplatin, carmustine, chlorambucil, cyclophosphamide
monohydrate, cyelophosphamide monohydrate ISOPAC , cis-diammineplatinum(II)
dichloride (Cisplatin), esculetin, melphalan, methoxyamine hydrochloride,
mitomycin
C, mitoxantrone dihydrochloride, oxaliplatin, and streptozocin.
Representative DNA synthesis inhibitors include ( )amethopterin
(methotrexate), 3 -amino- 1,2,4-benzotriazine 1,4-dioxide, aminopterin,
cytosine b-D-
arabinofuranoside (Ara-C), cytosine b-D-arabinofuranoside (Ara-C)
hydrochloride, 2-
fluoroadenine-9-b-D-arabinofuranoside (Fludarabine des-phosphate; F-ara-A), 5-
fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, 6-
mercaptopurine,
and 6-thioguanine.
Representative DNA/RNA transcription regulators include actinomycin D,
daunorubicin hydrochloride, 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside,
doxorubicin hydrochloride, homoharringtonine, and idarubicin hydrochloride.
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Representative enzyme activators and inhibitors include forskolin, DL-
aminoglutethimide, apicidin, Bowman-Birk Inhibitor, butein, (S)-(+)-
camptothecin,
curcumin, (-)-deguelin, (-)-depudecin, doxycycline hyclate, etoposide,
formestane,
fostriecin sodium salt, hispidin, 2-imino-l-imidazolidineacetic acid
(Cyclocreatine),
oxamflatin, 4-phenylbutyric acid, roscovitine, sodium valproate, trichostatin
A,
tyrphostin AG 34, tyrphostin AG 879, urinary trypsin inhibitor fragment,
valproic acid
(2-propylpentanoic acid), and XK469.
Representative gene regulators include 5-aza-2'-deoxycytidine, 5-azacytidine,
cholecalciferol (Vitamin D3), ciglitizone, cyproterone acetate, 15-deoxy-D12
14-
prostaglandin J2, epitestosterone, flutamide, glycyrrhizic acid ammonium salt
(glycyrrhizin), 4-hydroxytamoxifen, mifepristone, procainamide hydrochloride,
raloxifene hydrochloride, all trans-retinal (vitamin A aldehyde), retinoic
acid (vitamin
A acid), 9-cis-retinoic acid, 13-cis-retinoic acid, retinoic acid p-
hydroxyanilide, retinol
(Vitamin A), tamoxifen, tamoxifen citrate salt, tetradecylthioacetic acid, and
troglitazone.
Representative HSP-90 inhibitors include 17-(allylamino)-17-
demethoxygeldanamycin and geldanamycin.
Representative microtubule inhibitors include colchicines, dolastatin 15,
nocodazole, taxanes and in particular paclitaxel, podophyllotoxin, rhizoxin,
vinblastine sulfate salt, vincristine sulfate salt, and vindesine sulfate salt
and
vinorelbine (Navelbine) ditartrate salt.
Representative agents for performing phototherapy include photoactive
porphyrin rings, hypericin, 5-methoxypsoralen, 8-methoxypsoralen, psoralen and
ursodeoxycholic acid.
Representative agents used as therapy adjuncts include amifostine, 4-amino-
1,8-naphthalimide, brefeldin A, cimetidine, phosphomycin disodium salt,
leuprolide
(leuprorelin) acetate salt, luteinizing hormone-releasing hormone (LH-RH)
acetate
salt, lectin, papaverine hydrochloride, pifithrin-a, (-)-scopolamine
hydrobromide, and
thapsigargin.
The agents can also be anti-VEGF (vascuiar endothelial growth factor) agents,
as such are known in the art. Several antibodies and small molecules are
currently in
clinical trials or have been approved that function by inhibiting VEGF, such
as
Avastin (Bevacizumab), SU5416, SU11248 and BAY 43-9006. The agents can also
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be directed against growth factor receptors such as those of the EGF/Erb-B
family
such as EGF Receptor (Iressa or Gefitinib, and Tarceva or Erlotinib), Erb-B2,
receptor
(Herceptin or Trastuzumab), other receptors (such as Rituximab or
RituxanlMabThera), tyrosine kinases, non-receptor tyrosine kinases, cellular
5 serine/threonine kinases (including MAP kinases), and various other proteins
whose
deregulation contribute to oncogenesis (such as small/Ras family and
large/heterotrimeric G proteins). Several antibodies and small molecules
targeting
those molecules are currently at various stages of development (including
approved
for treatment or in clinical trials).
10 Some of the most commonly used antitumor agents currently in use or in
clinical trials include paclitaxel, docetaxel, tamoxifen, vinorelbine,
gemcitabine,
cisplatin, etoposide, topotecan, irinotecan, anastrozole, rituximab,
trastuzumab,
fludarabine, cyclophosphamide, gentuzumab, carboplatin, interferons, and
doxorubicin. The most commonly used anticancer agent is paclitaxel, which is
used
15 alone or in combination with other chemotherapy drugs such as: 5-FU,
doxorubicin,
vinorelbine, cytoxan, and cisplatin.
Combination therapy can be provided by combining two or more of the above
compounds.
20 c) Agents that Treat Chrohn's Disease
There are several therapeutic approaches for treating Chrohn's Disease. Most
people are first treated with drugs containing mesalamine, a substance that
helps
control inflammation. Sulfasalazine is the most commonly used of these drugs.
Patients who do not benefit from it or who cannot tolerate it may be put on
other
mesalamine-containing drugs, generally known as 5-ASA agents, such as Asacol,
Dipentum, or Pentasa. Corticosteroids are often administered to control
inflammation.
Immunosuppressive agents are also used to treat Crohn's disease. Most
commonly prescribed are 6-mercaptopurine and a related drug, azathioprine.
Immunosuppressive agents work by blocking the immune reaction that contributes
to
inflammation.
Patients can be treated with combinations of these agents, for example,
combinations of corticosteroids and immunosuppressive drugs.
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The U.S. Food and Drug Administration has approved the drug infliximab
(brand name, Remicade) for the treatment of moderate to severe Crohn's disease
that
does not respond to standard therapies (mesalamine substances,
corticosteroids,
immunosuppressive agents) and for the treatment of open, draining fistulas.
Infliximab is an anti-tumor necrosis factor alpha (TNF-alpha) antibody. This
and
other anti-TNF-alpha agents can be used to remove TNF-alpha from the colon,
thereby preventing inflammation, without the side effects that might result if
TNF-
alpha were removed from the blood stream outside of the colon.
Antidiarrheal agents are often also administered, including diphenoxylate,
loperamide, and codeine.
d) Agents that Treat Ulcerative Colitis
The agents that are used to treat ulcerative colitis overlap with those used
to
treat Chrohn's Disease. Examples include aminosalicylates, drugs that contain
5-
aminosalicyclic acid (5-ASA), to help control inflammation, such as
sulfasalazine,
olsalazine, mesalamine, and balsalazide. They also include corticosteroids
such as
prednisone and hydrocortisone, and immunomodulators such as azathioprine and 6-
mercapto-purine (6-MP), cytokines, interleukins, and lymphokines. Cyclosporine
A
may be used with 6-MP or azathioprine to treat active, severe ulcerative
colitis. Anti-
TNF-alpha agents, the thiazolidinediones or glitazones, including
rosiglitazone and
pioglitazone, can also be used.
eLgents that Treat Constipation/Irritable Bowel Syndrome
Constipation, such as that associated with irritable bowel syndrome, is often
treated using stimulant laxatives, osmotic laxatives such as Lactulose and
MiraLax,
stool softeners (such as mineral oil or Colace), bulking agents (such as
Metamucil or
bran). Agents such as Zelnorm (also called tegaserod) can be used to treat IBS
with
constipation. Additionally, anticholinergic medications such as Bentyl and
Levsin
have been found to be helpful in alleviating the bowel spasms of IBS.
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f) Protein and Peptide Drugs
The drug delivery systems can be used to orally administer proteins and
peptides that might otherwise be degraded if orally administered, and which
might
otherwise have to be administered intramuscularly or intravenously.
Examples of protein and peptide drugs useful in the present invention include:
Adrenocorticotropic hormone (ACTH) peptides including, but not limited to,
ACTH, human; ACTH 1-10; ACTH 1-13, human; ACTH 1-16, human; ACTH 1-17;
ACTH 1-24, human; ACTH 4-10; ACTH 4-11; ACTH 6-24; ACTH 7-38, human;
ACTH 18-39, human; ACTH, rat; ACTH 12-39, rat; beta-cell tropin (ACTH 22-39);
biotinyl-ACTH 1-24, human; biotinyl-ACTH 7-38, human; corticostatin, human;
corticostatin, rabbit; [Met(02)4, DLys8, Pheg] ACTH 4-9, human;
[Met(0)4,DLysg,
Phe9] ACTH 4-9, human; N-acetyl, ACTH 1-17, human; and ebiratide.
Adrenomedullin peptides including, but not limited to, adrenomedullin,
adrenomedullin 1-52, human; adrenomedullin 1-12, human; adrenomedullin 13-52,
human; adrenomedullin 22-52, human; pro-adrenomedullin 45-92, human; pro-
adrenomedullin 153-185, human; adrenomedullin 1-52, porcine; pro-
adrenomedullin
(N-20), porcine; adrenomedullin 1-50, rat; adrenomedullin 11-50, rat; and
proAM-
N20 (proadrenomedullin N-termina120 peptide), rat.
Allatostatin peptides including, but not limited to, allatostatin I;
allatostatin II;
allatostatin III; and allatostatin IV.
Amylin peptides including, but not limited to, acetyl-amylin 8-37, human;
acetylated amylin 8-37, rat; AC187 amylin antagonist; AC253 amylin antagonist;
AC625 amylin antagonist; amylin 8-37, human; amylin (IAPP), cat; amylin
(insulinoma or islet amyloid polypeptide(IAPP)); amylin amide, human; amylin 1-
13
(diabetes-associated peptide 1-13), human; amylin 20-29 (IAPP 20-29), human;
AC625 amylin antagonist; amylin 8-37, human; amylin (IAPP), cat; amylin, rat;
amylin 8-37, rat; biotinyl-amylin, rat; and biotinyl-amylin amide, human.
Amyloid beta-protein fragment peptides including, but not limited to,
Alzheimer's disease beta-protein 12-28 (SP17); amyloid beta-protein 25-35;
amyloid
beta/A4-protein prccursor 328-332; arriyloid beta/A4 protein precursor (APP)
319-
335; amyloid beta-protein 1-43; amyloid beta-protein 1-42; amyloid beta-
protein 1-40;
amyloid beta-protein 10-20; amyloid beta-protein 22-35; Alzheimer's disease
beta-
protein (SP28); beta-amyloid peptide 1-42, rat; beta-amyloid peptide 1-40,
rat; beta-
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amyloid 1-11; beta-amyloid 31-35; beta-amyloid 32-35; beta-amyloid 35-25; beta-
amyloid/A4 protein precursor 96-110; beta-amyloid precursor protein 657-676;
beta-
amyloid 1-38; [G1nl I]-Alzheimer's disease beta-protein; [Gln"]-beta-amyloid 1-
40;
[G1n22]-beta-amyloid 6-40; non-A beta component of Alzheimer's disease amyloid
(NAC); P3, (A beta 17-40) Alzheimer's disease amyloid 0-peptide; and SAP
(serum
amyloid P component) 194-204.
Angiotensin peptides including, but not limited to, A-779; Ala-Pro-Gly-
angiotensin Il; [I1e3,Va15]-angiotensin II; angiotensin III antipeptide;
angiogenin
fragment 108-122; angiogenin fragment 108-123; angiotensin I converting enzyme
inhibitor; angiotensin I, human; angiotensin I converting enzyme substrate;
angiotensin 11-7, human; angiopeptin; angiotensin II, human; angiotensin II
antipeptide; angiotensin 11 1-4, human; angiotensin 11 3-8, human; angiotensin
II 4-8,
human; angiotensin II 5-8, human; angiotensin III ([Des-Aspi]-angiotensin II),
human;
angiotensin III inhibitor ([Ile7]-angiotensin III); angiotensin-converting
enzyme
inhibitor (Neothunnus macropterus); [Asn', Val5]-angiotensin I, goosefish;
[Asn',
Va15, Asn9]-angiotensin I, salmon; [Asn', Va15, Gly9]-angiotensin I, eel;
[Asn', Va15]-
angiotensin I 1-7, eel, goosefish, salmon; [Asn', Va15] -angiotensin II;
biotinyl-
angiotensin I, human; biotinyl-angiotensin II, human; biotinyl-Ala-Ala-Ala-
angiotensin II; [Des-Aspl]-angiotensin I, human; [p-aminophenylalanine 6] -
angiotensin
II; renin substrate (angiotensinogen 1-13), human; preangiotensinogen 1-14
(renin
substrate tetradecapeptide), human; renin substrate tetradecapeptide
(angiotensinogen
1-14), porcine; [Sari]-angiotensin II, [Sari]-angiotensin II 1-7 amide; [Sari,
AlaB]-
angiotensin II; [Sarl, Ileg]-angiotensin II; [Sarl, Thr 8]-angiotensin lI;
[Sar', Tyr(Me)4]-
angiotensin II (Sarmesin); [Sari, Va15 , A1ag]-angiotensin II; [Sari, I1e7 ]-
angiotensin III;
synthetic tetradecapeptide renin substrate (No. 2); [Val4]-angiotensin III;
[Vals]-
angiotensin II; [Val5]-angiotensin 1, human; [Va15]-angiotensin I; [Val5,
Asn9]-
angiotensin 1, bullfrog; and [Va15, Ser4]-angiotensin I, fowl.
Antibiotic peptides including, but not limited to, Ac-SQNY; bactenecin,
bovine; CAP 37 (20-44); carbormethoxycarbonyl-DPro-DPhe-OBzi; CD36 peptide P
139-155; CD36 peptide P 93-110; cecropin A-melittin hybrid peptide [CA(I-7)M(2-
9)NH2]; cecropin B, free acid; CYS(Bzl)84 CD fragment 81-92; defensin (human)
HNP-2; dermaseptin; immunostimulating peptide, human; lactoferricin, bovine
(BLFC); and magainin spacer.
