Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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plyLy~ BpSgD pHARMACEUTICAL COMPOSITIONS FOR TARGETED DELIVERY OF BIOLOGICALLY
ACTIVE
AGENTS -
BACKGROUND OF THE INVENTION
to
FIELD OF THE INVENTION
This invention relates generally to polymeric constructs useful for the
delivery of
biologically active agents. In particular, it relates to polymeric constructs
useful for
delivery of biogenic amines, pharmaceutical compositions thereof, and methods
for
I5 treating disease states therewith.
RELATED ART
Various polymeric formulations of biologically active agents and methods for
their
preparation have been described.
2o U.S. Patent Nos. 3,773,919, 3,991,776, 4,076,779, 4,093,709, 4,118,470,
4,131,648, 4,138,344, 4,293,539 and 4,675,189, inter alia, disclose the
preparation and
use of biocompatible, biodegradable polymers, such as poly(lactic acid),
poly(giycolic
acid), copolymers of glycolic and lactic acids, poly(o-hydroxycarboxylic
acid),
polylactones, polyacetals, polyorthoesters and polyorthocarbonates, for the
encapsulation
25 of drugs and medicaments. These polymers mechanically entrap the active
constituents
and later provide controlled release of the active ingredient via polymer
dissolution or
degradation. Certain condensation polymers formed from divinyl ethers and
polyols are
described in Polymer Letters, ~,$., 293 (1980). Polymers have proven to be
successful
controlled-release drug delivery devices; however, their performance would
benefit from
3 o increased stability and integrity during storage and from increased target
specificity,
which should enhance the therapeutic index of an incorporated drug.
The disclosures of the foregoing patents and publications, as well as that of
all
other patents and publications referred to in this specification, are
expressly incorporated
herein by reference.
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2
SUMMARY OF THE INVENTION
It is an object of this invention to provide means for improving the storage
stability
and delivery efficiency of biologically active agents, such as biogenic
amines,
incorporated in polymeric carriers.
This invention thus provides a polymeric construct for delivering a
biologically
active agent to a host comprising a first and primary polymeric matrix, a
biologically
active agent entrapped within the first polymeric matrix, and a second
polymeric
component chemically bound to the biologically active agent, which second
polymer is
present in an amount effective to minimize leakage of the active agent from
the polymeric
1 o carrier. Optionally, the polymeric construct further comprises targeting
moieties
associated with its surface for directing the construct to the desired situs.
The second
polymeric component may optionally be copolymerized or otherwise bound to the
first
polymeric matrix.
Also provided are pharmaceutical compositions of the polymeric constructs with
pharmaceutically acceptable excipients and methods of treating mammalian
conditions
with the compositions. Treatment of conditions responsive to biogenic amines,
particularly the treatment of Type II diabetes with serotonin or a
serotonergic agonist is
particularly preferred.
2 o DETAILED DESCRIPTION OF THE INVENTION
In its broadest embodiment, this invention provides a polymeric construct for
delivering a biologically active agent to a host comprising a first and
primary polymeric
matrix, a biologically active agent entrapped within the first polymeric
matrix, and a
second polymeric component chemically bound to the biologically active agent,
which
2 5 second polymer is present in an amount effective to minimize leakage of
the active agent
from the first polymeric matrix. Optionally, the polymeric construct further
comprises
targeting moieties associated with its surface for directing the construct to
the desired
situs: The second polymeric component may optionally be copolymerized or
otherwise
bound to the first polymeric matrix.
3 o The particular biologically active agent or agents employed does not
impose any
significant limitation upon the scope of the invention, provided that the
agent may be
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encapsulated or otherwise entrapped within a polymeric matrix without
significant
diminution of its inherent biological activity.
The first polymeric matrix may be, but is not limited to,
poly(lactide/glycolide)
copolymers (PLGA), poly(lactic acid), poly(glycolic acid), poly(o-
hydroxycarboxylic
acids), poly(lactones), poly(acetals), poly(orthoesters),
poly(orthocarbonates), poly(amino
acids), chitosan glutamate, poly(acrylic acid), poly(divinyl glycol), albumin,
polycarbonates, polyamines, poly(hydroxybutyric acid), scleroglucans,
polyoxypropylene-
polyoxyethylenes, polygalacturonic acid (partially esterifled) and xanthan
gum. In some
embodiments of the invention, combinations of these primary polymers may be
used
1 o together.
The second polymeric component comprises a polymer that carries negatively
charged functional groups. In a preferred embodiment, the second polymeric
component
comprises a poly(amino acid) carrying a net negative charge. In another
preferred
embodiment, the second polymeric component comprises a copolymer of polylysine
with
other amino acids or compounds. Optionally, more than one anchoring secondary
polymeric component may be used in the polymeric construct of the present
invention.
In still another aspect, this invention comprises a method of making a
polymeric
construct comprising mixing a first biocompatible polymer, a second copolymer
of
polylysine-poly(glutamic acid/aspartic acid), a biogenic amine, and a
targeting moiety,
2 o whereby said first and second polymers form a composite polymeric carrier
containing the
biogenic amine and the targeting moiety.
In one embodiment the biologically active agent comprises a biogenic amine,
which includes the sympathomimetic amines, i.e. naturally occurring
catecholamines and
drugs that mimic their action, and autacoids that act at serotonin receptors
to elicit an
2 5 hepatic glucose storage response. See Goodman and Gilman, The
Pharmacological Basis
of Therapeutics, 9th ed. Macmillan Publishing Co. {1995). Representative
biogenic
amines include, but are not limited to, L-~3-3,4-dihydroxyphenylalanine (L-
DOPA), 3-(2-
aminoethyl)-5-hydroxyindole (5-hydroxytryptamine or serotonin), 2-(4-
imidazolyl)ethyl
amine (histamine), 4-[1-hydroxy-2-(methylamino)ethyl]-1,2-benzenediol
(epinephrine), 1-
3 0 [3,4-dihydroxyphenyl]-2-aminoethanol (norepinephrine), y-amino-n-butyric
acid,
acetylcholine and amino acids. In a preferred embodiment, the biogenic amine
is
serotonin, a serotonin analog, or a serotonergic agonist. As used herein, the
terms
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"serotonin analog" and "serotonergic agonist" refer to compounds which mimic
the
activity of serotonin, in particular compounds which act at 5-HT~,2c receptors
of
hepatocytes, which interaction is blocked by methysergide.
The targeting molecule attached to the surface of the polymeric construct
directs
the polymeric construct to an appropriate receptor, such as for example, a
hepatobiliary
receptor. Such target molecules for hepatobiliary receptors can be selected
from
substituted iminodiacetic acids, N'-substituted derivatives of ethylene
diamine N,N-
diacetic acid (EDDA), hepatobiliary dyes, hepatobiliary contrast agents, bile
salts,
hepatobiliary thiol complexes, and hepatobiiiary amine complexes.
