U.S. patent application number 14/617844 was filed with the patent office on 2015-05-28 for controlled-released peptide formulations.
The applicant listed for this patent is Corden Pharma, Ltd., SurModics Pharmaceuticals, Inc.. Invention is credited to Mimoun Ayoub, Kevin Burton, Thomas R. Tice, Torsten Woehr.
Application Number | 20150148295 14/617844 |
Document ID | / |
Family ID | 41266659 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150148295 |
Kind Code |
A1 |
Burton; Kevin ; et
al. |
May 28, 2015 |
Controlled-Released Peptide Formulations
Abstract
Described herein are methods and compositions for modulating the
release and/or drug loading characteristics of encapsulated
bioactive agents in polymer-based delivery systems via direct
modification of the isoelectric point and/or net charge of the
bioactive agent.
Inventors: |
Burton; Kevin; (Hoover,
AL) ; Woehr; Torsten; (Marblehead, MA) ; Tice;
Thomas R.; (Indian Springs, AL) ; Ayoub; Mimoun;
(Otelfingen, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SurModics Pharmaceuticals, Inc.
Corden Pharma, Ltd. |
Birmingham
Cork |
AL |
US
IE |
|
|
Family ID: |
41266659 |
Appl. No.: |
14/617844 |
Filed: |
February 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13060676 |
May 16, 2011 |
8951973 |
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PCT/US2009/055461 |
Aug 28, 2009 |
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14617844 |
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61093258 |
Aug 29, 2008 |
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Current U.S.
Class: |
514/11.9 ;
514/21.7 |
Current CPC
Class: |
A61P 19/10 20180101;
C07K 7/06 20130101; A61K 9/1647 20130101; C07K 14/585 20130101;
A61K 38/08 20130101; A61K 38/00 20130101; A61K 9/146 20130101; A61K
38/23 20130101 |
Class at
Publication: |
514/11.9 ;
514/21.7 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 38/08 20060101 A61K038/08; A61K 38/23 20060101
A61K038/23; C07K 14/585 20060101 C07K014/585; C07K 7/06 20060101
C07K007/06 |
Claims
1. A controlled-release pharmaceutical formulation comprising a
modified bioactive agent encapsulated by a polymer, wherein the
isoelectric point of said modified bioactive agent has been
increased relative to a parent molecule to increase drug loading
efficiency.
2. The controlled-release pharmaceutical formulation according to
claim 1, wherein the modified bioactive agent comprises additional
positive charge relative to a parent molecule.
3. The controlled-release pharmaceutical formulation according to
claim 2, wherein said modified bioactive agent is conjugated to a
positively-charged accessory molecule.
4. The controlled-release pharmaceutical formulation according to
claim 2, wherein said modified bioactive agent comprises
positively-charged amino acid substitutions.
5. The controlled-release pharmaceutical formulation according to
claim 3, wherein said modified bioactive agent is a peptide having
the amino acid sequence set forth as SEQ ID NO:2.
6. The controlled-release pharmaceutical formulation according to
claim 4, wherein said modified bioactive agent is a peptide having
the amino acid sequence set forth as SEQ ID NO:9.
7. A controlled-release pharmaceutical formulation comprising a
modified bioactive agent encapsulated by a biodegradable polymer,
wherein the isoelectric point of said modified bioactive agent has
been increased or decreased relative to a parent molecule to
increase the erosional release rate.
8. The controlled-release pharmaceutical formulation according to
claim 7, wherein the modified bioactive agent comprises additional
positive charge relative to a parent molecule.
9. The controlled-release pharmaceutical formulation according to
claim 8 wherein said modified bioactive agent is conjugated to a
positively-charged accessory molecule.
10. The controlled-release pharmaceutical formulation according to
claim 8, wherein said modified bioactive agent comprises
positively-charged amino acid substitutions.
11. The controlled-release pharmaceutical formulation according to
any one of claims 7 to 10, wherein said polymer is an
acid-terminated polymer.
12. A controlled-release pharmaceutical formulation comprising a
modified bioactive agent encapsulated by a polymer, wherein the
isoelectric point of said modified bioactive agent has been
decreased relative to a parent molecule to reduce the initial
diffusion rate of the agent from the polymer.
13. The controlled-release pharmaceutical formulation according to
claim 12, wherein the modified bioactive agent comprises additional
negative charge relative to a parent molecule.
14. The controlled-release pharmaceutical formulation according to
claim 13, wherein said modified bioactive agent is conjugated to a
negatively-charged accessory molecule.
15. The controlled-release pharmaceutical formulation according to
claim 13, wherein said modified bioactive agent comprises
negatively-charged amino acid substitutions.
16. The controlled-release pharmaceutical formulation according to
any one of claims 12 to 15, wherein said polymer is an
ester-terminated polymer.
17. A controlled-release pharmaceutical formulation comprising a
modified bioactive agent encapsulated by a polymer, wherein the
isoelectric point of said modified bioactive agent has been
increased relative to a parent molecule to increase the initial
diffusion rate of the agent from the polymer.
18. The controlled-release pharmaceutical formulation according to
claim 17, wherein the modified bioactive agent comprises additional
positive charge relative to a parent molecule.
19. The controlled-release pharmaceutical formulation according to
claim 18, wherein said modified bioactive agent comprises a
positively-charged accessory molecule.
20. The controlled-release pharmaceutical formulation according to
claim 18, wherein said modified bioactive agent comprises
positively-charged amino acid substitutions.
21. The controlled-release pharmaceutical formulation according to
claim 18, wherein said modified bioactive agent further comprises
an acetate counter ion.
22. The controlled-release pharmaceutical formulation according to
any one of claims 17 to 21, wherein said polymer is an
ester-terminated polymer.