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Antigenic polypeptides, which can elicit an enhanced immune response,
enhance an immune response and or cause an immunizingly effective response to
diseases and/or disease causing agents including, but not limited to,
adenoviruses;
anthrax; Bordetella pertussus; botulism; bovine rhinotracheitis; Branhamella
catarrhalis; canine hepatitis; canine distemper; Chlamydiae; cholera;
coccidiomycosis; cowpox; cytomegalovirus; Dengue fever; dengue toxoplasmosis;
diphtheria; encephalitis; enterotoxigenic Escherichia coli; Epstein Barr
virus; equine
encephalitis; equine infectious anemia; equine influenza; equine pneumonia;
equine
rhinovirus; Escherichia coli; feline leukemia; flavivirus; globulin;
haemophilus
influenza type b; Haemophilus influenzae; Haemophilus pertussis; Helicobacter
pylon; hemophilus ??; hepatitis ??; hepatitis virus A; hepatitis virus B;
Hepatitis virus
C; herpes viruses; HIV; HIV- 1 viruses; HIV-2 viruses; HTLV I; HTLV II; HTLV
III;
influenza ?; Japanese encephalitis; Klebsiellae species; Legionella
pneumophila;
leishmania; leprosy; lyme disease; malaria immunogen; measles; meningitis;
Meningococcus; Meningococcal polysaccharide group A; Meningococcal
polysaccharide group C; mumps; mumps virus; mycobacteria; Mycobacterium
tuberculosis=, Neisseria; Neisseria gonorrhoeae; Neisseria meningitidis; ovine
blue
tongue; ovine encephalitis; papilloma viruses; parainfluenza; paramyxoviruses;
Pertussis toxins; plague; pneumococcus; Pneumocystis carinii; pneumonia;
poliovirus; Proteus species; Pseudomonas aeruginosa; rabies; respiratory
syncytial
virus; rotavirus; rubella; Salmonellae; schistosomiasis; shigellae; simian
immunodeficiency virus; smallpox; Staphylococcus aureus; Staphylococcus
species;
Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus species;
Clostridium dif~cile; Clostridium species; swine influenza; tetanus; Treponema
pallidum; typhoid; vaccinia; varicella-zoster virus; and vibrio cholerae.
Anti-microbial peptides including, but not limited to, buforin I; buforin II;
cecropin A; cecropin B; cecropin Pl, porcine; gaegurin 2 (Rana rugosa);
gaegurin 5
(Rana rugosa); indolicidin; protegrin-(PG)-I; magainin 1; and magainin 2; and
T-22
[Tyr$'12, Lys']-poly-phemusin II peptide.
Apoptosis related peptides including, but not limited to, Alzheimer's disease
beta-protein (SP28); calpain inhibitor peptide; capsase-1 inhibitor V; capsase-
3,
substrate IV; caspase-l inhibitor I, cell-permeable; caspase-1 inhibitor VI;
caspase-3
substrate III, fluorogenic; caspase-1 substrate V, fluorogenic; caspase-3
inhibitor I,
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cell-permeable; caspase-6 ICE inhibitor III; [Des-Ac, biotin]-ICE inhibitor
HI; IL-1 B
converting enzyme (ICE) inhibitor II; IL-1 B converting enzyme (ICE) substrate
IV;
MDL 28170; and MG-132.
Atrial natriuretic peptides including, but not limited to, alpha-ANP (alpha-
5 chANP), chicken; anantin; ANP 1-11, rat; ANP 8-30, frog; ANP 11-30, frog;
ANP-21
(fANP-21), frog; ANP-24 (fANP-24), frog; ANP-30, frog; ANP fragment 5-28,
human, canine; ANP-7-23, human; ANP fragment 7-28, human, canine; alpha-atrial
natriuretic polypeptide 1-28, human, canine; A71915, rat; atrial natriuretic
factor 8-33,
rat; atrial natriuretic polypeptide 3-28, human; atrial natriuretic
polypeptide 4-28,
10 human, canine; atrial natriuretic polypeptide 5-27; human; atrial
natriuretic aeptide
(ANP), eel; atriopeptin I, rat, rabbit, mouse; atriopeptin II, rat, rabbit,
mouse;
atriopeptin III, rat, rabbit, mouse; atrial natriuretic factor (rANF), rat,
auriculin A (rat
ANF 126-149); auriculin B (rat ANF 126-150); beta-ANP (1-28, dimer,
antiparallel);
beta-rANF 17-48; biotinyl-alpha-ANP 1-28, human, canine; biotinyl-atrial
natriuretic
15 factor (biotinyl-rANF), rat; cardiodilatin 1-16, human; C-ANF 4-23, rat;
Des-[Cys105~
Cysl2 il-atrial natriuretic factor 104-126, rat; [Met(O)i2 ] ANP 1-28, human;
[Mpr7,DAlag]ANP 7-28, amide, rat; prepro-ANF 104-116, human; prepro-ANF 26-55
(proANF 1-30), human; prepro-ANF 56-92 (proANF 31-67), human; prepro-ANF
104-123, human; [Tyr ]-atriopeptin I, rat, rabbit, mouse; [Tyr ]-atriopeptin
II, rat,
20 rabbit, mouse; [Tyr ]-prepro ANF 104-123, human; urodilatin (CDD/ANP 95-
126);
ventricular natriuretic peptide (VNP), eel; and ventricular natriuretic
peptide (VNP),
rainbow trout.
Bag cell peptides including, but not limited to, alpha bag cell peptide; alpha-
bag cell peptide 1-9; alpha-bag cell peptide 1-8; alpha-bag cell peptide 1-7;
beta-bag
25 cell factor; and gamma-bag cell factor.
Bombesin peptides including, but not limited to, alpha-s 1 casein 101-123
(bovine milk); biotinyl-bombesin; bombesin 8-14; bombesin; [Leur3-psi
(CH2NH)Leu14]-bombesin; [D-Phe6, Des-Met14]-bombesin 6-14 ethylamide; [DPhel2]
bombesin; [DPhe12,Leu14]-bombesin; [Tyr4]-bombesin; and [Tyr4,DPhel2]-
bombesin.
Bone GLA peptides (BGP) including, but not limited to, bone GLA protein;
bone GLA protein 45-49; [Gl17 , G1a21'24]-osteocalcin 1-49, human;
myclopeptide -2
(MP-2); osteocalcin 1-49 human; osteocalcin 37-49, human; and [Tyr38, Phe42 46
] bone
GLA protein 38-49, human.
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Bradykinin peptides including, but not limited to, [Ala2 6, des-Pro3]-
bradykinin; bradykinin; bradykinin (Bowfin. Gar); bradykinin potentiating
peptide;
bradykinin 1-3; bradykinin 1-5; bradykinin 1-6; bradykinin 1-7; bradykinin 2-
7;
bradykinin 2-9; [DPhe7] bradykinin; [Des-Arg9]-bradykinin; [Des-Arg1 ]-Lys-
bradykinin ([Des-Arg10]-kallidin); [D-N-Me-Phe7]-bradykinin; [Des-Arg9, Leu8]-
bradykinin; Lys-bradykinin (kallidin); Lys-[Des-Arg9,Leug]-bradykinin ([Des-
Arg10,Leu9]-kallidin); [Lys -Hyp3]-bradykinin; ovokinin; [Lys , Ala3]-
bradykinin;
Met-Lys-bradykinin; peptide K12 bradykinin potentiating peptide; [(pCl)Phe5 g]-
bradykinin; T-kinin (Ile-Ser-bradykinin); [Thi5'8, D-Phe7]-bradykinin; [Tyr ]-
bradykinin; [Tyr5]-bradykinin; [Tyr 8]-bradykinin; and kallikrein.
Brain natriuretic peptides (BNP) including, but not limited to, BNP 32,
canine;
BNP-like Peptide, eel; BNP-32, human; BNP-45, mouse; BNP-26, porcine; BNP-32,
porcine; biotinyl-BNP-32, porcine; BNP-32, rat; biotinyl-BNP-32, rat; BNP-45
(BNP
51-95, 5K cardiac natriuretic peptide), rat; and [Tyr ]-BNP 1-32, human.
C-peptides including, but not limited to, C-peptide; and [Tyr ]-Gpeptide,
human.
C-type natriuretic peptides (CNP) including, but not limited to, C-type
natriuretic peptide, chicken; C-type natriuretic peptide-22 (CNP-22), porcine,
rat,
human; C-type natriuretic peptide-53 (CNP-53), human; C-type natriuretic
peptide-53
(CNP-53), porcine, rat; C-type natriuretic peptide-53 (porcine, rat) 1-29 (CNP-
53 1-
29); prepro-CNP 1-27, rat; prepro-CNP 30-50, porcine, rat; vasonatrin peptide
(VNP);
and [Tyr ]-C-type natriuretic peptide-22 ([Tyr ]-CNP-22).
Calcitonin peptides including, but not limited to, biotinyl-calcitonin, human;
biotinyl-calcitonin, rat; biotinyl-calcitonin, salmon; calcitonin, chicken;
calcitonin,
eel; calcitonin, human; calcitonin, porcine; calcitonin, rat; calcitonin,
salmon;
calcitonin 1-7, human; calcitonin 8-32, salmon; katacalcin (PDN-21) (C-
procalcitonin); and N-proCT (amino-terminal procalcitonin cleavage peptide),
human.
Calcitonin gene related peptides (CGRP) including, but not limited to, acetyl-
alpha-CGRP 19-37, human; alpha-CGRP 19-37, human; alpha-CGRP 23-37, human;
biotinyl-CGRP, human; biotinyl-CGRP II, human; biotinyl-CGRP, rat; beta-CGRP,
rat; biotinyl-beta-CGRP, rat; CGRP, rat; CGRP, human; calcitonin C-terminal
adjacent peptide; CGRP 1-19, human; CGRP 20-37, human; CGRP 8-37, human;
CGRP II, human; CGRP, rat; CGRP 8-37, rat; CGRP 29-37, rat; CGRP 30-37, rat;
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CGRP 31-37, rat; CGRP 32-37, rat; CGRP 33-37, rat; CGRP 31-37, rat;
([Cys(Acm)2'7]-CGRP; elcatonin; [Tyr ]-CGRP, human; [Tyr ]-CGRP 11, human;
[Tyr ]-CGRP 28-37, rat; [Tyr ]-CGRP, rat; and [Tyr22]-CGRP 22-37, rat.
CART peptides including, but not limited to, CART, human; CART 55-102,
human; CART, rat; and CART 55-102, rat.
Casomorphin peptides including, but not limited to, beta-casomorphin, human;
beta-casomorphin 1-3; beta-casomorphin 1-3, amide; beta-casomorphin, bovine;
beta-
casomorphin 1-4, bovine; beta-casomorphin 1-5, bovine; beta-casomorphin 1-5,
amide, bovine; beta-casomorphin 1-6, bovine; [DAla2]-beta-casomorphin 1-3,
amide,
bovine; [DAla2,Hyp4,Tyr5]-beta-casomorphin 1-5 amide; [DAla2,DPro4,Tyr5]-beta-
casomorphin 1-5, arnide; [DAla2,Tyr 5] -beta-casomorphin 1-5, amide, bovine;
[DAla2'4,Tyr5]-beta-casomorphin 1-5, amide, bovine; [DA1a2, (pC1)Phe3]-beta-
casomorphin, amide, bovine; [DAla2]-beta-casomorphin 1-4, amide, bovine;
[DAla2]-
beta-casomorphin 1-5, bovine; [DAla2]-beta-casomorphin 1-5, amide, bovine;
[DAIaZ,MetS]-beta-casomorphin 1-5, bovine; [DPro2]-beta-easomorphin 1-5,
amide,
bovine; [DAla2]-beta-casomorphin 1-6, bovine; [DPro2]-beta-casomorphin 1-4,
amide; [Des-Tyrl]-beta-casomorphin, bovine; [DAla2'4,Tyr5]-beta-casomorphin 1-
5,
amide, bovine; [DAla2, (pCl)Phe3]-beta-casomorphin, amide, bovine; [DAla2]-
beta-
casomorphin 1-4, amide, bovine; [DAla2]-beta-casomorphin 1-5, bovine; [DAla2]-
beta-casomorphin 1-5, amide, bovine; [DAIa2,Met5]-beta-casomorphin 1-5,
bovine;
[DPro2]-beta-casomorphin 1-5, amide, bovine; [DAla2]-beta-casomorphin 1-6,
bovine;
[DPro2]-beta-casomorphin 1-4, amide; [Des-Tyrl]-beta-casomorphin, bovine; and
[Va13]-beta-casomorphin 1-4, amide, bovine.
Chemotactic peptides including, but not limited to, defensin 1(human) HNP-1
(human neutrophil peptide-1); and N-formyl-Met-Leu-Phe.
Cholecystokinin (CCK) peptides including, but not limited to, caerulein;
cholecystokinin; cholecystokinin-pancreozymin; CCK-33, human; cholecystokinin
octapeptide 1-4 (non-sulfated) (CCK 26-29, unsulfated); cholecystokinin
octapeptide
(CCK 26-33); cholecystokinin octapeptide (non-sulfated) (CCK 26-33,
unsulfated);
choiecystokinin heptapeptide (CCK 27-33); cholecystokinin tetrapeptide (CCK 30-
33); CCK-33, porcine; CR 1 409, cholecystokinin antagonist; CCK flanking
peptide
(unsulfated); N-acetyl cholecystokinin, CCK 26-30, sulfated; N-acetyl
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28
cholecystokinin, CCK 26-31, sulfated; N-acetyl cholecystokinin, CCK 26-31, non-
sulfated; prepro CCK fragment V-9-M; and proglumide.
Colony-stimulating factor peptides including, but not limited to, colony-
stimulating factor (CSF); GM-CSF; M-CSF; and G-CSF.
Corticortropin releasing factor (CRF) peptides including, but not limited to,
astressin; alpha-helical CRF 12-41; biotinyl-CRF, ovine; biotinyl-CRF, human,
rat;
CRF, bovine; CRF, human, rat; CRF, ovine; CRF, porcine; [Cys2l]-CRF, human,
rat;
CRF antagonist (alpha-helical CRF 9-41); CRF 6-33, human, rat; [DPros]-CRF,
human, rat; [D-Phe12, N1e21'3g]-CRF 12-41, human, rat; eosinophilotactic
peptide;
[Met(0)21]-CRF, ovine; [N1e21,Tyr3z]-CRF, ovine; prepro CRF 125-151, human;
sauvagine, frog; [Tyr ]-CRF, human, rat; [Tyr ]-CRF, ovine; [Tyr ]-CRF 34-41,
ovine; [Tyr ]-urocortin; urocortin amide, human; urocortin, rat; urotensin I
(Catostomus commersoni); urotensin II; and urotensin II (Rana ridibunda).
Cortistatin peptides including, but not limited to, cortistatin 29;
cortistatin 29
(1-13); [Tyr ]-cortistatin 29; pro-cortistatin 28-47; and pro-cortistatin 51-
81.
Cytokine peptides including, but not limited to, tumor necrosis factor alpha
(TNF-a); and tumor necrosis factor-(3 (TNF-(3). Interleukins, including but
not limited
to IL-1 a, IL-10, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, and
IL-13.
Interleukin peptides including, but not limited to, interleukin-1 beta 165-
181, rat; and
interleukin-8 (IL-8, CINC/gro), rat. Chemokines including but not limited to
RANTES, MCP-1, MIP- l a, MIP-1(3.
Dermorphin peptides including, but not limited to, dermorphin and dermorphin
analog 1-4.