1 o In preferred embodiments, the first polymeric matrix is selected from
polymers
such as polylysine, poly(lactide/glycolide), chitosan glutamate, poly(acrylic
acid),
poly(divinyl glycol), albumin, polycarbonates, polyamines, poly(hydroxybutyric
acid),
scleroglucans, polyoxypropylene-polyoxyethylene, polygalacturonic acid
(partially
esterified) and xanthan gum and the second polymeric component is a copolymer
of
polylysine and poly(giutamiclaspartic acid).
The polymeric constructs and pharmaceutical compositions of this invention are
useful for the treatment of disease states responsive to biogenic amines,
including
sympathomimetic amines and autacoids. Such disease states may include, for
example;
hypertension, shock, cardiac failure, arrhythmias, asthma, allergy,
anaphylaxis and
2 o diabetes. Accordingly, the invention comprises a method of treating a
disease state in a
mammal by administering to the mammal a therapeutically effective amount of a
biogenic
amine in a pharmaceutical composition comprising a polymeric construct
containing the
biogenic amine and a second copolymer of polylysine and poly(glutamic/aspartic
acid).
This invention is particularly useful for delivery of biogenic amines, which
as
2 5 potent neurotransmitters, should be sequestered tightly within a polymeric
construct in
order to minimize or substantially eliminate interactions with receptors of
organs or
tissues other than those targeted by the construct. The biogenic amine
neurotransmitters
are water soluble and have polar chemical functionalities. The quaternary
amines, such as
epinephrine and acetylcholine, manifest a positive charge over a broad pH
range, while
3 o the other biogenic amine neurotransmitters are primary amines, positively
charged at
physiological pH and below.
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Because the biogenic amines evince a pronounced polarity contributed by either
a
positively charged quaternary amine group or a positively charged primary
amine group at
physiological pH, they demonstrate unusual water-activity. They are thus
difficult to
retain within a polymeric construct, resulting in poor storage stability and
undesirable,
5 and potentially toxic, leakage after administration. Biogenic amines
interact with a wide
variety of receptors on different cell.types, not necessarily associated with
the condition to
be treated. These cells include, inter alia, neurons, platelets, mast cells,
and
enterochromaffm cells.
This situation may be illustrated with the hormone and neurotransmitter
serotonin.
to In earlier studies, such as described in U.S. Patent No. 4,761,287, it was
shown
that in order to adequately neat non-insulin dependent diabetes mellitus (Type
II), it is
necessary to deliver serotonin to the cellular hepatocytes in the liver using
a targeted
liposomal construct. The delivery of this hormone, in conjunction with the
hormone
insulin, signals the liver to store blood glucose as glycogen. This action
results in the
reduction of the high circulating levels of glucose and lessens exposure of
other tissues
and organs in the body to high glucose levels. If the hormone were to leak
from its
carrier, it would be difficult to deliver the correct dosage to the liver and
the intended use
of the targeted delivery system would be compromised.
Therefore, in a preferred embodiment, this invention is directed to the
retention of
2 0 biogenic amines, such as serotonin or a serotonergic agonist, within a
targeted polymeric
construct. The sequestration of serotonin is required not only due to its
hormonal
function, but also because serotonin acts in vivo as a neurotransmitter in
many different
cell types not associated with the liver. Therefore if exogenous serotonin,
which has been
incorporated in a targeted polymer, is not strongly sequestered or retained by
the polymer
2 5 structure, it may leak from the polymer and elicit undesirable
pharmacological responses
with other cell types that manifest serotonin receptors.
The novel polymeric constructs of this .invention provide a means to deliver a
biologically active agent within a chemically stable carrier, due to strong
ionic bonding
and dipole-dipole interactions between the various carrier constituents. These
chemical
3 o interactions effectively lower the water activity of the biologically
active agent, e.g. the
biogenic amine, within the polymeric construct and thus minimize diffusion of
the agent
from the polymeric carrier. Also the chemical and physical interactions among
the active
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agents themselves (particularly when different agents are used together) are
decreased. As
a consequence of the overall lowering of water activity within the polymeric
construct,
there is less likelihood that individual constructs will fuse, coalesce,
aggregate and/or
precipitate. These interactions markedly increase shelf life stability and in
addition
protect the chemical integrity and stability of the biochemical cargo.
This invention provides a secondary polymeric anchor by which biologically
active
agents, including biogenic amines such as serotonin, can be effectively
sequestered within
a first polymeric matrix for an extended period of time to minimize their
leakage to the
external environment. In a preferred embodiment, the invention provides for
the
1 o sequestration of biogenic amines such as serotonin within a hepatobiliary
targeted
polymeric construct. In one embodiment, serotonin and the hepatobiliary target
molecule
are retained within the first polymeric matrix using a secondary copolymeric
anchor
comprising polylysine-poly(glutamic-aspartic acid).
The use of the polymeric constructs of this invention facilitates the
sequestering
and delivery of biogenic amines by specific functional group interactions
described in this
disclosure. For example, the carbonyl and amino functional groups of the
primary and
secondary polymers can react with biogenic amines by engaging in hydrogen bond
formation. Also, the negatively charged polymeric carboxyl groups can form
ionic
linkages with positively charged primary and quaternary biogenic amines. Thus
the
2 o targeting and cargo molecules may be retained more securely, which
effectively prevents
drug leakage into the external aqueous phase.
Among the polymers useful as polymeric carriers are copolymers of lactic and
glycolic acids (PLGA). These polymers are biocompatible, biodegradable and
approved
for human use. PLGA microspheres generally range in diameter from about 200 to
about
2 5 1500 Angstroms depending upon the degree of polymerization.
This invention involves three interacting aspects. The first is the
incorporation of
a copolymer of lysine and glutamic and aspartic acids (or glutamate and
aspartate) in the
first polymeric matrix as an anchoring molecule for biogenic amines. The
second aspect
is the use of hepatobiliary target molecules bound either to the first
polymeric matrix or,
3 0 optionally, to the anchoring polymer, for delivering the biogenic amine to
the hepatocytes
of the liver. Thus, the secondary copolymer may function both to chemically
anchor
hepatobiliary target molecules and also to retain the biogenic amine. The
third aspect
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relates to the interaction between the primary and secondary polymers which
contributes
to construct stability among the primary polymeric matrix, the secondary
polymeric
anchor, the active agent, and other construct constituents.