Dynorphin peptides including, but not limited to, big dynorphin (prodynorphin
209-240), porcine; biotinyl-dynorphin A (biotinyl-prodynorphin 209-225);
[DAla2,
DArg6]-dynorphin A 1-13, porcine; [D-Ala2]-dynorphin A, porcine; [D-A1a2]-
dynorphin A amide, porcine; [D-Ala2]-dynorphin A 1-13, amide, porcine; [D-
Ala2]-
dynorphin A 1-9, porcine; [DArg6]-dynorphin A 1-13, porcine; [DArg8] -
dynorphin A
1-13, porcine; [Des-Tyr1]-dynorphin A 1-8; [D-Pro10]-dynorphin A 1-11,
porcine;
dynorphin A amide, porcine; dynorphin A 1-6, porcine; dynorphin A 1-7,
porcine;
dynorphin A 1-8, porcine; dynorphin A 1-9, porcine; dynorphin A 1-10, porcine;
dynorphin A 1-10 amide, porcine; dynorphin A 1-11, porcine; dynorphin A 1-12,
porcine; dynorphin A 1-13, porcine; dynorphin A 1-13 amide, porcine; DAKLI
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29
(dynorphin A-analogue kappa ligand); DAKLI-biotin ([Argll'13]-dynorphin A(1-
13)-
Gly-NH(CI42)5NH-biotin); dynorphin A 2-17, porcine; dynorphin 2-17, amide,
porcine; dynorphin A 2-12, porcine; dynorphin A 3-17, amide, porcine;
dynorphin A
3-8, porcine; dynorphin A 3-13, porcine; dynorphin A 3-17, porcine; dynorphin
A 7-
17, porcine; dynorphin A 8-17, porcine; dynorphin A 6-17, porcine; dynorphin A
13-
17, porcine; dynorphin A (prodynorphin 209-225), porcine; dynorphin B 1-9;
[MeTyr1, MeArg7, D-Leug]-dynorphin 1-8 ethyl amide; [(nMe)Tyrl] dynorphin A 1-
13, amide, porcine; [Phe7]-dynorphin A 1-7, porcine; [Phe7]-dynorphin A 1-7,
amide,
porcine; and prodynorphin 228-256 (dynorphin B 29) (leumorphin), porcine.
Endorphin peptides including, but not limited to, alpha-neo-endorphin,
porcine; beta-neo-endorphin; Ac-beta-endorphin, camel, bovine, ovine; Ac-beta-
endorphin 1-27, camel, bovine, ovine; Ac-beta-endorphin, human; Ac-beta-
endorphin
1-26, human; Ac-beta-endorphin 1-27, human; Ac-gamma-endorphin (Ac-beta-
lipotropin 61-77); acetyl-alpha-endorphin; alpha-endorphin (beta-lipotropin 61-
76);
alpha-neo-endorphin analog; alpha-neo-endorphin 1-7; [Arg&]-alpha-neo-
endorphin 1-
8; beta-endorphin (beta-lipotropin 61-91), camel, bovine, ovine; beta-
endorphin 1-27,
camel, bovine, ovine; beta-endorphin, equine; beta-endorphin (beta-lipotropin
61-91),
human; beta-endorphin (1-5) +(16-31), human; beta-endorphin 1-26, human; beta-
endorphin 1-27, human; beta-endorphin 6-31, human; beta-endorphin 18-31,
human;
beta-endorphin, porcine; beta-endorphin, rat; beta-lipotropin 1-10, porcine;
beta-
lipotropin 60-65; beta-lipotropin 61-64; beta-lipotropin 61-69; beta-
lipotropin 88-91;
biotinyl-beta-endorphin (biotinyl-beta-lipotropin 61-91); biocytin-beta-
endorphin,
human; gamma-endorphin (beta-lipotropin 61-77); [DAla2]-alpha-neo-endorphin 1-
2,
amide; [DAla2]-beta-lipotropin 61-69; [DAIa2]-gamma-endorphin; [Des-Tyrl]-beta-
endorphin, human; [Des-Tyrl]-gamma-endorphin (beta-lipotropin 62-77); [Leu5]-
beta-
endorphin, camel, bovine, ovine; [Met5, Lys6]-alpha-neo-endorphin 1-6; [Met5,
Lys6'7]-alpha-neo-endorphin 1-7; and [Met5, Lys6, Arg?]-alpha-neo-endorphin 1-
7.
Endothelin peptides including, but not limited to, endothelin-1 (ET-1);
endothelin-1 [Biotin-Lys9]; endothelin-1 (1-15), human; endothelin-1 (1-15),
amide,
human; Ac-endothelin-1 (16-21), human; Ac-[DTrp16]-endothelin-1 (16-21),
human;
[Ala3'll]-endothelin-1; [Dprl, Asp15]-endothelin-1; [Ala2]-endothelin-3,
human;
[Alai8]-endothelin-1, human; [Asnl&]-endothelin-1, human; [Res-701-1]-
endothelin B
receptor antagonist; Suc-[Glu9, A1a11'1s]-endothelin-1 (8-21), IRL-1620;
endothelin-C-
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terminal hexapeptide; [D-Va122] -big endothelin-1 (16-38), human; endothelin-2
(ET-
2), human, canine; endothelin-3 (ET-3), human, rat, porcine, rabbit; biotinyl-
endothelin-3 (biotinyl-ET-3); prepro-endothelin-1 (94-109), porcine; BQ-518;
BQ-
610; BQ-788; endothelium-dependent relaxation antagonist; FR139317; IRL-1038;
5 JKC-30 1; JKC-302; PD-145065; PD 142893; sarafotoxin S6a (atractaspis
engaddensis); sarafotoxin S6b (atractaspis engaddensis); sarafotoxin S6c
(atractaspis
engaddensis); [Lys4]-sarafotoxin S6c; sarafotoxin S6d; big endothelin-1,
human;
biotinyl-big endothelin-1, human; big endothelin-1 (1-39), porcine; big
endothelin-3
(22-41), amide, human; big endothelin-1 (22-39), rat; big endothelin-1 (1-39),
bovine;
10 big endothelin-1 (22-39), bovine; big endothelin-1 (19-38), human; big
endothelin-1
(22-38), human; big endothelin-2, human; big endothelin-2 (22-37), human; big
endothelin-3, human; big endothelin-1, porcine; big endothelin-1 (22-39)
(prepro-
endothelin-1 (74-91)); big endothelin-1, rat; big endothelin-2 (1-38), human;
big
endothelin-2 (22-38), human; big endothelin-3, rat; biotinyl-big endothelin-1,
human;
15 and [Tyr123]-prepro-endothelin (110-130), amide, human.
ETa receptor antagonist peptides including, but not limited to, [BQ-123];
[BE18257B]; [BE-18257A]/[W-7338A]; [BQ-485]; FR139317; PD-151242; and
TTA-386.
ETb receptor antagonist peptides including, but not limited to, [BQ-3020];
20 [RES-701-3]; and [IRL-1720].
Enkephalin peptides including, but not limited to, adrenorphin, free acid;
amidorphin (proenkephalin A (104-129)-NH2), bovine; BAM-12P (bovine adrenal
medulla dodecapeptide); BAM-22P (bovine adrenal medulla docosapeptide);
benzoyl-
Phe- Ala-Arg; enkephalin; [D-Ala2, D-Leu5]-enkephalin; [D-A1a2, D-Met5]-
25 enkephalin; [DAla2]-Leu-enkephalin, amide; [DAla2,Leu5,Arg6]-enkephalin;
[Des-
Tyr1,DPen2'5]-enkephalin; [Des-Tyr1,DPen2,Pen5]-enkephalin; [Des-Tyrl ]-Leu-
enkephalin; [D-Pen2'5]-enkephalin; [DPen2, Pen5]-enkephalin; enkephalinase
substrate; [D-Pen2, pCI-Phe4, D-Pen5]-enkephalin; Leu-enkephalin; Leu-
enkephalin,
amide; biotinyl-Leu-enkephalin; [D-A1a2]-Leu-enkephalin; [D-Ser2]-Leu-
enkephalin-
30 Thr (delta-receptor peptide) (DSLET); [D-Thr 2] -Leu-enkephalin-Thr
(DTLET);
[Lys6]-Leu-enkephalin; [Met5,Arg6]-enkephalin; [Met5,Arg6]-enkephalin-Arg;
[Met5,Arg6,Phe7]-enkephalin, amide; Met-enkephalin; biotinyl-Met-enkephalin;
[D-
Ala2]-Met-enkephalin; [D-Ala2]-Met-enkephalin, amide; Met-enkephalin-Arg-Phe;
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Met-enkephalin, amide; [Ala2]-Met-enkephalin, amide; [DMet2,Pro5]-enkephalin,
amide; [DTrp2]-Met-enkephalin, amide, metorphinamide (adrenorphin); peptide B,
bovine; 3200-Dalton adrenal peptide E, bovine; peptide F, bovine;
preproenkephalin
B 186-204, human; spinorphin, bovine; and thiorphan (D, L, 3-mercapto-2-
benzylpropanoyl-glycine).
Ephrin B, its analogues and antagonists.
Fibronectin peptides including, but not limited to platelet factor-4 (58-70),
human; echistatin (Echis carinatus); E, P,L selectin conserved region;
fibronectin
analog; fibronectin-binding protein; fibrinopeptide A, human; [Tyr ]-
fibrinopeptide A,
human; fibrinopeptide B, human; [Glul]-fibrinopeptide B, human; [TyrI5]-
fibrinopeptide B, human; fibrinogen beta-chain fragment of 24-42; fibrinogen
binding
inhibitor peptide; fibronectin related peptide (collagen binding fragment);
fibrinolysis
inhibiting factor; FN-C/H-1 (fibronectin heparin-binding fragment); FN-C/H-V
(fibronectin heparin-binding fragment); heparin-binding peptide; laminin penta
peptide, amide; Leu-Asp-Val-NH2 (LDV-NH2), human, bovine, rat, chicken;
necrofibrin, human; necrofibrin, rat; and platelet membrane glycoprotein IIB
peptide
296-306.
Galanin peptides including, but not limited to, galanin, human; galanin 1-19,
human; preprogalanin 1-30, human; preprogalanin 65-88, human; preprogalanin 89-
123, human; galanin, porcine; galanin 1-16, porcine, rat; galanin, rat;
biotinyl-galanin,
rat; preprogalanin 28-67, rat; galanin 1-13-bradykinin 2-9, amide; M40,
galanin 1-13-
Pro-Pro-(Ala-Leu) 2-Ala-amide; C7, galanin 1-13-spantide-amide; GMAP 1-41,
amide; GMAP 16-41, amide; GMAP 25-41, amide; galantide; and entero-kassinin.
Gastrin peptides including, but not limited to, gastrin, chicken; gastric
inhibitory peptide (GIP), human; gastrin I, human; biotinyl-gastrin I, human;
big
gastrin-1, human; gastrin releasing peptide, human; gastrin releasing peptide
1-16,
human; gastric inhibitory polypeptide (GIP), porcine; gastrin releasing
peptide,
porcine; biotinyl-gastrin releasing peptide, porcine; gastrin releasing
peptide 14-27,
porcine, human; little gastrin, rat; pentagastrin; gastric inhibitory peptide
1-30,
porcine; gastric inhibitory peptide 1-30, amide, porcine; [Tyr ]-gastric
inhibitory
peptide 23-42, human; and gastric inhibitory peptide, rat.
Glucagon peptides including, but not limited to, [Des-His1,Glu9]-glucagon,
extendin-4, glucagon, human; biotinyl-glucagon, human; glucagon 19-29, human;
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glucagon 22-29, human; Des-Hisl-[GIu9]-glucagon, amide; glucagon-like peptide
1,
amide (preproglucagon 72-107, amide); glucagon-like peptide 1(preproglucagon
72-
108), human; glucagon-like peptide 1 (7-36) (preproglucagon 78-107, amide);
glucagon-like peptide II, rat; biotinyl-glucagon-like peptide-1 (7-36)
(biotinyl-
preproglucagon 78-107, amide); glucagon-like peptide 2 (preproglucagon 126-
159),
human; oxyntomodulin/glucagon 37; and valosin (peptide VQY), porcine.
Gn-RH associated peptides (GAP) including, but not limited to, Gn-RH
associated peptide 25-53, human; Gn-RH associated peptide 1-24, human; Gn-RH
associated peptide 1-13, human; Gn-RH associated peptide 1-13, rat;
gonadotropin
releasing peptide, follicular, human; [Tyr ]-GAP ([Tyr ]-Gn-RH Precursor
Peptide
14-69), human; and proopiomelanocortin (POMC) precursor 27-52, porcine.
Growth factor peptides including, but not limited to, cell growth factors;
epidermal growth factors; tumor growth factor; TGF-alpha, human; TGF-alpha,
from
other mammalian species TGF-beta; alpha-TGF 34-43; human EGF (epidermal
growth factor); acidic fibroblast growth factor; basic fibroblast growth
factor; basic
fibroblast growth factor 13-18; basic fibroblast growth factor 120-125; brain
derived
acidic fibroblast growth factor 1-11; brain derived basic fibroblast growth
factor 1-24;
brain derived acidic fibroblast growth factor 102-111; [Cys(Acm20'31)]-
epidermal
growth factor 20-31; epidermal growth factor receptor peptide 985-996; insulin-
like
growth factor (IGF)-I, chicken; IGF-I, rat; IGF-I, human; Des (1-3) IGF-I,
human; R3
IGF-I, human; R3 IGF-I, human; long R3 IGF-I, human; adjuvant peptide analog;
anorexigenic peptide; Des (1-6) IGF-II, human; R6 IGF-II, human; IGF-I
analogue;
IGF I(24-41); IGF I(57-70); IGF I(30-41); IGF II; IGF II (33-40); [Tyr ]-IGF
II (33-
40); liver cell growth factor; midkine; midkine 60-121, human; N-acetyl, alpha-
TGF
34-43, methyl ester, rat; nerve growth factor (NGF), mouse; platelet-derived
growth
factor; platelet-derived growth factor antagonist;.ligands for the receptors
of the Erb-B
family.
Growth hormone peptides including, but not limited to, growth hormone
(hGH), human; growth hormone 1-43, human; growth hormone 6-13, human; growth
hormone releasing factor, hu~-~nan; growtl-i hormone releasing factor, bovine;
growth
hormone releasing factor, porcine; growth hormone releasing factor 1-29,
amide, rat;
growth hormone pro-releasing factor, human; biotinyl-growth hormone releasing
factor, human; growth hormone releasing factor 1-29, amide, human; [D-A1a2]-
growth
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hormone releasing factor 1-29, amide, human; [N-Ac-Tyri, D-Arg2]-GRF 1-29,
amide;
[His', N1e27]-growth hormone releasing factor 1-32, amide; growth hormone
releasing
factor 1-37, human; growth hormone releasing factor 1-40, human; growth
hormone
releasing factor 1-40, amide, human; growth hormone releasing factor 30-44,
amide,
human; growth hormone releasing factor, mouse; growth hormone releasing
factor,
ovine; growth hormone releasing factor, rat; biotinyl- growth hormone
releasing
factor, rat; GHRP-6 ([His', Lys6]-GHRP); hexarelin (growth hormone releasing
hexapeptide); and [D-Lys3]-GHRP-6.