Representative hepatobiliary targeting molecules include substituted
iminodiacetic
acids such as N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid, N-
(2,6-
diethylphenyl-carbamoyl methyl)iminodiacetic acid, N-(2,6-
dimethylphenylcarbamoylmethyl)iminodiacetic acid, N-(4-
isopropylphenylcarbamoylmethyl)iminodiacetic acid, N-(4-butylphenylcarbamoyl-
methyl)
iminodiacetic acid, N-(2,3-dimethylphenylcarbamoylmethyl)iminodiacetic acid, N-
(3-
1 o butylphenyl-carbamoylmethyl)iminodiacetic acid, N-(2-
butylphenylcarbamoylmethyl)
iminodiacetic acid, N-(4-t-butylphenylcarbamoylmethyl)iminodiacetic acid, N-(3-
butoxyphenylcarbamoylmethyl)imino-diacetic acid, N-(2-
hexyloxyphenylcarbamoylmethyl) iminodiacetic acid, N-(4-hexyloxyphenyl-
carbamoylmethyl)iminodiacetic acid; azo substituted iminodiacetic acid,
iminodicarboxymethyl-2-naphthyl ketone, phthalein complexone, N-(5-pregnene-3-
~-0l-2-
oylcarbamoylmethyl)imino-diacetic acid, 3a: '7a 12a: trihydroxy-24-norchol:
amyl-23-
iminodiacetic acid, N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl)
iminodiacetic
acid, benzimidazole-methyliminodiacetic acid, N-(3-cyano-4,5-dimethyl-2-
pyrrylcarbamoylmethyl)iminodiacetic acid, other derivatives of N-(3-cyano-4-
methyl-2-
2 o pyrryl carbamoylmethyl)imninodiacetic acid, ethylenediamine-N,N-bis(-2-
hydroxy-5-
bromophenyl) acetate, N'-acyl and N'-sulfonylethylenediamine-N,N-diacetic
acid; N'-
substituted derivatives of ethylenediamine-N,N-diacetic acid (EDDA) such as N'-
acetyl
EDDA, N'-benzoyl EDDA, N'-(p-toluenesulfonyl) EDDA, N'-(p-t-butylbenzoyl)
EDDA,
N'-(benzenesulfonyl) EDDA, N'-(p-chlorobenzenesulfonyl) EDDA, N'-(p-
ethylbenzenesulfonyl) EDDA, N'-(p-n-propylbenzenesulfonyl) EDDA, N'-
(naphthalene-2-
sulfonyl) EDDA, N'-(2,5-dimethylbenzenesulfonyl) EDDA; N-(2-
acetylnaphthyl)iminodiacetic acid; N-(2-naphthylmethyl)-iminodiacetic acid;
hepatobiliary
dyes such as rose bengal, Congo red, bromosulphthalein, bromophenol blue,
phenolphthalein, toluidine blue, indocyanine green; hepatobiliary contrast
agents such as
3 o iodipamide, ioglycamic acid; bile salts such as bilirubin,
cholyglycyliodohistamine,
thyroxineglucuronide; hepatobiliary thiol complexes such as penicillamine, ~i-
mercaptoisobutyric acid, dihydroehioctic acid, 6-mercaptopurine, kethoxal-
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bis(thiosemicarbazone); hepatobiliary amine complexes such as 1-
hydrazinophthalazine-
(hydralazine)sulfonyl urea; hepatobiliary amino acid Schiff Base complexes
such as
pyridoxylidene glutamate, pyridoxylidene isoleucine, pyridoxylidene
phenylalanine,
pyridoxylidene tryptophan, pyridoxylidene 5-methyl tryptophan; additional
pyridoxylidene
aminates; 3-hydroxy-4-formyl-pyridene glutamic acid; and miscellaneous
hepatobiliary
complexes such as tetracycline, 7-carboxy-~i-hydroxyquinoline,
phenolphthalexon, eosin,
and verograffin.
The use of a primary polymeric matrix in conjunction with a secondary
polylysine
poly(glutamiclaspartic acid) copolymer provides a polymeric construct which
anchors not
l o only biogenic amines, but also hepatobiliary target molecules. This
construct manifests a
complex synergy of interaction that maintains the structural integrity of the
entire targeted
polymeric carrier system. Several polymers have been employed to illustrate
the concept
and benefits of the dual polymer construct and to delineate the specialized
interactions that
occur when different primary polymeric matrices are mixed with secondary
copolymers.
One embodiment of a polymeric construct of this invention comprises a
polylysine
primary polymeric matrix and a secondary anchoring copolymer of polylysine-
poly(glutamic-aspartic acids). Because a single s-amino functional group
accompanies
every amino acid residue of polylysine, amino groups occur far more frequently
and are
chemically more reactive than the amino functionalities generally found in
naturally
2 0 occurring proteins. Therefore, chemical derivatization or subsequent
chemical interaction
may be manipulated with a high degree of precision. The rich cluster of amino
groups of
polylysine creates a favorable positively charged ionic environment that
facilitates
interaction between positively charged amino groups on the primary polymer
matrix and
the negatively charged carboxyl group functionalities on the polyglutamic-
aspartic acid
2 5 portion of the secondary polymeric construct under physiological
conditions. Thus the
likelihood of developing significant polymer-to-polymer binding between the
polylysine
and the secondary copolymeric construct is markedly increased. The ionic
interaction
between the negatively charged y- and ~i-carboxyl groups of glutamic and
aspartic acids,
respectively, with the positively charged amine groups of biogenic amines
enhances
3 0 retention of the active agent. Likewise, ionic bonding between the
carboxyl groups of
the hepatobiliary targeting molecule and the amino group of polylysine
enhances retention
of the targeting molecule on the surface of the polymeric carrier.
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Another embodiment of the invention uses PLGA as the primary polymeric matrix.
By varying the molar ratio of glycolide-to-lactide the hydrophilicity of the
copolymeric
construct may be regulated. The primary polymeric matrix thus provides
numerous
carbonyl functional groups that can participate in hydrogen bond formation.
The
polylysine-poly(glutamic/aspartic) acid secondary copolymeric anchor provides
multiple
binding sites for all constituents of the carrier. Furthermore, ionic
interaction Between the
positively charged nitrogen atoms of the biogenic amines and the ~i-carboxyl
groups of
aspartic acid and the y-carboxyl groups of glutamic acid enhances stability.
A third embodiment of a polymeric construct of this invention employs chitosan
1 o glutamate as the primary polymeric matrix. Chitosan glutamate is a
deacylated derivative
of chitin and may be prepared by mixing chitosan and giutamic acid together. A
key
distinction between chitosan glutamate and native chitosan is that chitosan
glutamate is
water soluble, whereas native chitosan is generally only soluble in dilute
organic acids.
Chitosan glutamate is a polymeric amino sugar with a free amino functional
group on
carbon #2 of every glucosamine residue which is glycosidically linked ~i-(1-->
4) in linear
sequence to every other glucosamine residue in the polymer. Chitosan glutamate
has
strong hydrogen bonding functional groups such as hydroxyl and amino groups.
The
molecule manifests an array of positive charges at physiological pH and below,
a high
molecular weight, good polymer chain flexibility and surface energy properties
which
2 o favor molecular interaction with other kinds of polymers, including the
secondary
copolymer of polylysine-poly(glutamic/aspartic acid). This construct has a
polysaccharide
primary polymer backbone.
The three aforementioned polymer constructs are representative members of
distinct polymer classes and illustrate the diversity of polymer carriers.
Other
2 5 representative first and primary polymers include, but are not limited to,
polycarbophil, a
high molecular weight copolymer of acrylic acid cross-linked with divinyl
glycol,
albumin, polycarbonates, polyamines and polyhydroxybutyric acid. Nonionic
polymers
include polysaccharides such as scleroglucans, homopolysaccharides having
glucose
subunits, e.g. dextran, starch and hydroxyethyl starch. Also, polyoxyethylene
polymers
3 o and copolymers may be used as the primary polymers in this invention.
Anionic polymers
suitable as primary polymers include some polymeric sugars and are represented
by pectin
and xanthan gum.
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The polymeric constructs of this invention are useful for administering an
active
agent to a host. Accordingly, the constructs of this invention are useful as
pharmaceutical
compositions in combination with pharmaceutically acceptable carriers.
Administration of
the constructs described herein can be via any of the accepted modes of
administration for
5 the biologically active substances drat are desired to be administered.
These methods
include oral, topical, parenteral, ocular, transdermal, nasal and other
systemic or aerosol
forms.
Depending on the intended mode of administration, the compositions used may be
in the form of solid, semi-solid or liquid dosage forms, such, as for example,
tablets,
1 o suppositories, pills, capsules, powders, liquids, suspensions, or the
like, preferably in unit
dosage forms suitable for single administration of precise dosages. The
pharmaceutical
compositions will include the polymeric construct as described and a
pharmaceutically
acceptable excipient, and, optionally, may include other active agents,
carriers, adjuvants,
etc.
Topical formulations composed of the polymeric constructs hereof, penetration
enhancers, and other ingredients may be applied in various ways. The solution
can be
applied dropwise, from a suitable delivery device, to the appropriate area of
skin or
mucous membranes and rubbed in by hand or simply allowed to air dry. A
suitable
gelling agent can be added to the solution and the preparation can be applied
to the
2 o appropriate area and rubbed in. Alternatively, the solution formulation
can be placed into
a spray device and be delivered as a spray. This type of drug delivery device
is
particularly well suited for application to large areas of skin, to highly
sensitive skin, or to
the nasal or oral cavities.
Parenteral administration is generally characterized by injection, either
subcutaneously, intramuscularly or intravenously. Injectables can be prepared
in
conventional forms, either as liquid solutions or suspensions, solid forms
suitable for
solution or suspension in liquid prior to injection, or as emulsions. Suitable
excipients
are, for example, water, saline, dextrose, glycerol, ethanol or the. like. In
addition, if
desired, the pharmaceutical compositions to be administered may also contain
minor
3 0 amounts of non-toxic auxiliary substances such as wetting or emulsifying
agents, pH
buffering agents and the like, such as for example, sodium acetate, sorbitan
monolaurate,
triethanolaine oleate, etc.
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The amount of active compound administered will of course, be dependent on the
subject being treated, the type and severity of the affliction, the manner of
administration-
and the judgment of the prescribing physician. In addition, if the dosage form
is intended
for sustained release, the total dose will be integrated over the total time
period of the
sustained release device in order to compute the appropriate dose required.
Although
effective dosage ranges for specific biologically active substances of
interest are dependent
upon a variety of factors, and are generally known to one of ordinary skill in
the art, some
dosage guidelines can be generally defined. For most forms of administration,
the
polymeric component will be suspended in an aqueous solution and generally not
exceed
30% (w/v) of the total formulation. The drug component of the formulation will
most
likely be less than 20 % (w/v) of the formulation and generally greater than
0.01 % (w/v)
In general, topical formulations are prepared in gels, creams or solutions
having an
active ingredient in the range of from 0.001 % to 10% (w/v), preferably 0.01
to 5%, and
most preferably about 1 % to about 5 % . (Of course, these ranges are subject
to variation
depending upon the potency of the biogenic amine, and could in appropriate
circumstances
fall within a range as broad as from 0.001 % to 20 % . ) In all of these
exemplary
formulations, as will other topical formulations, the total dose given will
depend upon the
size of the affected area of the skin and the number of doses per day.
For oral administration, a pharmaceutically acceptable, non-toxic composition
is
2 o formed by the incorporation of any of the normally employed excipients,
such as, for
example, manrlitol, lactose, starch, magnesium stearate, sodium saccharine,
talcum,
cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium
carbonate, and
the like. Such compositions include solutions, suspensions, tablets,
dispersible tablets,
pills, capsules, powders, sustained release formulations and the like.
2 5 Preferably the compositions will take the form of a pill or tablet. Thus
the
composition will contain along with the active ingredient: a diluent such as
lactose,
sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium
stearate or the
like; and a binder such as starch, gum acacia, gelatin, polyvinylpyrolidine,
cellulose and
derivatives thereof, and the like.
3 o Liquid pharmaceutically administrable compositions can, for example, be
prepared
by dispersing or suspending the polymeric construct as described above and
optional
pharmaceutical adjuvants in a carrier, such as, for example, water, saline,
aqueous
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dextrose, glycerol, glycols, ethanol, and the like, to thereby form a
suspension. If
desired, the pharmaceutical composition to be administered may also contain
minor
amounts of nontoxic auxiliary substances such as wetting agents, emulsifying
agents, or
solubilizing agents, pH buffering agents and the like, for example, acetate,
sodium citrate,
cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium
acetate,
triethanolamine oleate, etc. Actual methods of preparing such dosage forms are
known,
or will be apparent, to those skilled in this art; for example, see Remington:
The Science
and Practice of Pharmacy, 19th ed., 1995 (Mack Publishing Co., Easton, PA).
The
composition or formulation to be administered will, in any event, contain a
quantity of the
1 o active biogenic amine in an amount sufficient to effectively treat the
disorder or disease of
the subject being treated.
Dosage forms or compositions containing active ingredient in the range of
0.005
to 95 % with the balance made up from non-toxic carrier may be prepared. The
exact
composition of these formulations may vary widely depending on the particular
properties
of the biogenic amine in question. However, they will generally comprise from
0.01 % to
95 % , and preferably from 0.05 % to 10 % active ingredient for highly potent
agents, and
from 40-85 % for moderately active agents.
For a solid dosage form, the suspension, in for example propylene carbonate,
vegetable oils or triglycerides, is preferably encapsulated in a gelatin
capsule. Such
2 o suspensions and the preparation and encapsulation thereof, can be prepared
by methods
that are disclosed in U.S. Patents Nos. 4,328,245; 4,409,239; and 4,410,545.
For a
liquid dosage form, the suspension may be diluted with a sufficient quantity
of a
pharmaceutically acceptable liquid carrier, e.g. water, to be easily measured
for
administration.