GTP-binding proteins and fragment peptides thereof including, but not limited
to, [Arg8]-GTP-binding protein fragment, Gs alpha; GTP-binding protein
fragments,
of the G beta family; GTP-binding protein fragments, of the Ggamma family; GTP-
binding protein fragment, Galpha; GTP-binding protein fragments, Go alpha a
and b;
GTP-binding protein fragment, Gs alpha; and GTP-binding protein fragments, G
alpha il, G alpha i2, G alpha i3; GTP-binding protein fragment, Golf alpha;
GTP-
binding protein fragment, Gz alpha; GTP-binding protein fragment, Gq alpha.
Guanylin peptides including, but not limited to, guanylin, human; guanylin,
rat; and uroguanylin.
Inhibin peptides including, but not limited to, inhibin, bovine; inhibin,
alpha-
subunit 1-32, human; [Tyr ]-inhibin, alpha-subunit 1-32, human; seminal plasma
inhibin-like peptide, human; [Tyr ]-seminal plasma inhibin-like peptide,
human;
inhibin, alpha-subunit 1-32, porcine; and [Tyr ]-inhibin, alpha-subunit 1-32,
porcine.
Interferon peptides including, but not limited to, alpha interferon species
(e.g.,
alphal, alpha2, alpha2a, alpha2b, alpha2c, alpha2d, alpha3, alpha4, alpha4a,
alpha4b,
alpha5, alpha6, alpha74, alpha76, alphaA, alphaB, alphaCõ alphaC 1, alphaD,
alphaE,
alphaF, alphaG, alphaG, alphaH, alphal, alphaJl, alphaJ2, alphaK, alphaL);
interferon
beta species (e.g., betala); interferon gamma species (e.g., gammala,
gammalb);
interferon epsilon; interferon tau; interferon omega or any analogues of
interferon
omega. Various analogs of gamma interferon are described in Pechenov et al.
"Methods for preparation of recombinant cytokine proteins V. mutant analogues
of
human interferon-gamma with higher stability and activity" Protein Expr.
Turif:
24:173-180 (2002), which is incorporated herein by reference in its entirety
for
teachings directed to preparation and testing of interferon analogues.
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Insulin peptides including, but not limited to, insulin, human; insulin,
porcine;
IGF-I, human; insulin-like growth factor 11 (69-84); pro-insulin-like growth
factor II
(68-102), human; pro-insulin-like growth factor II(105-128), human; [AspB2g]-
insulin, human; [LysB28]-insulin, human; [LeuB28]-insulin, human; [Va1B28]-
insulin,
human; [AlaB2&]-insulin, human; [AspB28, ProB29]-insulin, human; [LysB28,
ProB29]-
insulin, human; [LeuB28, Pro$29]-insulin, human; [Va1B28, ProB29]-insulin,
human;
[Alag28, Pro$29]-insulin, human; [Glya21]-insulin, human; [G1yA21 G1nB3]-
insulin,
human; [AlaA2 ']-insulin, human; [AlaA2I G1nB3]-insulin, human; [G1nB3]-
insulin,
human; [Glns3o]-insulin, human; [GlyA21 GIuB'o]-insulin, human; [GlyA2l GInB3
Glus3o]-insulin, human; [GInB3 GluB3o]-insulin, human; B22-B30 insulin, human;
B23-B30 insulin, human; B25-B30 insulin, human; B26-B30 insulin, human; B27-
B30 insulin, human; B29-B30 insulin, human; the A chain of human insulin, and
the
B chain of human insulin.
Laminin peptides including, but not limited to, laminin; alphal (I)-CB3 435-
438, rat; and laminin binding inhibitor.
Leptin peptides including, but not limited to, leptin 93-105, human; leptin 22-
56, rat; Tyr-leptin 26-39, human; and leptin 116-130, amide, mouse.
Leucokinin peptides including, but not limited to, leucomyosuppressin (LMS);
leucopyrokinin (LPK); leucokinin I; leucokinin II; leucokinin lIl; leucokinin
IV;
leucokinin VI; leucokinin VII; and leucokinin VIII.
Luteinizing hormone-releasing hormone peptides including, but not limited to,
antide; Gn-RI-I II, chicken; luteinizing hormone-releasing hormone (LH-RH)
(GnRH);
biotinyl-LH-RH; cetrorelix (D-20761); [D-Ala ]-LH-RH; [Gin8]-LH-RH (Chicken
LH-RH); [DLeu6, Val?] LH-RH 1-9, ethyl amide; [D-Lys6]-LH-RH; [D-Phe2, Pro3, D-
Phe6]-LH-RH; [DPhe2, DAla6] LH-RH; [Des-Gly10]-LH-RH, ethyl amide; [D-Ala6,
Des-Gly10]-LH-RH, ethyl amide; [DTrp6]-LH-RH, ethyl amide; [D-Trp6, Des-G1yIo]-
LH-RH, ethyl amide (Deslorelin); [DSer(But)6, Des-Gly10]-LH-RH, ethyl amide;
ethyl
amide; leuprolide; LH-RH 4-10; LH-RH 7-10; LH-RH, free acid; LH-RH, lamprey;
LH-RH, salmon; [LysB]-LH-RH; [Trp?,Leug] LH-RI-I, free acid; and [(t-Bu)DSer6,
(Aza)Gly10]-L
Mastoparan peptides including, but not limited to, mastoparan; mas7; mas8;
mas 17; and mastoparan X.
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Mast cell degranulating peptides including, but not limited to, mast cell
degranulating peptide HR-1; and mast cell degranulating peptide HR-2.
Melanocyte stimulating hormone (MSH) peptides including, but not limited to,
[Ac-Cys4,DPhe7,Cys10] alpha-MSH 4-13, amide; alpha-melanocyte stimulating
5 hormone; alpha-MSH, free acid; beta-MSH, porcine; biotinyl-alpha-melanocyte
stimulating hormone; biotinyl-[Nle4 , D-Phe?] alpha-melanocyte stimulating
hormone;
[Des -Acetyl]-alpha-MSH; [DPhe7]-alpha-MSH, amide; gamma-l-MSH, amide;
[Lys ]-gamma-l-MSH, amide; MSH release inhibiting factor, amide; [N1e4]-alpha-
MSH, amide; [Nlea, D-Phe7]-alpha-MSH; N-Acetyl, [NIe4,DPhe7 ] alpha-MSH 4-10,
10 amide; beta-MSH, human; and gamma-MSH.
Morphiceptin peptides including, but not limited to, morphiceptin (beta-
casomorphin 1-4 amide); [D-Pro4] -morphiceptin; and [N-MePhe3,D-Pro4]-
morphiceptin.
Motilin peptides including, but not limited to, motilin, canine; motilin,
15 porcine; biotinyl-motilin, porcine; and [Leu13]-motilin, porcine.
Neuro-peptides including, but not limited to, Ac-Asp-Glu; achatina cardio-
excitatory peptide-1 (ACEP-1) (Achatina fulica); adipokinetic hormone (AKH)
(Locust); adipokinetic hormone (Heliothis zea and Manduca sexta); alytesin;
Tabanus
atratus adipokinetic hormone (Taa-AKH); adipokinetic hormone II (Locusta
20 migratoria); adipokinetic hormone II (Schistocera gregaria); adipokinetic
hormone III
(AKH-3); adipokinetic hormone G(AKH-G) (Gryllus bimaculatus); allatotropin
(AT)
(Manduca sexta); allatotropin 6-13 (Manduca sexta); APGW amide (Lymnaea
stagnalis); buccalin; cerebellin; [Des-Seri]-cerebellin; corazonin (American
Cockroach Periplaneta americana); crustacean cardioactive peptide (CCAP);
25 crustacean erythrophore; DF2 (Procambarus clarkii); diazepam-binding
inhibitor
fragment, human; diazepam binding inhibitor fragment (ODN); eledoisin related
peptide; FMRF amide (molluscan cardioexcitatory neuro-peptide); Gly-Pro-Glu
(GPE), human; granuliberin R; head activator neuropeptide; [His7]-corazonin;
stick
insect hypertrehalosaemic factor II; Tabanus atratus hypotrehalosemic hormone
(Taa-
30 HoTH); isoguvacine hydrochloride; bicuculline methiodide; piperidine-4-
sulphonic
acid; joining peptide of proopiomelanocortin (POMC), bovine; joining peptide,
rat;
KSAYMRF amide (P. redivivus); kassinin; kinetensin; levitide; litorin; LUQ 81-
91
(Aplysia califomica); LUQ 83-91 (Aplysia californica); myoactive peptide I
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(Periplanetin CC-1) (Neuro-hormone D); myoactive peptide II (Periplanetin CC-
2);
myomodulin; neuron specific peptide; neuron specific enolase 404-443, rat;
neuropeptide FF; neuropeptide K, porcine; NEI (prepro-MCH 131-143)
neuropeptide,
rat; NGE (prepro-MCH 110-128) neuropeptide, rat; NFl (Procambarus clarkii);
PBAN-1 (Bombyx mori); Hez-PBAN (Heliothis zea); SCPB (cardioactive peptide
from aplysia); secretoneurin, rat; uperolein; urechistachykinin I;
urechistachykinin lI;
xenopsin-related peptide 1; xenopsin-related peptide II; pedal peptide (Pep),
aplysia;
peptide Fl, lobster; phyllomedusin; polistes mastoparan; proctolin;
ranatensin; Ro I
(Lubber Grasshopper, Romalea microptera); Ro II (Lubber Grasshopper, Romalea
microptera); SALMF amide 1(Sl); SALMF amide 2 (S2); and SCPA.
Neuropeptide Y (NPY) peptides including, but not limited to, [Leu31,Pro34]-
neuropeptide Y, human; neuropeptide F (Moniezia expansa); B 1 BP3226 NPY
antagonist; Bis (31/31') {[Cys31, Trp32, Nva34] NPY 31-36}; neuropeptide Y,
human,
rat; neuropeptide Y 1-24 amide, human; biotinyl-neuropeptide Y; [D-Tyr2l'36, D-
Thr3']-NPY 27-36; Des 10-17 (cyclo 7-21) [Cys7 2i, Pro34]-NPY; C2-NPY; [Leu31,
Pro34] neuropeptide Y, human; neuropeptide Y, free acid, human; neuropeptide
Y,
free acid, porcine; prepro NPY 68-97, human; N-acetyl-[Leu28, Leu31] NPY 24-
36;
neuropeptide Y, porcine; [D-Trp32]-neuropeptide Y, porcine; [D-Trp32] NPY 1-
36,
human; [Leul?,DTrp32] neuropeptide Y, human; [Leu31, Pro34]-NPY, porcine; NPY
2-
36, porcine; NPY 3-36, human; NPY 3-36, porcine; NPY 13-36, human; NPY 13-36,
porcine; NPY 16-36. porcine; NPY 18-36, porcine; NPY 20-36; NFY 22-36; NPY 26-
36; [Pro34]-NPY 1-36, human; [Pro34]-neuropeptide Y, porcine; PYX-l; PYX-2; T4-
[NPY(33-36)]4; and Tyr(OMe)ZI]-neuropeptide Y, human.
Neurotropic factor peptides including, but not limited to, glial derived
neurotropic factor (GDNF); brain derived neurotropic factor (BDNF); and
ciliary
neurotropic factor (CNTF).
Ligands of the Notch receptor including, but not limited to the Delta-like-1,
Delta-like-2, Delta-like-3, Delta-like-4, Jagged-1 and Jagged-2 proteins, and
fragments thereof.
Orexin peptides including, but not limited to, orexin A; orexin B, human;
orexin B, rat, mouse.
Opioid peptides including, but not limited to, alpha-casein fragment 90-95;
BAM-18P; casomokinin L; casoxin D; crystalline; DALDA; dermenkephalin
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(deltorphin) (Phylomedusa sauvagei); [D-Ala2]-deltorphin I; [D-Ala2]-
deltorphin Il;
endomorphin-1; endomorphin-2; kyotorphin; [DArg2]-kyotorphin; morphin
tolerance
peptide; morphine modulating peptide, C-terminal fragment; morphine modulating
neuropeptide (A-18-F-NH2); nociceptin [orphanin FQ] (ORLI agonist); TIPP; Tyr-
MIF-1; Tyr-W-MIF-1; valorphin; LW-hemorphin-6, human; Leu-valorphin-Arg; and
Z-Pro-D-Leu.
Oxytocin peptides including, but not limited to, [Asu6]-oxytocin; oxytocin;
biotinyl-oxytocin; [Thr4, G1y7]-oxytocin; and tocinoic acid ([I1e3]-pressinoic
acid).
PACAP (pituitary adenylating cyclase activating peptide) peptides including,
but not limited to, PACAP 1-27, human, ovine, rat; PACAP (1-27)-Gly-Lys-Arg-
NH2, human; [Des-Glny6]-PACAP 6-27, human, ovine, rat; PACAP38, frog;
PACAP27-NH2, human, ovine, rat; biotinyl-PACAP27-NH2, human, ovine, rat;
PACAP 6-27, human, ovine, rat; PACAP38, human, ovine, rat; biotinyl-PACAP38,
human, ovine, rat; PACAP 6-38, human, ovine, rat; PACAP27-NH2, human, ovine,
rat; biotinyl-PACAP27-NH2, human, ovine, rat; PACAP 6-27, human, ovine, rat;
PACAP38, human, ovine, rat; biotinyl-PACAP38, human, ovine, rat; PACAP 6-38,
human, ovine, rat; PACAP3 8 16-38, human, ovine, rat; PACAP3 8 31-38, human,
ovine, rat; PACAP38 31-38, human, ovine, rat; PACAP-related peptide (PRP),
human; and PACAP-related peptide (PRP), rat.
Pancreastatin peptides including, but not limited to, chromostatin, bovine;
pancreastatin (hPST-52) (chromogranin A 250-301, amide); pancreastatin 24-52
(hPST-29), human; chromogranin A 286-301, amide, human; pancreastatin,
porcine;
biotinyl-pancreastatin, porcine; [N1eg]-pancreastatin, porcine; [Tyr ,N1e8]-
pancreastatin, porcine; [Tyr ]-pancreastatin, porcine; parastatin 1-19
(chromogranin A
347-365), porcine; pancreastatin (chromogranin A 264-314-amide, rat; biotinyl-
pancreastatin (biotinyl-chromogranin A 264-314-amide; [Tyr ]-pancreastatin,
rat;
pancreastatin 26-51, rat; and pancreastatin 33-49, porcine.
Pancreatic polypeptides including, but not limited to, pancreatic polypeptide,
avian; pancreatic polypeptide, human; C-fragment pancreatic polypeptide acid,
human; C-fragment pancreatic polypeptide amide, human; pancreatic polypeptide
(Rana temporaria); pancreatic polypeptide, rat; and pancreatic polypeptide,
salmon.