2 5 Alternatively, liquid or semi-solid oral formulations may be prepared by
dissolving
or dispersing the polymeric construct in vegetable oils, glycols,
triglycerides, propylene
glycol esters (e.g. propylene carbonate) and the like, and encapsulating these
solutions or
suspensions in hard or soft gelatin capsule shells.
Other useful formulations include those set forth in U.S. Patents Nos. Re.
28,819
3 o and 4,358,603.
Parenteral administration is generally characterized by injection, either
subcutaneously, intramuscularly or intravenously. Injectables can be prepared
in
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conventional forms, either as liquid solutions or suspensions, solid forms
suitable for
solution or suspension in liquid prior to injection, or as emulsions. Suitable
excipients
are, for example, water, saline, dextrose, glycerol, ethanol or the like. In
addition, if
desired, the pharmaceutical compositions to be administered may also contain
minor
amounts of non-toxic auxiliary substances such as wetting or emulsifying
agents, pH
buffering agents, solubility enhancers, and the like, such as for example,
sodium acetate,
sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.
A more recently devised approach for parenteral administration employs the
implantation of a slow-release or sustained-release system, such that a
constant level of
to dosage is maintained. See, e.g., U.S. Patent No. 3,710,795.
The percentage of active agent contained in parenteral compositions is highly
dependent on the specific nature thereof, as well as the activity of the
compound and the
needs of the subject. However, percentages of active ingredient of 0.01 ~ to
10 l in
solution are employable, and will be higher if the composition is a solid
which will be
subsequently diluted to the above percentages. Preferably the composition will
comprise
0.2 - 2 % of the active agent in solution.
Nasal suspensions of the polymeric construct alone or in combination with
pharmaceutically acceptable excipients can also be administered.
Formulations of the polymeric construct may also be administered to the
2 o respiratory tract as an aerosol for a nebulizer. In such a case, the
particles of the
formulation have diameters of less than 50 microns, preferably less than 10
microns.
EXAMPLES
The following specific Examples are intended to illustrate the invention and
should
2 5 not be construed as limiting the scope of the claims.
All of the following binding studies were performed in solutions buffered with
HEPES at pH 7Ø
Example 1. Association of serotonin HCl with bovine serum albumin.
3 o Experiment A. A bovine serum albumin solution was prepared by dissolving
40 mg
of albumin in 10 ml of 10 mM HEPES buffer, pH 7Ø A serotonin solution was
prepared
by adding 10 mg of serotonin HCl (5-hydroxytryptamine HCl) to 10 ml of 10 mM
HEPES
*rB
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buffer, pH 7.0, and then adding a minute quantity (50 izl) of radiolabeled 5-
hydroxytryptamine creatinine sulfate to the solution. 1.0 ml of the albumin
solution was
mixed with 1.0 ml of the serotonin solution and vortexed for 20 seconds. A 25
p.l aliquot
and a 50 ~,1 aliquot were taken of this mixture for use as standards. The
final
concentration of albumin in the mixed solution was 2 mg/ml (0.03 mM). The
final
concentration of serotonin in the mixed solution was 0.5 mg/ml (2.4 mM).
The remaining 1.925 ml solution was placed in a clear Centricon tube having a
filter with a molecular weight cut-off of 30,000. The tube was spun for 30
minutes at
6500 rpm. Three 192.5 ~l aliquots of the filtrate were counted on a
scintillation counter.
1 o The scintillation counter results indicated that 924 wg serotonin HCl was
in the
filtrate (95.9% of 963 p.g/1.925 ml). Therefore, 4.1 % of the serotonin
appeared to have
been retained on the filter, presumably due to binding with the albumin.
Experiment B. One ml of the albumin solution (4 mg/ml) from Experiment A was
mixed with 20 ~1 of the serotonin solution (1 mg/ml) from Experiment A and
vortexed for
20 seconds. A 20 pl aliquot was taken of this mixture for use as standards.
The final
concentration of albumin in the mixed solution was 4 mg/ml (0.06 mM). The
final
concentration of serotonin HCl in the mixed solution was 0.020 mg/ml (0.094
mM).
The 1.0 mi of the solution was placed in a green Centricon tube having a
filter
with a molecular weight cut-off of 10,000. The sample was then spun for 30
minutes at
2 0 6500 rpm. Two 100 p,l aliquots of the filtrate were counted on the
scintillation counter.
The results indicated that 16.3 p.g of the serotonin HCl remained in the
filtrate
(81.5 % of 20 p.g/1 ml) while 18.5 % of the serotonin was retained on the
Centricon
filter.
Experiment C. In this experiment, an albumin solution (40 mg/ml) was prepared
2 5 by dissolving 120 mg of albumin in 3.0 ml of 10 mM HEPES buffer, pH 7Ø
The
serotonin solution (1 mg/ml) from Experiment A was used. 1.0 ml of this
albumin
solution was mixed with 20 ~1 of the serotonin solution and vortexed for 20
seconds. A
~,1 aliquot was taken of this mixture for use as a standard. This mixture was
allowed to
stand for 20 minutes. The final concentrations of albumin and serotonin HCl in
the
3 o mixture were 40 mg/ml (0.61 mM) and 0.02 mg/ml (0.094 mM), respectively.
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1.0 ml was placed in a clear Centricon tube having a filter with a molecular
weight
cut-off of 30,000. The sample was then spun for 30 minutes at 6500 rpm. Two
100 pl
aliquots of the filtrate were counted on the scintillation counter.
The results indicated that 9.3 p,g of serotonin HCl was in the filtrate (47.5
% of 20
5 ~,g/m 1), whereas 52.5 % of the serotonin was retained by the Centricon
filter.
The results of Experiments A, B, and C are summarized in the following table:
Molar Ratios Serotonin Retained
Sero nin Albumin
10 Experiment A: 80 moles 1 mole 4.1
Experiment B: 3 moles 1 mole 18.5 %
Experiment C: 1 mole 6.5 moles52.5
Example 2. Association of serotonin with phytic acid, poly-lysine, poly-lysine-
succinyl,
15 and N-2,6-(diisopropylphenylcarbamoylmethyl)iminodiacetic acid (DIDA).
A solution of phytic acid was prepared by mixing 5.6 mg with 10 ml of 10 mM
HEPES buffer, pH 7Ø The poly-lysine solution contained 5.6 mg in 10 ml of
HEPES
buffer, pH 7Ø A poly-lysine-succinyl solution also was prepared with 5.6 mg
in 10 ml
of HEPES buffer, pH 7Ø The DIDA solution contained 8.4 mg in 10 mI HEPES
buffer,
2 o pH 7Ø 20 pl of the serotonin solution from Example 1, Experiment A, (1
mg/ml) was
added to 1.0 ml of the phytic acid solution (0.56 mg/ml) and vortexed. 1.0 ml
of the
poly-lysine solution (0.56 mg/ml} was then added and vortexed. 1.0 ml of the
poly-
lysine-succinyl solution (0.56 mg/ml) was added and the mixture again
vortexed. Finally,
wl of the DIDA solution (0.84 mg/ml) was added to the mixture.
2 5 Final concentrations of the components
of the mixture were as follows:
serotonin HCI: 0.007 mglml (0.033
mM)
phytic acid: 0.18 mg/ml (0.20
mM)
poly-lysine: 0.18 mg/ml (1.4 mM)
poly-lysine-succinyl: 0.18 mg/ml (0.80
mM)
3 o DIDA: 0.006 mg/ml (0.02
mM)
1.0 ml of this mixture was placed in a yellow Centricon tube having a filter
with a
molecular weight cut-off of 3,000. The sample was then centrifuged for two
hours at
*rB
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16
6500 rpm. A 100 pl aliquot of the original mixture and two 100 p.l aliquots of
the filtrate-
were counted on the scintillation counter.
The amount of serotonin HCl in the filtrate was 5.0 ~g (75.8 % of 20 wg/3.040
ml). This indicates that 24.2 % of the serotonin was retained by the Centricon
filter in
this experiment, presumably due to ionic and/or hydrogen bonding interactions
between
the serotonin and the polymeric components of the solution mixture.
Example 3. Association of serotonin .with chitosan (Mr=70kD), poly-Glu-Lys,
and N-
(2,6-diisopropylphenylcarbamoylmetbyl) iminodiacetic acid (DIDA).
10 mg of the chitosan polymer was dispersed in 10 ml of distilled water and
titrated with 0.6 ml of glacial acetic acid to facilitate dissolution. A poly-
GIu-Lys solution
was prepared by dissolving 10 mg into 10 ml of 10 mM HEPES buffer, pH 7.0 and
sonicating for 30 seconds. 20 p.l of the serotonin HCl solution from Example
1,
Experiment 3, (1 mg/ml) was added to 1.0 ml of the poly-Glu-Lys solution (1.0
mg/ml)
and vortexed. Next, 10 p.l of the DIDA solution (0.84 mg/ml) was added and
vortexed.
500 ~1 of the chitosan solution (0.94 mg/ml) was also added to the mixture,
followed by
vortexmg.
The final concentrations in the mixed solution were as follows:
chitosan: 0. 31 mg/ml ( 1. 9 mM)
2 0 poly-Glu-Lys: 0.65 mg/ml (2.7 mM)
DIDA: 0.006 mg/ml (0.002 mM)
serotonin HCI: 0.013 mg/ml (0.061 mM)
A 1.0 m1 aliquot was placed in a Centricon tube having a filter with a
molecular
weight cut-off of 30,000. The sample was then centrifuged for 30 minutes. 100
~1
2 5 aliquots were taken from the original sample and from the filtrate to be
counted on the
scintillation counter.
11.4 pg of the serotonin HCl was found in the filtrate (85.1 % of 20 p.g/1.53
ml).
Therefore, 14.9 % of the serotonin HCl was retained on the filter in this
experiment,
presumably due to association with the polymers present in the mixture.
Example 4. Association of serotonin with poly(L-Iactide acid-co-glycolide);
poly-Glu-
Lys, and N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetic acid (DIDA).
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The poly(L-lactide acid-co-glycolide) polymer (PLGA) does not dissolve with
acetic acid, ethanol or NaOH, but does dissolve in dimethyl sulfoxide (DMSO).
10 mg of
PLGA -was dissolved in 0.5 ml of DMSO at 60°C for 30 seconds. Then 9.5
ml of 10 mM
HEPES, pH 7.0 was added to the polymer/DMSO and the mixture vortexed. In a
clean
test tube, one ml of the Poly-Glu-Lys solution (1 mg/ml) from Example 3 was
mixed with
20 pl of the serotonin HCl solution (1 mg/ml) from Example 1, Experiment A,
and
warmed to 60°C and vortexed. A 10 wl aliquot a DIDA solution (0.84
mg/ml) was added
and the mixture again vortexed. Finally, 0.5 ml of a poly(L-lactide acid-co-
glycolide)
solution (1 mg/ml) was added. The entire mixture was warmed and vortexed.
1 o The final concentrations of the mixture were as follows:
poly (L-lactide acid-co-glycolide): 0.33 mg/ml (1.0 mM)
poly-Glu-Lys: 0.65 mg/ml (2.7 mM)
serotonin HCI: 0.013 mg/ml (0.061 mM)
DIDA: 0.005 mg/ml (0.0014 mM)
1.0 ml of the solution mixture was placed in a green Centricon tube having a
filter
with a molecular weight cut-off of 10,000. The sample was then centrifuged at
6500 rpm
for 1 hour. 100 ~,1 aliquots were taken of the original sample and of the
filtrate and
counted on the scintillation counter.
11.5 lzg of serotonin HCl was detected in the filtrate (87.8 % of 20 pg/1.530
ml),
2 o indicating that 12.2 % of the serotonin HCl in the mixed solution was
retained on the
filter.
Example 5. Association of serotonin with polyoxypropylene-polyoxyethylene
(Pluronic~ F-127), poly-Glu-Lys, poly(Tyr-Glu)Ala-Lys, and N-(2,6-
2 5 diisopropylphenylcarbamoylmethyl) iminodiacetic acid (DIDA) .
1.0 ml of a poly-Glu-Lys solution (1 mg/ml) was heated to 100°C for two
minutes,
and then 20 pl of the 1 mg/ml serotonin HCl solution from Example 1,
Experiment A,
was added. In a separate test tube, 1.0 ml of a poly(Tyr-Glu)Ala-Lys solution
(1 mglml)
was heated to 100°C and then added to the above-mentioned solution.
Next, 10 ~1 of a
3 o DIDA solution (0.84 mg/ml) was mixed into the solution mixture. The entire
mixture
was vortexed and allowed to cool to room temperature and then chilled to
4°C. 0.5 ml of
a cold lmg/ml solution of polyoxypropylene-polyoxyethylene (Pluronic~ F-27)
was then
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18
added to the solution mixture. The solution mixture was vortexed and stored at
4°C for
64 hours.
The final concentrations of the components of the solution mixture were as
follows:
poly-Glu-Lys: 0.40 mg/ml (1.7 mM)
poly(Tyr-Glu)Ala-Lys: 0.40 mg/ml (0.94 ~nM)
serotonin HCI: 0.008 mg/ml (0.04 mM)
DIDA: 0.003 mg/ml (0.001 mM)
Pluronic~ F-127: 0.20 mg/ml (1.7 mM)
1.0 ml of the solution mixture was placed in a green Centricon tube having a
filter
with a molecular weight cut-off of 30,000. The sample was centrifuged for 1
hour. 100
wl aliquots were taken from the filtrate for counting in the scintillation
counter.