Parathyroid hormone peptides including, but not limited to, [Asp76]-
parathyroid hormone 39-84, human; [Asp76]-parathyroid hormone 53-84, human;
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38
[Asn76]-parathyroid hormone 1-84, hormone; [Asn76]-parathyroid hormone 64-84,
human; [Asn8, Leulg]-parathyroid hormone 1-34, human; [Cys5'28]-parathyroid
hormone 1-34, human; hypercalcemia malignancy factor 1-40; [Leu18]-parathyroid
hormone 1-34, human; [Lys(biotinyl) 13, Nle8' I8, Tyr34]-parathyroid hormone 1-
34
amide; [Nle8'18, Tyr34]-parathyroid hormone 1-34 amide; [Nle8'18, Tyr34]-
parathyroid
hormone 3-34 amide, bovine; [Nleg'Ig, Tyr 34] -parathyroid hormone 1-34,
human;
[Nleg'ig, Tyr34]-parathyroid hormone 1-34 amide, human; [Nleg'1 g, Tyr34]-
parathyroid
hormone 3-34 amide, human; [Nle8'18, Tyr34]- parathyroid hormone 7-34 amide,
bovine; [Nleg'21, T~r34]-parathyroid hormone 1-34 amide, rat; parathyroid
hormone
44-68, human; parathyroid hormone 1-34, bovine; parathyroid hormone 3-34,
bovine;
parathyroid hormone 1-31 amide, human; parathyroid hormone 1-34, human;
parathyroid hormone 13-34, human; parathyroid hormone 1-34, rat; parathyroid
hormone 1-38, human; parathyroid hormone 1-44, human; parathyroid hormone 28-
48, human; parathyroid hormone 39-68, human; parathyroid hormone 39-84, human;
parathyroid hormone 53-84, human; parathyroid hormone 69-84, human;
parathyroid
hormone 70-84, human; [Pro34]-peptide YY (PYY), human; [Tyr ]-hypercalcemia
malignancy factor 1-40; [Tyr ]-parathyroid hormone 1-44, human; [Tyr ]-
parathyroid
hormone 1-34, human; [Tyr']-parathyroid hormone 1-34, human; [Tyr27]-
parathyroid
hormone 27-48, human; [Tyr34]-parathyroid hormone 7-34 amide, bovine; [Tyr43]-
parathyroid hormone 43-68, human; [Tyr52, Asn76]-parathyroid hormone 52-84,
human; and [Tyr63]-parathyroid hormone 63-84, human.
Parathyroid hormone (PTH)-related peptides including, but not limited to,
PTHrP ([Tyr36]-PTHrP 1-36 amide), chicken; hHCF-(1-34)-NH2 (humoral
hypercalcemic factor), human; PTH-related protein 1-34, human; biotinyl-PTH-
related
protein 1-34, human; [Tyr ]-PTH-related protein 1-34, human; [Tyr34]-PTH-
related
protein 1-34 amide, human; PTH-related protein 1-37, human; PTH-related
protein 7-
34 amide, human; PTH-related protein 38-64 amide, human; PTH-related protein
67-
86 amide, human; PTH-related protein 107-111, human, rat, mouse; PTH-related
protein 107-111 free acid; PTH-related protein 107-138, human; and PTH-related
protein 109-111, human.
Peptide T peptides including, but not limited to, peptide T; [D Alal]-peptide
T; and [D-Alal]-peptide T amide.
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Prolactin-releasing peptides including, but not limited to, prolactin-
releasing
peptide 31, human; prolactin-releasing peptide 20, human; prolactin-releasing
peptide
31, rat; prolactin-releasing peptide 20, rat; prolactin-releasing peptide 31,
bovine; and
prolactin-releasing peptide 20, bovine.
Peptide YY (PYY) peptides including, but not limited to, PYY, human; PYY
3-36, human; biotinyl-PYY, human; PYY, porcine, rat; and [Leu31, Pro34]-PYY,
human.
Renin substrate peptides including, but not limited to, acetyl,
angiotensinogen
1-14, human; angiotensinogen 1-14, porcine; renin substrate tetradecapeptide,
rat;
[Cys&]-renin substrate tetradecapeptide, rat; [Leug]-renin substrate
tetradecapeptide,
rat; and [Valg]-renin substrate tetradecapeptide, rat.
Secretin peptides including, but not limited to, secretin, canine; secretin,
chicken; secretin, human; biotinyl-secretin, human; secretin, porcine; and
secretin, rat.
Somatostatin (GIF) peptides including, but not limited to, BIM-23027;
biotinyl-somatostatin; biotinylated cortistatin 17, human; cortistatin 14,
rat; cortistatin
17, human; [Tyr ]-cortistatin 17, human; cortistatin 29, rat; [D-Trp8]-
somatostatin;
[DTrp8,DCys14]-somatostatin; [DTrp8,Tyrl 1]-somatostatin; [D-Trpl I]-
somatostatin;
NTB (Naltriben); [Nleg]-somatostatin 1-28; octreotide (SMS 201-995);
prosomatostatin 1-32, porcine; [Tyr ]-somatostatin; [Tyr']-somatostatin;
[Tyrl]-
somatostatin 28 (1-14); [Tyril]-somatostatin; [Tyr , D-TrpB]-somatostatin;
somatostatin; somatostatin antagonist; somatostatin-25; somatostatin-28;
somatostatin
28 (1-12); biotinyl-somatostatin-28; [Tyr ]-somatostatin-28; [Leu8, D-Trp22,
Tyr25]-
somatostatin-28; biotinyl-[Leug, D-Trp22, Tyr25]-somatostatin-28; somatostatin-
28 (1-
14); and somatostatin analog, RC-160.
Substance P peptides including, but not limited to, G protein antagonist-2; Ac-
[Argd, Sar9, Met(02)1 l]-substance P 6-11; [Arg3] -substance P; Ac-Trp-3,5-
bis(trifluoromethyl) benzyl ester; Ac-[Arg6, Sar9, Met(02)11]-substance P 6-
11; [D-
A1a4]-substance P 4-11; [Tyr6, D-Phe', D-His9] -substance P 6-11 (sendide);
biotinyl-
substance P; biotinyl-NTE[Arg3]-substance P; [Tyrg] -substance P; [Sar9,
Met(02)11]-
substance P; [D-Pro2, D-Trp?'9]-substance P; [D-Pro4, 0-Trp7'9]-substance P 4-
11;
substance P 4-11; [DTrp2'7'9]-substance P; [(Dehydro)Pro2'4, Pro9]-substance
P;
[Dehydro-Pro4] -substance P 4-11; [G1p5,(Me)Pheg,Sar9]-substance P 5-11;
[G1p5,Sar9]-substance P 5-11; [Glp5] -substance P 5-11; hepta-substance P
(substance
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P 5-11); hexa-substance P(substance P 6-11); [MePhe8,Sar9]-substance P; [Nle"]-
substance P; Octa-substance P(substance P 4-11); [pGlul]-hexa-substance P
([pGlu6]-
substance P 6-11); [pGlu6, D-Pro9] -substance P 6-11; [(pNO2)Phe7Nleil]-
substance P; penta-substance P (substance P 7-11); [Pro9] -substance P;
GR73632, substance P 7-
5 11; [Sar4] -substance P 4-11; [Sar9] -substance P; septide ([pGlu6, Pro9]-
substance P 6-
11); spantide 1; spantide II; substance P; substance P, cod; substance P,
trout;
substance P antagonist; substance P-Gly-Lys-Arg; substance P 1-4; substance P
1-6;
substance P 1-7; substance P 1-9; deca-substance P (substance P 2-11); nona-
substance P (substance P 3-11); substance P tetrapeptide (substance P 8-11);
10 substance P tripeptide (substance P 9-11); substance P, free acid;
substance P methyl
ester; and [TyrB,Nle11] substance P.
Tachykinin peptides including, but not limited to, [Ala5, beta-Ala8]
neurokinin
A 4-10; eledoisin; locustatachykinin I(Lom-TK-I) (Locusta migratoria);
locustatachykinin II (Lom-TK-II) (Locusta migratoria); neurokinin A 4-10;
neurokinin
15 A (neuromedin L, substance K); neurokinin A, cod and trout; biotinyl-
neurokinin A
(biotinyl-neuromedin L, biotinyl-substance K); [Tyr ]-neurokinin A; [Tyr6]-
substance
K; FR64349; [Lys3, Gly8-(R)-gamma-lactam-Leu9]-neurokinin A 3-10; GR83074;
GR87389; GR94800; [Beta-Alag]-neurokinin A 4-10; [Nle10]-neurokinin A 4-10;
[Trp7 , beta-Ala8]-neurokinin A 4-10; neurokinin B (neuromedin K); biotinyl-
20 neurokinin B (biotinyl-neuromedin K); [MePhe7]-neurokinin B; [Pro7]-
neurokinin B;
[Tyr ]-neurokinin B; neuromedin B, porcine; biotinyl-neuromedin B, porcine;
neuromedin B-30, porcine; neuromedin B-32, porcine; neuromedin B receptor
antagonist; neuromedin C, porcine; neuromedin N, porcine; neuromedin (U-8),
porcine; neuromedin (U-25), porcine; neuromedin U, rat; neuropeptide-gamma
25 (gamma-preprotachykinin 72-92); PG-KII; phyllolitorin; [Leug]-phyllolitorin
(Phyllomedusa sauvagei); physalaemin; physalaemin 1-11; scyliorhinin II,
amide,
dogfish; senktide, selective neurokinin B receptor peptide; [Ser2]-neuromedin
C; beta-
preprotachykinin 69-91, human; beta-preprotachykinin 111-129, human;
tachyplesin I;
xenopsin; and xenopsin 25 (xenin 25), human.
30 Thyrotropin-releasing hormone (TRH) peptides including, but not limited to,
biotinyl-thyrotropin-releasing hormone; [Glul]-TRH; His-Pro-diketopiperazine;
[3-
Me-His2]-TRH; pGlu-Gln-Pro-amide; pGlu-His; [Phe2]-TRH; prepro TRH 53-74;
prepro TRH 83-106; prepro-TRH 160-169 (Ps4, TRH-potentiating peptide); prepro-
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TRH 178-199; thyrotropin-releasing hormone (TRH); TRH, free acid; TRH-SH Pro;
and TRH precursor peptide.
Toxin peptides including, but not limited to, omega-agatoxin TK; agelenin,
(spider, Agelena opulenta); apamin (honeybee, Apis mellifera); calcicudine
(CaC)
(green mamba, Dedroaspis angusticeps); calciseptine (black mamba, Dendroaspis
polylepis polylepis); charybdotoxin (ChTX) (scorpion, Leiurus quinquestriatus
var.
hebraeus); chlorotoxin; conotoxin GI (marine snail, Conus geographus);
conotoxin GS
(marine snail, Conus geographus); conotoxin MI (Marine Conus magus); alpha-
conotoxin El, Conus ermineus; alpha-conotoxin SIA; alpha-conotoxin Iml; alpha-
conotoxin SI (cone snail, Conus striatus); micro-conotoxin GIIIB (marine
snail, Conus
geographus); omega-conotoxin GVIA (marine snail, Conus geographus); omega-
conotoxin MVIIA (Conus magus); omega-conotoxin MVIIC (Conus magus); omega-
conotoxin SVIB (cone snail, Conus striatus); endotoxin inhibitor;
geographutoxin I
(GTX-I) (g-Conotoxin GIIIA); iberiotoxin (IbTX) (scorpion, Buthus tamulus);
kaliotoxin 1-37; kaliotoxin (scorpion, Androct-onus mauretanicus
mauretanicus);
mast cell-degranulating peptide (MCD-peptide, peptide 401); margatoxin (MgTX)
(scorpion, Centruriodes Margaritatus); neurotoxin NSTX-3 (pupua new guinean
spider, Nephilia maculata); PLTX-II (spider, Plectreurys tristes); scyllatoxin
(leiurotoxin I); and stichodactyla toxin (ShK); diphtheria toxin; ricin A;
Pseudomonas
aeruginosa exotoxin A.
Immunotoxins consist in toxins covalently linked to an antibody which acts as
a homing system, specifically targeting the toxin to those cells which one
wishes to
eliminate by the means of an antibody (polyclonal or monoclonal) directed
against a
molecule, or a group of molecules, carried at the surface of the targeted
cells. Toxins
including, but not limited to those cited above, can be used to this effect.
The
invention described in this patent application may be used to deliver such
immunotoxins to the colon. In some cases, the antibody may be replaced by a
small
molecule that similarly acts to target the toxin to a chosen group of cells.
Vasoactive intestinal peptides (VIP/PHI) including, but not limited to, VIP,
human, porcine, rat, ovine; VIP-GIy-Lys-Arg-NH2; biotinyl-PHI (biotinyl-PHI-
27),
porcine; [Glp16] VIP 16-28, porcine; PHI (PHI-27), porcine; PHI (PHI-27), rat;
PHM-
27 (PHI), human; prepro VIP 81-122, human; preproVlP/PHM 111-122; prepro
VIP/PHM 156-170; biotinyl-PHM-27 (biotinyl-PHI), human; vasoactive intestinal
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contractor (endothelin-beta); vasoactive intestinal octacosa-peptide, chicken;
vasoactive intestinal peptide, guinea pig; biotinyl-VIP, human, porcine, rat;
vasoactive
intestinal peptide 1-12, human, porcine, rat; vasoactive intestinal peptide 10-
28,
human, porcine, rat; vasoactive intestinal peptide 11-28, human, porcine, rat,
ovine;
vasoactive intestinal peptide (cod, Gadus morhua); vasoactive intestinal
peptide 6-28;
vasoactive intestinal peptide antagonist; vasoactive intestinal peptide
antagonist ([Ac-
Tyr', D-Phe2]-GHRF 1-29 amide); vasoactive intestinal peptide receptor
antagonist
(4-Cl-D-Phe6 , Leu17]-VIP); and vasoactive intestinal peptide receptor binding
inhibitor, L-8-K.
Vasopressin (ADH) peptides including, but not limited to, vasopressin;
[Asul'6,Argg]-vasopressin; vasotocin; [Asul'6,Argg]-vasotocin; [Lysg]-
vasopressin;
pressinoic acid; [Argg]-desamino vasopressin desglycinamide; [Arg8]-
vasopressin
(AVP); [Argg]-vasopressin desglycinamide; biotinyl-[Arg8]-vasopressin
(biotinyl-
AVP); [D-Arg8] -vasopressin; desamino-[Arg8]-vasopressin; desamino-[D-Arg8]-
vasopressin (DDAVP); [deamino-[D-3-(3'-pyridyl-Ala)]-[Arg8]-vasopressin; [1-
(beta-
Mereapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine]-
[Arg8]-vasopressin; vasopressin metabolite neuropeptide [pGlu4, Cys6];
vasopressin
metabolite neuropeptide [pGlu4, Cys6]; [Lysg]-deamino vasopressin
desglycinamide;
[Lys8]-vasopressin; [Mpr',Va14,DArg8]-vasopressin; [Phe2, Ile3, Orn8]-
vasopressin
([Phe2, Orn8]-vasotocin); [Arg8]-vasotocin; and [d(CH2)5, Tyr(Me)2, Orn8]-
vasotoein.