The concentration of serotonin HCl in the filtrate was found to be 7.4 wg/ml
(92.5
% of 20 pg/2.530 ml). This result indicates that approximately 7.5 % of the
serotonin
HCI in the original solution mixture was retained on the filter, presumably as
part of a
complex with the polymers of the solution.
Example 6. Association of serotonin with ATP, poly-Glu-Lys, poly-lysine, and N-
(2,6-
diisopropylphenylcarbamoylmethyl)iminodiacetic acid (DIDA).
2 o The ATP solution was prepared by weighing 10 mg in 10 ml of 10 mM HEPES
buffer, pH 7Ø In a separate test tube, 20 ~1 of the 1 mg/ml serotonin HCl
solution from
Example 1, Experiment A, was added to 1.13 ml of a poly-Glu-Lys solution (1
mg/ml)
and vortexed. Then, 26 pl of a solution of ATP (1 mg/ml) was added and
vortexed. 1.07
ml of a poly-Lys solution (0.56 mg/ml) was added next. 10 pl of a DIDA
solution (0.84
2 5 mg/ml) was then added to the mixture and vortexed.
The final concentrations of the components in the solution mixture were as
follows:
serotonin HCI: 0.009 mg/ml (0.04
mM)
poly-Glu-Lys: 0.50 mg/ml (2.0
mM)
3 o poly-Lys: 0.27 mg/ml (2.0
mM)
ATP-Na2 : 0.012 mg/ml (0.022
mM)
DIDA: 0.0037 mg/ml (0.01
mM)
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1.0 ml of the mixed solution was placed into a green Centricon tube having a
filter
with a molecular weight cut-off of 30,000. The sample was centrifuged for 1
hour. 100
pl aliquots of the filtrate were taken to be counted on the scintillation
counter.
The results indicated that serotonin HCl was present in the filtrate at a
concentration of 7.4 p.g/ml (82 % of 0. 02 mg/2.256 ml) . Therefore,
approximately 18
of the serotonin had been retained at the filter, presumably as part of an
ionic and/or
hydrogen bonding complex with the polymers of the solution mixture.
Example 7. Association of serotonin with poly(acrylic acid), poly-Glu-Lys, and
N-(2,6-
to diisopropylphenyicarbamoylmethyl)iminodiacetic acid (DIDA).
1.3 ml of poly(acrylic acid) solution (25 % aqueous) was added to 20 pl of the
1.0
mg/ml solution of serotonin HCl from Example 1, Experiment A, and vortexed for
20
seconds. In a separate vial, 1.13 ml of a 1.0 mg/ml solution of poly-Glu-Lys
was added
to 10 p,l of an 0.84 mg/ml solution of DIDA and vortexed for 20 seconds. The
two vials
were vortexed together and two 200 ~1 aliquots were taken for use as standards
in the
scintillation counter.
The final concentrations of the reaction mixture components were as follows:
poly(acrylic acid): 0.13 mg/ml (1.9 mM)
poly-Glu-Lys: 0.46 mg/ml (1.9 mM)
2 0 serotonin -HCI: 0.01 mg/ml (0.04 mM)
DIDA: < 0.01 mg/ml ( < 0.01 mM)
0.5 ml of this solution mixture was placed in a Centricon tube having a filter
with
a molecular weight cut-off of 10,000. The tube was centrifuged for 30 minutes.
One 200
p,l aliquot was taken from the filtrate and counted on the scintillation
counter.
The results indicated that 74% of the serotonin was present in the filtrate.
Therefore, approximately 26 % of the serotonin appeared to have been retained
by the
Centricon filter, presumably due to ionic association and/or hydrogen bonding
with the
polymeric components of the solution mixture.
3 o Example 8. Association of serotonin with polyvinyl sulfonic acid), poly-
Glu-Lys, and
N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid (DIDA).
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WO 99104824 PCT/US98/15457
3.3 ml of a polyvinyl sulfonic acid) solution (15% aqueous) was added to 20 ~1
of
the 1.0 mglml serotonin HCl solution from Example 1, Experiment A, and
vortexed for
20 seconds. In a separate vial, 1:13 ml of a 1.0 mg/ml solution of poly-Glu-
Lys was
added to 10 ~.1 of 0.84 mg/ml solution of DIDA and vortexed for 20 seconds.
The two
5 vials were then vortexed together and three 200 pl aliquots were taken for
use as
standards in the scintillation counter.
The final concentrations of the components of the solution mixture were as
follows:
polyvinyl sulfonic acid): 0.10 mg/ml (1.0 mM)
to poly-Glu-Lys: 0.25 mg/ml (1.0 mM)
serotonin HCI; 0.0004 mg/ml (0.02 mM)
DIDA: < 0.01 mg/ml ( < 0.01 mM)
0.5 ml of this mixture was placed in a Centricon tube having a filter with a
molecular weight cut-off of 10,000. The sample was centrifuged for 30 minutes.
One
15 200 ~,1 aliquot was taken from the filtrate and counted on the
scintillation counter.
The results from the scintillation counter indicated that approximately 88 %
of the
original serotonin was present in the filtrate. It is assumed that 12% of the
serotonin was
retained on the filter of the Centricon tube due to association of the
serotonin with a
polymeric complex in the solution mixture.
Example 9. Association of serotonin with poly(aspartic acid), poly-Glu-Lys,
and N-
(2,6-diisopropylphenylcarbamoylmethyl}iminodiacetic acid (DIDA).
0.62 ml of a poly(aspartic acid) solution (1.0 mg/ml} was added to 20 p,l of
1.0
mg/ml serotonin HCI from Example 1, Experiment A, and vortexed for 20 seconds.
In a
2 5 separate vial, 1.13 ml of a 1.0 mg/ml solution of poly-Glu-Lys was added
to 10 p,l of a
0.84 mg/ml solution of DIDA and vortexed for 20 seconds. The two vials were
vortexed
together and two 200 ~,1 aliquots were taken for use as standards.
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The final concentrations of the individual components of the vortexed solution
mixture were as follows:
poly(aspartic acid) 0.35 mg/ml (2.6 mM)
poly-Glu-Lys 0.63 mg/ml (2.6 mM)
serotonin HCl 0.01 mg/ml (0.05 mM)
DIDA < 0.01 mg/ml ( < 0.01 mM)
0.5 ml of this mixture was placed in a Centricon tube having a filter with a
molecular weight cut-off of 10,000. The sample was centrifuged for 30 minutes.
Two
200 ~1 aliquots were taken from the filtrate and counted on the scintillation
counter.
1 o Approximately 92 % of the original serotonin was found in the filtrate.
Approximately 8 % was assumed to have been retained on the filter of the
Centricon tube
due to ionic interactions and/or hydrogen bonding with the polymers in the
solution
mixture.
15 Example 10. Association of serotonin with poly(lysine), phytic acid, poly-
Glu-Lys, and
N-(2,6-diisopropylphenylcarbamoylmethy)iminodiacetic acid (DIDA).
In this experiment, 1.05 ml of poly(lysine) solution (0.56 mg/ml) was added to
10
p,l of a 0.84 mg/inl solution of DIDA and vortexed for 20 seconds. In a
separate vial,
1.28 ml of a 0.56 mg/ml solution of phytic acid added to 20 ~1 of the 1.0
mg/ml serotonin
2 o HCl solution from Example 1, Experiment A. 1.13 ml of a 1.0 mg/ml solution
of poly-
Glu-Lys was also added to the second vial. The contents of the second vial
were vortexed
for 20 seconds. The two vials were then vortexed together and two 200 ~1
aliquots were
taken for use as standards.
The final concentrations of the various components of the mixed solution were
as
2 5 follows:
poly(lysine): 0.17 mg/ml (1.3 mM)
phytic Acid: 0.21 mglml (0.2 mM)
poly-Glu-Lys: 0.32 mglml (1.3 mM)
serotonin HCI: < 0.01 mglml ( < 0.01
mM)
3 o DIDA: < 0.01 mg/ml ( < 0.01
mM)
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0.5 ml of this mixture was placed in a Centricon tube having a filter with a
molecular weight cut-off of 10,000. The tube was centrifuged for 30 minutes.
Two 200
p,l aliquots were then taken from the filtrate and counted on the
scintillation counter.
The data from the scintillation counter indicated that 86 % of the original
serotonin
was in the filtrate. This result suggests that approximately 14 % of the
original serotonin
was bound to the polymers of the solution mixture and was therefore retained
on the filter.
Example 11. Association of serotonin with poly(glutamic acid), chitosan, and N-
(2,6-
diisopropylphenylcarbamoylmethy)iminodiacetic acid (DIDA).
1 o In this experiment, 1.06 ml of 0.5 mg/ml poly(glutamic acid) was added to
20 ~.1
of the 1.0 mg/ml solution of serotonin HCI from Example l, Experiment A, and
vortexed
for 20 seconds. Then 0.76 of a 1.0 mg/ml solution of low molecular weight (MW)
chitosan was added to 10 ~l of a 0.84 mg/ml solution of DIDA and vortexed for
20
seconds. The two vials were vortexed together, and two 200 pl aliquots were
taken for
use as standards in the scintillation counter.
Final concentrations of the individual components in the solution mixture were
as
follows:
poly(glutamic acid): 0.29 mg/ml (2.5 mM)
chitosan (low MW): 0.41 mg/ml {2.5 mM)
2 o serotonin -HCI: 0.01 mg/ml (0.05 mM)
DIDA: < 0.01 mglml ( < 0.01 mM)
0.5 ml of this mixture was placed in a Centricon tube having a filter with a
molecular weight cut-off of 10,000, and the tube was then centrifuged for 30
minutes.
Two 200 ~1 aliquots were taken from the filtrate and counted on the
scintillation counter.
2 5 The results indicated that the filtrate contained 95 % of the original
serotonin. 5 %
of the original serotonin was retained by the filter.
Example 12. Association of serotonin with poly(galacturonic acid), poly-Glu-
Lys, and
N-(2,6-diisopropylphenylcarbamoylmethy)iminodiacetic acid ~(DIDA).
30 20 pl of the serotonin HCl solution from Example 1, Experiment A, (lmglml)
was
added to 0.370 ml of poly{galacturonic acid) solution (2mg/ml) and heated for
a few
seconds at 60°C and vortexed. Then 1.0 ml of the poly-Glu-Lys solution
(1 mg/ml) was
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23
added to the above mixture and vortexed. 10 ~1 of DIDA (0.84 mg/ml) was then
added.
The solutions were mixed by again vortexing.
Concentrations in the final solution mixture were as follows:
poly(galacturonic acid): 0.53 mg/ml (3 mM)
poly-Glu-Lys: 0.71 mg/ml (3 mM)
serotonin HCI: 0.014 mg/ml (0.07 mM)
DIDA: 0.0006 mg/ml (0.02 mM)
1 ml of this mixture was placed into a green Centricon tube having a filter
with a
molecular weight cut-off of 10,000. The sample was centrifuged for 1 hour at
6,500 rpm
on a Sorvall refrigerated centrifuge at 10°C. 100 ~l aliquots of the
sample, before and
after filtration, were counted on the scintillation counter.
The data from the scintillation counter indicated that the filtrate contained
90 % of
the serotonin that was in the mixed solution. Approximately 10 % of the
original
serotonin appeared to have been retained on the Centricon filter, presumably
due to
binding of serotonin to the polymeric components of the solution mixture.
Example 13. Failure of serotonin to bind with poly (L-lactide acid-co-
glycolide),
chitosan (Mr=70kD), and N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic
acid
(DIDA) in the absence of an anchoring secondary polymer.
In a first vial, 1.49 ml of the poly (L-lactide acid-co-glycolide) solution
(1.0
mg/ml) from Example 4 was added to 20 ~1 of the 1.0 mg/ml serotonin HCl
solution from
Example l, Experiment A, and vortexed for 20 seconds. A solution of DIDA was
prepared by weighing out 8.4 mg of DIDA and dissolving it in 10 ml of 10 mM
HEPES
buffer, pH 7Ø In a second vial, 0.85 ml of the low molecular weight chitosan
solution
2 5 (1.0 mg/ml) from Example 3 was added to 10 ~1 of the DIDA solution, and
the mixture
was vortexed for 20 seconds. The two vials were mixed together and two 100 ~1
aliquots
were taken for use as standards
The final concentrations of the various components of the solution mixture
were as
follows:
3 o Poly (L-lactide acid-co-glycolide): 0.68 mg/ml {2.1 mM)
chitosan (Mr=70kD): 0.39 mg/ml (2.4 mM)
serotonin HCI: 0.01 mg/ml (0.04 mM)
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24
DIDA: < 0.01 mg/ml ( < 0.01 mM)
1.0 ml of this mixture was placed in a yellow Centricon tube having a filter
with a
molecular weight cut-off of 3,000. The tube and contents were centrifuged for
one hour.
Two 100 pl aliquots were taken from the filtrate and counted on the
scintillation counter.
The data from the scintillation counter indicated that the filtrate contained
100 % of
the serotonin originally in the solution mixture. None of the serotonin
appeared to have
been retained on the Centricon filter. This result suggests that without the
anchoring
presence of a suitable secondary polymer such as poly-Glu-Lys, serotonin may
be unable
to form a stable association with primary polymeric matrices such as poly(L-
lactide acid-
1 o co-glycolide) and chitosan.
While the invention has been described with respect to the specific
embodiments
described herein, it is understood that modifications thereto and equivalents
and variations
thereof will be apparent to one skilled in the art and are intended to be and
are included
within the scope of the claims appended hereto.