Virus related peptides including, but not limited to, fluorogenic human CMV
protease substrate; HCV core protein 59-68; HCV NS4A protein 18-40 (JT
strain);
HCV NS4A protein 21-34 (JT strain); hepatitis B virus receptor binding
fragment;
hepatitus B virus pre-S region 120-145; [Ala127]-hepatitus B virus pre-S
region 120-
131; herpes virus inhibitor 2; HIV envelope protein fragment 254-274; HIV gag
fragment 129-135; HIV substrate; P 18 peptide; peptide T; [3,5 diiodo-Tyr7]
peptide
T; R15K HIV-1 inhibitory peptide; T20; T21; V3 decapeptide P 18-110; and virus
replication inhibiting peptide.
Proteins of the Wnt family, and fragments thereof.
While certain analogs, fragments, and/ar analog fragments of the various
polypeptides have been described above, it is to be understood that other
analogs,
fragments, and/or analog fragments that retain all or some of the activity of
the
particular polypeptide, or on the contrary that act as an antagonist thereby
preventing
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43
its action, may also be useful in embodiments of the present invention.
Analogs may
be obtained by various means, as will be understood by those skilled in the
art. For
example, certain amino acids may be substituted for other amino acids in a
polypeptide without appreciable loss of interactive binding capacity with
structures
such as, for example, antigen-binding regions of antibodies or binding sites
on
substrate molecules. As the interactive capacity and nature of a polypeptide
drug
defines its biological functional activity, certain amino acid sequence
substitutions can
be made in the amino acid sequence and nevertheless remain a polypeptide with
like
properties, or on the contrary confer to this analogue antagonistic activity
that
interferes with or blocks the action of the natural product. Furthermore,
small
molecules, whether peptidomimetic or not, natural or synthetic, may be able to
substitute for the proteins and peptides cited above and have similar activity
by
binding to their receptors. On the contrary, such small molecules may block or
interfere with the activity of the proteins and peptides cited above by
various
mechanisms, including, but not limited to, preventing their interaction with
their
cognate receptors. Additionally, many of the proteins cited above act as
initiators of
signaling pathways. An embodiment of this invention is the use of chemical
molecules
(peptides, peptidomimetics, or any other natural or synthetic molecule of any
chemical
nature) as activators or inhibitors of these signaling pathways. Examples of
this
strategy are the use of inhibitors of gamma-secretase to inhibit the Notch
signaling
pathway, or inhibitors of the interaction between beta-catenin and Tcf
transcription
factors to inhibit the Wnt-beta-catenin pathway, both of which are involved in
colorectal cancer.
g) Oligonucleotide Agents
The active agents can also be in the form of oligonucleotides, including
oligoribonucleotides, oligodeoxyribonucleotides and derivatives thereof useful
for
prophylactic, palliative or therapeutic purposes, including gene therapy and
the
treatment of cancer, such as colon cancer.
An oligor~ucleotide is a polymer of a repeating unit generically known as a
nucleotide. An unmodified (naturally occurring) nucleotide has three
components: (1)
a nitrogen-containing heterocyclic base linked by one of its nitrogen atoms to
(2) a 5-
pentofuranosyl sugar and (3) a phosphate esterified to one of the 5' or 3'
carbon atoms
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of the sugar. When incorporated into an oligonucleotide chain, the phosphate
of a first
nucleotide is also esterified to an adjacent sugar of a second, adjacent
nucleotide via a
3'-5' phosphate linkage. Nucleotides are nucleosides that further include a
phosphate
group covalently linked to the sugar portion of the nucleoside. In forming
oligonucleotides, the phosphate groups covalently link adjacent nucleosides to
one
another to form a linear polymeric compound. The respective ends of this
linear
polymeric structure can be further joined to form a circular structure,
however, within
the context of the invention, open linear structures are generally preferred.
Oligonucleotides can include nucleotide sequences sufficient in identity and
number to effect specific hybridization with a particular nucleic acid. Such
oligonucleotides which specifically hybridize to a portion of the sense strand
of a gene
are commonly described as "antisense." In the context of the invention,
"hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen
or
reversed Hoogsteen hydrogen bonding, between complementary nucleotides. For
example, adenine and thymine are complementary nucleobases which pair through
the
formation of hydrogen bonds. "Complementary," as used herein, refers to the
capacity
for precise pairing between two nucleotides. For example, if a nucleotide at a
certain
position of an oligonucleotide is capable of hydrogen bonding with a
nucleotide at the
same position of a DNA or RNA molecule, then the oligonucleotide and the DNA
or
RNA are considered to be complementary to each other at that position. The
oligonucleotide and the DNA or RNA are complementary to each other when a
sufficient number of corresponding positions in each molecule are occupied by
nucleotides which can hydrogen bond with each other.
The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term
includes
oligonucleotides composed of naturally-occurring nucleobases, sugars and
covalent
intersugar (backbone) linkages as well as oligonucleotides having non-
naturally-
occurring portions which function similarly. They may be single or double
stranded.
Generally, oligonucleotides formulated in the compositions of the invention
may be
from about 8 to about 100 nucleotides in length, more preferably from about 10
to
about so nucleotides in length, and most preferably from about 10 about 25
nucleotides in length.
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Oligonucleotides that are formulated in the compositions of the invention
include antisense compounds and other bioactive oligonucleotides. A discussion
of
antisense oligonucleotides and some desirable modifications can be found in De
Mesmaeker et al. (Acc. Chem. Res., 1995, 28, 366).
5 As used herein, antisense compounds include antisense oligonucleotides,
antisense peptide nucleic acids (PNAs), small interfering RNAs, short hairpin
RNAs,
ribozymes and external guide sequences (EGSs). In antisense modulation of
messenger RNA (mRNA), hybridization of an antisense compound with its mRNA
target interferes with the normal role of mRNA and causes a modulation of its
10 function in cells. The functions of mRNA to be interfered with include all
vital
functions such as translocation of the RNA to the site for protein
translation, actual
translation of protein from the RNA, splicing of the RNA to yield one or more
mRNA
species, turnover or degradation of the mRNA and possibly even independent
catalytic
activity which may be engaged in by the RNA. The overall effect of such
interference
15 with mRNA function is modulation of the expression of a protein, wherein
"modulation" means either an increase (stimulation) or a decrease (inhibition)
in the
expression of the protein. In the context of the present invention, inhibition
is the
preferred form of modulation of gene expression.
Antisense compounds can exert their effect by a variety of means. One such
20 means is the antisense-mediated direction of an endogenous nuclease, such
as RNase
H in eukaryotes or RNase P in prokaryotes, to the target nucleic acid (Chiang
et al., J.
Biol. Chem., 1991, 266, 18162; Forster et al., Science, 1990, 249, 783).
The sequences that recruit RNase P are known as External Guide Sequences,
hence the abbreviation "EGS" (Guerrier-Takada et al., Proc. Natl. Acad. Sci.
USA,
25 1997, 94, 8468). Another means involves covalently linking a synthetic
moiety having
nuclease activity to an oligonucleotide having an antisense sequence, rather
than
relying upon recruitment of an endogenous nuclease. Synthetic moieties having
nuclease activity include, but are not limited to, enzymatic RNAs, lanthanide
ion
complexes, and the like (Haseloff et al., Nature, 1988, 334, 585; Baker et
al., J. Am.
30 Chem. Soc., 1997, 119, 8749).
As used herein, the term "antisense compound" also includes ribozymes,
synthetic RNA molecules and derivatives thereof that catalyze highly specific
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endoribonuclease reactions (see, generally, U.S. Pat. No. 5,543,508 to
Haseloff et al.
and U.S. Pat. No. 5,545,729 to Goodchild et al.).
In addition, the term "antisense compound" includes RNAs (or DNAs that
encode such RNAs) leading to the modulation of gene expression by the
mechanism
of RNA interference. Such molecules include, but are not limited to, short
interfering
RNAs, consisting of double stranded RNAs of less than 50 base pairs, typically
21 or
29 nucleotides in length with the addition at either of their extremities of
other
chemical molecules (including deoxyribonucleotides, natural or modified), as
well as
short hairpin RNAs (or DNA molecules including plasmids and viruses of any
nature
leading to their production, in vitro or in vivo) that act by RNA
interference. This also
includes any DNA or RNA molecule, single or double strand, that leads in cells
to
RNA interference.
The antisense compounds formulated in the compositions of the invention (1)
can be from about 8 to about 100 nucleotides in length, more preferably from
about 10
to about 30 nucleotides in length, (2) single or double stranded, (3) are
targeted to a
nucleic acid sequence required for the expression of a gene from a mammal,
including
a human, and (4), when contacted with cells expressing the target gene,
modulate its
expression. Due to the biological activity of the gene product encoded by the
target
gene, modulation of its expression has the desirable result of providing
specific
prophylactic, palliative and/or therapeutic effects.
It is understood in the art that the nucleobase sequence of an oligonucleotide
or
other antisense compound need not be 100% complementary to its target nucleic
acid
sequence to be specifically hybridizable. An antisense compound is
specifically
hybridizable to its target nucleic acid when there is a sufficient degree of
complementarity to avoid non-specific binding of the oligonucleotide to non-
target
sequences under conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or therapeutic
treatment, or, in
the case of in vitro assays, under assay conditions.
Other bioactive oligonucleotides include aptamers and molecular decoys. As
used herein, the terin is meant to refer to any oligonucleotide (including a
peptide-
nucleic acid or PNA) that (1) provides a prophylactic, palliative or
therapeutic effect
to an animal in need thereof and (2) acts by a non-antisense mechanism, i.e.,
by some
means other than by hybridizing to a nucleic acid.
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The name aptamer has been coined by Ellington et al. (Nature, 1990, 346, 818)
to refer to nucleic acid molecules that fit and therefore bind with
significant specificity
to non-nucleic acid ligands such as peptides, proteins and small molecules
such as
drugs and dyes. Because of these specific ligand binding properties, nucleic
acids and
oligonucleotides that may be classified as aptamers may be readily purified or
isolated
via affinity chromatography using columns that bear immobilized ligand.
Aptamers
may be nucleic acids that are relatively short to those that are as large as a
few
hundred nucleotides. For example, RNA aptamers that are 155 nucleotides long
and
that bind dyes such as Cibacron Blue and Reactive Blue 4 with good selectivity
have
been reported (Ellington et al., Nature, 1990, 346, 818). While RNA molecules
were
first referred to as aptamers, the term as used in the present invention
refers to any
nucleic acid or oligonucleotide that exhibits specific binding to small
molecule
ligands including, but not limited to, DNA, RNA, DNA derivatives and
conjugates,
RNA derivatives and conjugates, modified oligonucleotides, chimeric
oligonucleotides, and gapmers (see, e.g., U.S. Pat. No. 5,523,3B9, to Ecker et
al.,
issued Jun. 4, 1996 and incorporated herein by reference).
Molecular decoys are short double-stranded nucleic acids (including single-
stranded nucleic acids designed to "fold back" on themselves) that mimic a
site on a
nucleic acid to which a factor, such as a protein, binds. Such decoys are
expected to
competitively inhibit the factor; that is, because the factor molecules are
bound to an
excess of the decoy, the concentration of factor bound to the cellular site
corresponding to the decoy decreases, with resulting therapeutic, palliative
or
prophylactic effects. Methods of identifying and constructing decoy molecules
are
described in, e.g., U.S. Pat. No. 5,716,780 to Edwards et al.
Another type of bioactive oligonucleotide is an RNA-DNA hybrid molecule
that can direct gene conversion of an endogenous nucleic acid (Cole-Strauss et
al.,
Science, 1996, 273, 1386).
Preferred modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphoro-dithioates,
phosphotriesters,
aminoalkylphosphotriesters, methyl and other a1ky1 phosphonates including 3`-
alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates
including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkyiphosphonates, thionoalklyphosphotriesters,
and
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boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these,
and
those having inverted polarity wherein the adjacent pairs of nucleoside units
are linked_
3'-5' to 5'-3' or 2'-5' to 5'-2. Various salts, mixed salts and free acid
forms are also
included.
Any of the preceding bioactive oligonucleotides can be formulated into the
drug delivery system of the invention and used for prophylactic or therapeutic
purposes. The oligonucleotides can be stabilized through complexation, for
example,
with cationic lipids such as Lipoplexe or cationic polymers such as Polyplexe.
h) Diagnostic Agents
Medical imaging is the non-invasive or non-surgical visualization of internal
organs or processes. Representative diagnostic methods include X-rays,
magnetic
resonance imaging (MRI), radionuclides or nuclear medicine, and ultrasound.
Radionuclides are nuclei that decay by dissipating excess energy (parent) to
become stable (daughter) by energy emission in form of particulate or
electromagnetic
radiation. Fluoroscopy is a fluorescent screen that detects gamma or X-rays,
which
are imaged by a TV camera to afford real time images of organs in motion by
using
contrast agents, such as PCTA. CAT - Computed axial tomography - takes
advantage of small differences in tissue radiographic density to create an
image. The
colon is often imaged using a lower GI series of a barium enema to conduct a
radiographic study of the large bowel colon and rectum.
Technetium is a common radiolabel. Other radiolabeled compounds include
iodine radiolabels, such as iobenguane sulfate 131I, sodium 123iodine, sodium
131iodine,
and indium labels, such as 111In radiolabels, indium chloride, and indium
satumomabpendetide. Imaging contrast agents include iron-containing contrast
agents
such as ferumoxides and dentritic gadolinium.
The present invention can be used to deliver to the colon agents that enable
or
facilitate the visualization of structures, lesions, cells caM-ing defined
cell surface or
intracellular molecules by any irrlag?mg technique including, but not
restricted to,
radiographya radio-tornt_,(r,t:'eY, resonance imaging (MRI), ultrasonic,
positron emission tomography (PET scan), or any other form of irnaging
technique
using radio-magnetic waves of wEiatever wavclength. For example, small
molecules or
antibodies that recognize cell-surface structures of colon cancer cells can be
labeled
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with radionuclides such as 99":I'eclanecium aiid used to detect tumor cells
and
metastases of various sizes incliiding micro-metastases.
IL Methods for Prepar: ie Pectin Beads
Pectin beads can be prepared using methods known to those of skill in the art,
including by mixing the active agent(s) in a pectin solution, and gelling the
pectin
anionic moieties with a divalent cation such as divalent zinc, for example, in
the form
of a zinc acetate solution.
The gellation is typically done by stirring a solution, suspension or
dispersion
of the active agent, in one embodiment, (3-lactamase L1, and pectin, adjusting
the pH
of the solution if necessary, and adding this solution dropwise to a zinc
acetate
solution under agitation. In some embodiments, where the active agent(s) are
not
adversely affected by other metal ions, divalent or trivalent metal ions other
than zinc
can be used.
Suitable technologies for adding the pectin solution dropwise to the zinc
acetate solution are known to those of skill in the art; and include the multi-
nozzle
system from Nisco Engineering AG and other relevant technologies to produce
drops
from a pectin solution.
The pectin drops undergo a gelification process, ideally during a
predetermined time to obtain the best encapsulation yield and subsequent
release
efficiency.
The concentration of the pectin solution is advantageously from around 4 to
around 10% (w/v), preferably around 4 to around 7%, the metal cation, such as
zinc
acetate, solution is advantageously from about 2 to about 20% (w/v),
preferably from
about 5 to about 15%. More preferably, the pectin solution is about 5% (w/v),
the zinc
acetate solution is about 12% (w/v).
The pectin beads are advantageously stirred in the metal cation, such as zinc
acetate, solution, at a pH of about 6, at room temperature, under slow
agitation, for at
least around 12 minutes up to around 20 hours, preferably from around 20
minutes to
around 2 hours.
The beads can then then be recollected and rinsed in distilled water, ideally
until the conductivity of the rinsing solution reaches a plateau. Rinsing is
preferably
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done at least twice or under a continuous process to minimize the amount of
residual
zinc acetate recovered in the rinsing solution.
The rinsed beads can then be collected and can be subjected to a drying
process using methods known to those of skill in the art, including heated
incubator or
5 fluidized bed technologies.
The beads are typically dried at a temperature of between around 20 and
around 40 C for around 30 min to around 24 hours, preferably at around 35 C
overnight. Drying is preferably performed until the weight of the beads
reaches a
plateau.
10 The diameter of the particles can be finely tuned using needles of
appropriate
internal diameter to form the pectin drops added to the zinc acetate solution.
The
beads are preferably between about 600 and 1500 p.m in diameter.
When the active agent is (3-lactamase L1, the encapsulation yields are
typically
between 50 and 100%, measured in terms of enzymatic activity.
III. Formation of Drug Delivery Systems Including Pectin Beads
The pectin beads can be collected, and combined with appropriate excipients
and formulated into a variety of oral drug delivery systems. For example, the
beads
can be combined with a solid excipient, and tableted, or included in a
capsule.
The pectin beads can also be combined with liquid/gel excipients which do not
degrade the pectin beads, and the mixture/dispersion can be incorporated into
a
capsule, such as a gel-cap.
The tablets or capsules can be coated, if desired, with a suitable enteric
coating
so as to assist in passing through the stomach without degradation. The pH in
the
stomach is of the order of 1 to 3 but it increases in the small intestine and
the colon to
attain values close to 7 (Hovgaard L. et al. (1996) Current Applications of
Polysaccharides in Colon Targeting, Critical Reviews in Therapeutic Drug
Carrier
Systems, 13, 185). The drug delivery systems, in the form of tablets, gelatin
capsules,
spheroids and the like, can reach the colon, without being exposed to these
variations
in pH, by coating them with a pH-dependent polymer, insoluble in acidic pH but
soluble in neutral or alkaline pH (Kinget et al. op. cit.). The polymers most
currently
used for this purpose are derivatives of inethacrylic acid, Eudragit L and S
(Ashford
M. et al. (1993), An in vivo investigation of the suitability of pH-dependent
polymers
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for colonic targeting, International Journal of Pharmaceutics, 95, 193 and 95,
241;
and David A. et al. (1997) Acrylic polymers for colon-specific drug delivery,
S. T. P.
Pharma Sciences, 7, 546) and more recently Eudragit FS.
The drug delivery systems are administered in an effective amount suitable to
provide the adequate degree of treatment or prevention of the disorders for
which the
compounds are administered. The efficient amounts of these compounds are
typically
below the threshold concentration required to elicit any appreciable side
effects. The
compounds can be administered in a therapeutic window in which some the
disorders
are treated and certain side effects are avoided. Ideally, the effective dose
of the
compounds described herein is sufficient to provide the desired effects in the
colon
but is insufficient (i.e., is not at a high enough level) to provide
undesirable side
effects elsewhere in the body.
Most preferably, effective doses are at very low concentrations, where
maximal effects are observed to occur, with minimal side effects, and this is
optimized by targeted colonic delivery of the active agents. The foregoing
effective
doses typically represent that amount administered as a single dose, or as one
or more
doses administered over a 24-hour period.
IV. Methods of Treatment Using the Drug Delivery Systems Described
Herein
The drug delivery systems described herein can be used to treat those types of
conditions and disorders for which colonic delivery is appropriate. In one
embodiment, the disorders are those that result from exposure of the colon to
antibiotics, such as diarrhea, modification of the commensal flora and the
development of bacterial resistance to antibiotics. In this embodiment, the
drug
delivery systems contain agents which inactivate antibiotics, and the active
principles
can be administered in a therapeutically effective dosage to a patient who has
been, is
being, or will be treated with one or several antibiotics.
When the metallo-dependent enzyme is an enzyme other than one which
inactivates antibiotics, such enzyme can be administered to treat the specific
disorders
treated by such enzymes.
In another embodiment, the drug delivery systems are administered to a patient
who suffers from colon cancer. In this embodiment, the drug delivery systems
include
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one or more antitumor agents, and the systems are administered in a
therapeutically
effective dosage to a patient who is suffering from colon cancer.
Alternatively, the
cancer can be present at another location in the body, and the drug delivery
systems
can be used to by-pass the stomach and its concomitant degradation of certain
antitumor agents, so as to avoid the need to use intramuscular or intravenous
administration of these agents.
In another embodiment, the drug delivery systems are administered to a patient
who suffers from a colonic disorder such as Chrohn's disease, ulcerative
colitis,
irritable bowel syndrome, diarrhea, or constipation. In this embodiment, the
drug
delivery systems include agents which treat or prevent these disorders, and
the
systems can be administered in a therapeutically effective dosage to a patient
who is
suffering from such a disorder.
In still another embodiment, the drug delivery systems are used to administer
peptide or protein-based active agents, such as insulin, antibodies, and the
like, or
oligonucleotide-based therapeutics, such as antisense or RNA interference
therapy, so
that the agents pass through the stomach without being digested. In this
embodiment,
the drug delivery systems include these protein/peptide/oligonucleotide-based
agents,
and the systems can be administered in a therapeutically effective dosage to a
patient
in need of treatment with these agents, without the need to administer these
agents via
subcutaneous or intravenous injection.
In a further embodiment, the drug delivery systems are used to administer
diagnostic agents to the colon. In this embodiment, the drug delivery systems
include
diagnostic agents, such as imaging contrast agents, and the systems are
administered
in a diagnostically effective dosage to a patient who will be subjected to a
diagnostic
assay for diagnosis of a colonic disorder.
The present invention will be further understood with reference to the
following non-limiting examples.
Example 1: Development of a Sensitive, Quantitative and Specific Assay for ~3-
lactamase L1
Hydrolysis of nitrocefin is a well known technique used to quantify
penicillinase activity. However, the usual format is in single tubes and is
not adapted
for analysis of a large number of samples. This example describes the
development
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53
and fit for purpose qualification of this assay in 96 wells microplate format
A stock solution of nitrocefin was obtained by dissolving nitrocefin dried
powder at a concentration of 10 mM in dimethylsulfoxide (DMSO). The stock
solution was stored at -20 C and diluted 100-fold immediately prior to use in
50 mM
sodium phosphate buffer (Hepes buffer) pH 7.0 containing 0.1 mg/ml bovine
serum
albumin (BSA). Buffer selection is described in Table I.
20 l containing the solution to be analyzed were added to 180 ~tl of diluted
nitrocefin. Kinetics of nitrocefin hydrolysis were followed at 37 C with
absorbance
measured at 492 nm each 30 seconds using a Multiskan Ascent (Thermo
Labsystems)
plate reader.
The slope (difference in absorbance/second) was calculated using Excel Adds
In Cellula (Prism Technologies, Cambridge UK).
t3-lactamase L1 (Eurogentec, Belgium, approx. 10mg/mL was determined by
BCA assay) was diluted 500x, 1000x, 2000x and 4000x in each solubilization
buffer
and reaction was initiated by adding 20 l of solution containing enzyme to
180 gl of
buffers containing nitrocefin at 100 p.M.
Activity of (3-lactamase Ll was tested in 10 mM Hepes, 145 mM NaCI buffer
pH 7.4. The interference of EDTA with the activity of the metallo-dependent
enzyme
and the need for a carrier protein (Bovine Serum Albumin, abbreviated as BSA)
were
tested. As illustrated in Table 2, EDTA (which can be used to solubilize beads
in vitro
to assay their contents) should be avoided. The inclusion of BSA or other
carrier
proteins is beneficial.
Table 2: Selection of buffer composition for /3-lactamase LI activity
quantifieation
Buffer Slope Yield
10 mM Hepes, 145 mM NaCI pH 7.4 0.142 100%
10 mM Hepes, 145 mM NaC1, 1 lo EDTA pH 7.4 0.026 18.8%
10 mM Hepes, 145 mM NaCI, 0.1 mg/ml BSA pH 7.4 0.167 118.2%
10 mM Hepes, 145 mM NaCI, 0.1 mg/ml BSA, 1% 0.084 59.0%
EDTA pH 7.4
As illustrated in Table 1, EDTA interferes with the enzymatic activity assay,
and BSA enhances the recovery of enzymatic activity.
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Example 2: Instability of P-lactamase Ll In Original Pectin Mix and Effect of
Metallic Counter-Ion
0.3 ml of (3-lactamase L 1 (Eurogentec, Belgium, approx. l Omg/mL as
determined by BCA assay) was mixed to 10g of a 6% pectin solution (Low
methoxylated amidated pectin (Unipectine), Texturant Systems, cat# OG175C)
made
in water; the pH of the pectin solution was not adjusted.
The pectin/13-lactamase Ll mixture was added drop-wise over a period of 2
minutes using a peristaltic pump and a needle of 0.8 mm inner diameter to a
beaker
containing 40 ml of calcium chloride (6%) under agitation (200 rpm) at room
temperature.
After further incubation to allow equilibration between free and bound calcium
ions, beads were recovered by filtration and washed 3 times in 200 ml of
purified
water to eliminate excess of free calcium. At this stage, beads are referred
to as
"gelled beads".
Beads were dried 2 hours at 37 C in an oven, yielding dried beads.
2 x 5 droplets and 2 x 15 droplets were sampled at the exit of the needle to
measure the initial P-lactamase Ll activity. Protein-free beads were also
prepared as
negative controls.
The P-lactamase L1 enzymatic activity (nitrocefin hydrolysis) was quantified
with and without Zn ions (0.1 mM ZnC12) as described in example 1.
As illustrated in Table 3, no enzymatic activity was found in the (3-lactamase
L 1/ pectin mix while significant activity was recovered in the beads assayed
in buffer
containing Zn "2.
30
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Table 3 : Inactivation of ALactamase LI in non pH-adjusted pectin solution
Slope/min Without ZnAc With ZnAc
Before mix with pectin 0.105 0.103
(100.0%) (100.0%)
P-Lactamase/pectin mix 0.000 0.000
(0.0%) (0.0%)
Gelled milli particles 0.0078 0.042
(7.4%) (40.7%)
Example 3: Optimization of Metallic Ion Used to Gel the Pectin, and the Effect
of
5 pH of the Pectin Solution.
In order to determine the effects of the pectin solution parameters and zinc
ions, an experiment comparing four formulations was performed. The design was
built according to factorial design, Design Expert 6Ø10, Stat-Ease,
Minneapolis. Two
parameters were tested:
10 (a) pH of the pectin solution: 4.0 and 7.0
(b) the metallic cation in the gelification bath: Ca2+ (CaC12) or Zn2 + (Zinc
acetate
abbreviated ZnAc)
Beads were prepared as described in Example 2. However, the concentration
of the pectin solution was decreased from 6% to 4% due to the decrease in
solubility
15 of pectin with increased pH.
The encapsulation yield was measured by assaying the enzymatic activity of ~3-
Lactamase L 1 as described in Example 1.
5 beads were solubilized in 20 ml of 10 mM Hepes, 145 mM NaCl, 0.1 mg/ml
BSA at a pH of 7.4, in the presence or absence 1% pectinase (Pectinases from
20 Aspergillus Aculeatus, Pectinex SP-L Ultra (SIGMA, France) overnight at 4
C.
The positive control was prepared by diluting the same amount of 13-lactamase
L 1 as should be contained in 5 beads in 20 ml of 10 mM Hepes, 145 mM NaCI,
0.1
mg/ml BSA pH 7.4. As illustrated in Table 4, (3-lactamase Ll was inactivated
irrespective of the cation used for pectin gelification when the pectin
solution was at
25 pH 4.0 (4.3 % residual activity in calcium and 3.8 % in zinc), whereas
nearly full
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activity was retained after buffering the pectin solution to pH 7.0 (86.7% in
calcium
and 64.0% in zinc).
Table 4 : Effect of cation used for gelifzcation and pH of pectin on stability
(recovery of ALactamase activity)
Sample CaCl2, pH4 CaCl2, pH7 ZnAc, pH4 ZnAc, pH7
Before mix 0.102 0.090 0.108 0.090
(100%) (100%) (100%) (100%)
Gelled beads 0.004 0.072 0.004 0.072
(4.3%) (80.0%) (3.8%) (80.0%)
Dried beads 0.003 0.078 0.037 0.058
(31.7%) (86.7%) (35.0%) (64.0%)
Example 4: Determination of Critical Parameters to Formulate (3-Lactamase Ll
for Colon-Specific Delivery and Optimization of These Parameters
Five parameters were tested:
(a) Concentration of the pectin solution (Low methoxylated amidated pectin
(Unipectine), Texturant Systems, cat# OG175C): 4% and 5% (w/v)
(b) Cation for gelification : Ca2k or Zn2+
(c) Secondary coating of the gelled beads with polyethyleneimine (PEI)
solution (PEI, High molecular weight, water-free (SIGMA-ALDRICH, France))
(d) pH of the PEI solution: 7 and 11 (original non pH-adjusted solution).
(e) Solubilization of the beads to assay the encapsulated enzymatic activity
with and without 1% pectinase.
Table 5 summarizes the experimental design
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Table 5 : Experimental design.for the optimization qf'critical parameters
involved
in ,(3-Lactamase LI formulation
A: D:
Pectin B: C: pH of E:
Run (%) Ion PEI Coating PEI Pectinase
1 5 Zn~+ Yes 11 Yes
2 4 Zn2+ Yes 7 Yes
3 5 Ca2+ No Yes
4 4 Ca2+ Yes 7 No
5 Ca2 + Yes 7 Yes
6 4 Ca2+ No Yes
7 4 Zn2} Yes Il No
8 5 Ca2+ Yes 11 No
9 4 Ca2" Yes 11 Yes
4 Zn2+ No No
11 5 Zn2} No Yes
12 4 Zn2+ No Yes
13 5 Zn2+ Yes 7 No
14 5 Ca2+ No No
4 Ca2+ No No
16 5 Zn2+ No No
These 16 experiments were performed in duplicate (32 results).
5 Run 13 replicated (34 results).
The pH of the 4% and 5% pectin solutions were adjusted to 7Ø However, it
was determined that the pH of the 5% pectin solution was unstable and
decreased to
pH 5.4 by the end of the experiments. A 5% pectin solution was therefore also
adjusted to pH 8.5 for comparison.
10 Finally, the 48 results were analyzed using Factorial Design.
Beads were prepared as described in example 2 except that the gelification
time in the cation bath was reduced from 20 min to 10 min to allow a smart
timing of
the experiments.
Samples (5 beads) were solubilized overnight at 4 C in 20 ml of 10 mM
15 Hepes, 145 mM NaCl, 0.1 mg/ml BSA pH 7.4 with and without 1% pectinase
before
measuring enzymatic activity (nitrocefin hydrolysis as described in example
1).
Tale 6 summarizes the experimental results obtained.
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Table 6: Full results of Experimental design for
optimizing critical parameters involved in fl-Lactamase LI formulation
Run %pectin pH pectin ion PEI pH of PEI pectinase yield
1 5 5.4 Zn2+ yes 11 yes 1.201
17 5 5.4 Zn2+ yes 11 yes 1.13
3b 5 5.4 Zn2+ yes 11 yes 1.39
11 5 5.4 Zn2+ no yes 1.272
27 5 5.4 Zn2 " no yes 1.36
2b 5 5.4 zn2+ no yes 1.044
lb 5 5.4 Zn2+ yes 7 yes 1.045
13 5 5.4 Zn2+ yes 7 no 0.687
29 5 5.4 Zn2+ yes 7 no 0.72
33 5 5.4 Zn2" yes 7 no 0.661
34 5 5.4 Zn2+ yes 7 no 0.691
16 5 5.4 Zn2} no no 0.762
32 5 5.4 Zn2+ no no 0.788
45 5 8.5 Zn2 no yes 0.951
38 5 8.5 Zn2+ no yes 0.818
41 5 8.5 Zn2+ no no 0.245
48 5 8.5 Zn2+ no no 0.363
46 5 8.5 Zn2+ yes 7 no 0.815
39 5 8.5 Zn2 } yes 7 no 0.826
2 4 7 Zn2+ yes 7 yes 1.01
18 4 7 Zn2+ yes 7 yes 1.162
12 4 7 Zn2} no yes 1.165
28 4 7 Zn'`} no yes 1.148
7 4 7 Zn2+ yes 11 no 0.727
23 4 7 Zn2+ yes 11 no 0.679
4 7 Zn'+ no no 0.674
26 4 7 Zn2+ no no 0.659
5
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Table 6 (continued): Full results of Experimental design for optimizing
critical parameters involved in fl-Lactamase LI formulation
Run %pectin pH pectin ion PEI pH of PEI pectinase yield
3 5 5.4 Ca2+ yes 7 yes 0.094
5 5.4 Ca2+ yes 7 yes 0.031
19 5 5.4 Ca'+ yes 7 yes 0.108
21 5 5.4 Ca2} yes 7 yes 0.039
8 5 5.4 Ca2+ yes 11 no 0.047
24 5 5.4 Ca2+ yes 11 no 0.066
14 5 5.4 Ca2+ no no 0.488
30 5 5.4 Ca2T no no 0.512
35 5 8.5 Ca2+ yes 7 yes 0.35
36 5 8.5 Ca2+ yes 7 yes 0.379
42 5 8.5 Ca2+ yes 7 yes 0.363
43 5 8.5 Ca2+ yes 7 yes 0.394
4b 5 8.5 Ca2+ yes 7 yes 0.53
7b 5 8.5 Ca2+ yes 7 no 0.704
37 5 8.5 Ca2+ yes 11 no 0.029
44 5 8.5 Ca2+ yes 11 no 0.029
9b 5 8.5 CW+ yes 11 no 0.737
40 5 8.5 Ca2{ no 0.322
47 5 8.5 Ca2+ no 0.656
6b 5 8.5 Ca~+ yes 11 yes 0.517
5b 5 8.5 Ca2+ no yes 0.656
8b 5 8.5 Ca2+ no no 0.967
Simple mono-variate statistical analysis (decreasing yield of encapsulation
5 sorting) highlighted that an optimal formulation of (3-lactamase Ll was
obtained using
the following parameters:
(a) A pectin concentration of 5% (maximum solubility at pH 5.4)
(b) A pectin solution neutralized to a pH of at least 5.4
(c) A zinc ion should be used
(d) A secondary coating may be further evaluated with other type of
polymers
(e) A pectinase should be used to quantify formulated (3-lactamase Ll.
Example 5: Improvement of Stability of the Beads Comprising (3-Lactamase Li
in Simulated Intestinal Media (SIM) by Increased Zinc Ion Concentration and
Duration of Drying
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Beads containing (3-lactamase L 1 were prepared as described in example 4.
Increasing zinc acetate concentrations (6, 8, 10 and 12%) were tested. Further
coating
with or without PEI were compared.
Drying of beads was also increased from 2 hours to overnight.
5 Efficiency of washing to remove excess metallic ion used for gelification
was
also monitored by measuring the conductivity of the water rinsing solution.
As illustrated in Figure 1, efficient washing was obtained after washing the
beads in three water washes.
As illustrated in Table 7, the higher concentration of zinc acetate increased
10 stability in SIM (Simulated Intestinal Medium, US Pharmacopeia 26) of the
beads
containing (3-lactamase Ll while PEI secondary coating decreased their
stability.
Table 7: Effect of Zinc acetate concentration and PEI secondary coating on
stability of beads containing ALactamase LI in SI1V.f
SIM
Run# % Zn PEI 1 h 2 h 3 h 4 h 5 h
8 10% N + + + + +
3 12% Y + + + + +
4 12% N + + + + +
2 8% N + + + + +
5 6% Y - - - - -
1 8% Y + + - - -
7 10% Y +
+ : stable beads
- : dissolved beads
Y with PEI secondary coating
N without PEI secondary coating
Example 6: Effect of Zinc Concentration and Drying Time on the Stability of
Beads in Simulated Intestinal Media (SIM)
Beads containing (3-lactamase L1 were prepared as previously described, and
gelled with 6 or 12 % Zinc acetate solutions (see Example 5).
The effect of drying time was also tested by drying beads for 2, 4 and 16 h at
C (temperature preferred to 37 C for industrialization purposes). Only beads
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61
gelled in the 12% zinc solution and dried for more than 4 h were stable in SIM
after
5h incubation at 37 C. The stability of the beads in SIM for 5h @ 37 C was
measured, and the results are shown in Table 8.
Table 8: Stability of beads in Simulated Intestinal Medium for S h at 37 C.
The numbers represent the number of beads still apparently intact in solution.
III c, i" ,-i c,11 ait 211 ch-z-in, 4 11 dryir}g C1ver1light
:~7 C"d,l;
t~
I 5 0
milli-paa=ticles ~ 1 0 0
in 6% Zyi 3 1 0 0
4 1 0 0
0 0
i ~ 5 5
Il3iili-pat ticTes 2 5 i 5
ial 12~ o Zii 3 5 5 5
4 4 5 5
5 3 4 5
After washing and further incubation in Simulated colonic medium (SCM): 10
mM Hepes, 145 mM NaC1(stock solution). 1% pectinase, 0.1 mg/ml BSA were added
just before use; pH was adjusted to pH 6.0 with NaOH 1 M, 63% of the initial
(3-
lactamase activity (nitrocefin hydrolysis) was recovered.
Example 7: Effect of Gelification Time, Rinsing Process, and Drying Time on
Recovery of (i-Lactamase Ll Activity
Different batches of beads were prepared using a mutli-nozzle system from
Nisco Engeneering AG. The beads underwent various gelification times, rinsing
process and time and drying process type and time.
It appears clearly that the best encapsulation efficiency and enzyme activity
are
obtained when gelification time is less than 20 hours and when rinsing is
performed
such as to eliminate residual Zinc acetate from the beads. Results are
presented in
Figure 2.
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Example 8: Development of a Sensitive, Quantitative and Specific Assay for (3-
lactamase L1
Hydrolysis of CENTA is a well known technique used to quantify P-lactamase
activity. However, the usual format is in single tubes and is not adapted for
analysis of
a large number of samples. This example describes the development and fit for
purpose qualification of this assay in 96 wells microplate format
A stock solution of CENTA was obtained by solubilization of the CENTA
dried powder at a concentration of 25 mM in water; it was stored in 25 q.l
aliquots at -
20 C. The assay mix was done by diluting 22 l of CENTA stock solution in the
following assay buffer:10 ml 30 mM Hepes buffer pH 7.5 containing 50 m ZnC12,
hence yielding a CENTA concentration of 110 M. For the assay, 20 l
containing the
enzyme to be assayed were added to 180 l of assay mix, hence using a final
concentration of 100 M CENTA In the assay. Kinetics of CENTA hydrolysis were
followed at 37 C with a measure of absorbance at 405 nm each 9 seconds using a
Multiskan Ascent (Thermo Electron Corporation) plate reader. The slope
(difference
in absorbance/second) was calculated using Ascent Software for Multiskan
Ascent
version 2.6.
P-lactamase L1 (Eurogentec, Belgium, approx. l0mg/mL as determined by
BCA assay) was diluted to 0.2, 0.5, 1.0 and 2.0 glml in assay buffer and the
reaction was initiated by addition of 20 l of enzyme-containing solution to
180 l of
assay mix. As shown in Figure below, the assay was linear in 3 independent
assays
with respect to enzyme concentration in that range. Standard deviation was
less than
10%.
Example 9. Release of P-lactamase Ll from uncoated beads, and Eudragit-coated
beads with or without HPMC pre-coating.
A batch of pectin beads containing P-lactamase Ll was manufactured under
the following conditions: beads were formed by adding dropwise through a 0.5
mm
internal diameter needle a solution of 5% pectin containing 300 mg/l purified
recombinant P-lactamase Ll (Eurogentec, Belgium) to a 12% bath of Zn acetate,
2H?O. Beads were gelified for 90 min in the Zn acetate bath, collected, washed
with
water untill the water conductivity had reached a stable plateau, signifying
that rinsing
is optimal and finally dried at 35 C under vacuum. Dried beads obtained were
0.8-
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63
1.25 mm diameter, weighed on average 0.6 mg and contain approx 5 to 6 g (3-
laetamase L 1 per mg of beads. They were either left uncoated, or coated using
a Glatt
GPC 1.1 with Top spray according to the following formulas shown in Table 9.
Table 9
Raw materials Amount ~nm
Amount ~ Amount Ãg Amount Amount Amount
(g) (g) ~ (g) (g) (g) (g)
Batch 83 Batch Batch 82 Batch 99 Batch 81 Batch 97
à 100
q. m. ,.. . . . ~..... . . . .i.,...,....a ..... .... . .. ....~ w..:. .. . .
..,. ... _ . ., . .. . . . . .
L30D 55 ~~ 1600~0 149.5 0..,.U .0 31,9
Eudrait 3-
. m .w..,.. .. ...._..~~ __..~ .... _. . .~ _.. ~. ~~M. .~_..__-..~
Eudragit NE 30 D e ...w...em_._ 71000 744
i. .~...~...w.~~._.. _w. ,...._-.~...~
m~~..~._.~~...._.~
Eudragit FS30D $ ~ 800.0 8~.0
r_....... ... --._._~,._,..-...-..-w...,_.. M .,. _. ,_ .
..._ .m.r..., ..._.. .. ,........-w ~.mk
GMS (Glycerol 24.0 2.2 15.0 1.6 12.0 1.3
monostearate) _.. -... -. ..,_ ~ w ..
Sodimn Hydroxide 28.8 2.7 30.4 1.9 1.5
l ween.._.. ...b.n.. -... 80 48.0 2.2 18.0 . ~...._.-.- ...e- . ._ ,w.p.....
~ a m 1.6 14.4 1.3
(polysorbate)
33% Aqueous
x solution
.._ .~. _.rv.~...._.. ....-.4w w. ..~~ ~ ...w_.. _ -.._m.
11 07. 2-.
4 ~ itrate
94 5 4.50 67._2 10.0 25.2
Water
w mmm v, ~~
1600.0 149.5 565.7 1600.0 5()5.6 85.0
Pre-coating w~th 5% NO YES NO~~~ YESN(> YES
f g g
1 HPMC
._......~ ~ ....... _.......... .. ..... ..~._.~.. .~....~..~..........,..~~..
..._.~_. m . .. ......,. .. .. __.. .~
Pre-coating of beads was performed with HPMC using same material as for
the coating with Eudragit.
Scanning electron micrographs (SEMs) of Eudragit-coated beads are shown in
Figure 4. A cross-section shows the relative thickness of the Eudragit
coating.
I In order to assess the release of (3-lactamase L1, coated and uncoated beads
were incubated under gentle mixing at 37 C in 50 mM Hepes buffer pH 7.4
containing 0.1 M NaCI and 100 PG/ml pectinases from Aspergillus aculeatus
(Sigma
Aldrich). Medium was withdrawn at various times and assayed for P-lactamase
activity using the nitrocephin assay described in Example 1.
Release kinetics were measured using the coated and uncoated beads, and the
results are shown in Figure 5.
Example 10 : Efficiency of released L1 to hydrolyze antibiotics in vitro.
In order to assess whether coated beads would actually be able to hydrolyze
antibiotics when they reach the colon, they were successively incubated for lh
in
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64
simulated gastric medium (0.1N HCI), 3h at 37 C in simulated intestinal medium
(50
mM Na/K phosphate buffer pH 6.8 containing 0.1 M NaCI) and finally for the
indicated amounts of time in simulated colonic medium (50 mM Hepes buffer pH
7.4,
0.1 M NaCI) containing 100 PG/mi pectinases from Aspergillus aculeatus (Sigma
Aldrich) and 2 mg/ml amoxicillin. Medium was withdrawn at various times and
the
amount of residual amoxicillin was measure by HPLC and UV absorption. The
procedure was performed using a Bio-Diss III apparatus (Varian). Uncoated
beads
were only incubated in the simulated colonic medium with pectinases and
amoxicillin.
The results are shown in Figure 6.
Example 11: Effect of (3-lactamase L1 containing beads on the emergence of
bacterial resistance in piglets treated with amoxicillin.
6-7 week old piglets were either untreated, or orally treated with 20 mg/kg
amoxicillin per day for 7 days. Half of the treated animals received, together
with the
daily dose of antibiotics, a gelatin capsule filled with 320 mg pectin beads
containing
(3-lactamase LI, pre-coated with 5% HPMC and coated with 40% Eudragit L30D-55
(batch 100); the other half received similarly coated placebo pectin beads.
Feces were
collected 3 days before the onset of treatment, and each day during 7 days of
treatment
and analyzed for their content of total and amoxicillin-resistant
enterobacteria on
MacConkey agar plates containing 0 or 100 g/ml amoxicillin. As shown in
Figure 7,
the feces of untreated animals contained a minimal proportion of amoxicillin-
resistant
bacteria (<5%), whereas this proportion rapidly increased in animals treated
with
amoxicillin, reaching a value between 50 and 80% after 7 days. In contrast,
animals
receiving (3-lactamase containing beads together with amoxicillin only
exhibited a
transient and limited increase in antibiotic-resistant bacteria. This
experiment shows
that the co-administration of Eudragit-coated pectin beads containing P-
lactamase L1
protected piglets against the emergence of antibiotic resistant bacteria
induced by the
treatment of animals with amoxicillin.
All patents and publications disclosed herein are incorporated by reference in
their entirety. Modifications and variations of the present invention will be
obvious to
those skilled in the art from the foregoing detailed description of the
invention.