23. The controlled-release pharmaceutical formulation according to
any one of claims 1 to 6, and 12 to 22, wherein said polymer is a
non-biodegradable polymer.
24. The controlled-release pharmaceutical formulation according to
any one of claims 1 to 6, and 12 to 22, wherein said polymer is a
biodegradable polymer.
25. The controlled-release pharmaceutical formulation according to
any one of claims 2 to 4, 8 to 11, and 18 to 22, wherein said
additional positive charge is distributed uniformly across the
bioactive agent.
26. The controlled-release pharmaceutical formulation according to
any one of claims 2 to 4, 8 to 11, and 18 to 22, wherein said
additional positive charge is distributed non-uniformly across the
bioactive agent.
27. The controlled-release pharmaceutical formulation according to
any one of claims 13-16, wherein said additional negative charge is
distributed uniformly across the bioactive agent.
28. The controlled-release pharmaceutical formulation according to
any one of claims 13-16, wherein said additional negative charge is
distributed non-uniformly across the bioactive agent.
29. The controlled-release pharmaceutical formulation according to
any one of claims 1 to 28, wherein said bioactive agent is a
peptide molecule.
30. The controlled-release pharmaceutical formulation according to
any one of claims 1 to 29, wherein said controlled-release
pharmaceutical formulation is a microparticle.
31. A method for increasing bioactive agent loading efficiency in a
polymer-based delivery system, comprising modifying the isoelectric
point of the bioactive agent prior to encapsulation in the
polymer-based delivery system.
32. The method according to claim 31, wherein the isoeletric point
of the bioactive agent is increased such that it carries a more
positive charge in the environment of the polymer-based delivery
system.
33. A method for modulating the erosional release rate of a
bioactive agent from a polymer-based delivery system, comprising
modifying the isoelectric point of the agent prior to encapsulation
in the polymer-based delivery system.
34. The method according to claim 33, wherein the erosional release
rate is increased by quantitatively increasing or decreasing the
isoelectric point of the bioactive agent such that it carries a
greater net positive or negative charge, respectively, compared to
the parent molecule in the environment of the polymer-based
delivery system.
35. The method according to claim 34, wherein the isoelectric point
of the bioactive agent is increased or decreased by adding
additional positive or negative charge to a parent molecule to
produce a stoichiometric increase or decrease in net charge
relative to the parent molecule.
36. The method according to claim 35, wherein additional positive
charge is added to a neutral or cationic parent molecule to
increase the erosional release rate.
37. The method according to claim 35, wherein additional negative
charge is added to a neutral or anionic parent molecule to increase
the erosional release rate.
38. A method for modulating the initial diffusional release of a
bioactive agent from a polymer-based delivery system, comprising
modifying the isoelectric point of the agent prior to encapsulation
in the polymer-based delivery system.
39. The method according to claim 38, wherein the isoelectric point
of the bioactive agent is increased or decreased such that it
carries a greater net positive or negative charge, respectively,
relative to the parent molecule in the environment of the desired
polymer-based delivery system.
40. The method according to claim 39, wherein the initial
diffusional release rate is increased by adding additional positive
charge to the parent molecule to produce a stoichiometric increase
in net charge relative to the parent molecule.
Description
FIELD
[0001] The invention relates to peptide formulations, and more
specifically to methods and compositions for modifying the release
and/or drug-loading characteristics of such formulations.
BACKGROUND OF THE INVENTION
[0002] The importance of biocompatible and/or biodegradable
polymers as carriers for active therapeutic agents is well
established. Biocompatible, biodegradable, and relatively inert
polymers such as poly(lactide) (PL) or poly(lactide-co-glycolide)
(PLGA) containing a bioactive agent are commonly utilized in
controlled-release delivery systems (for review, see M. Chasin,
Biodegradable polymers for controlled drug delivery. In: J. O.
Hollinger Editor, Biomedical Applications of Synthetic
Biodegradable Polymers CRC, Boca Raton, Fla. (1995), pp. 1-15; T.
Hayashi, Biodegradable polymers for biomedical uses. Prog. Polym.
Sci. 19 4 (1994), pp. 663-700; and Harjit Tamber, Pal Johansen,
Hans P. Merkle and Bruno Gander, Formulation aspects of
biodegradable polymeric microspheres for antigen delivery, Advanced
Drug Delivery Reviews, Volume 57, Issue 3, 10 Jan. 2005, Pages
357-376).
[0003] With respect to the delivery of therapeutic peptides in
particular, however, there still exist many challenges to the
design of effective controlled-release, polymer-based delivery
systems. A basic requirement for such delivery systems is
appropriate control over the release of the encapsulated active
agent, an objective which is complicated by variations in the
release kinetics of polymer systems. Generally, an initial
diffusional or burst release phase from the intact polymer system
is followed by a slower lag phase leading to an erosional release
phase as the polymer system begins to degrade. It is important to
maintain the concentration of the peptide molecule within a
therapeutically effective window throughout both of the principal
peptide release phases and to avoid excessive concentrations, and
particularly an initial burst during the diffusional release phase,
which may lead to adverse side effects or untoward results. In this
respect, however, wide variation in the size, charge and
conformation of different peptide molecules has thus far prevented
a more uniform approach to their effective encapsulation.
[0004] The prior art describes various strategies for improving
controlled-release delivery from polymer-based delivery systems
including the use of new polymeric materials and polymer blends,
and/or the incorporation of additives in such systems to help
facilitate drug release. U.S. Pat. No. 7,326,425, for example,
describes a blended polymer-based delivery system having a first
polymer capable of forming hydrogen bonds with a desired bioactive
agent to prevent bursts, and a second polymer the degradation
products of which trigger the release of the active agent from the
first polymer. Alternatively, U.S. Patent Publication No.
2007/0092574 describes the addition of certain organic ions to
polymer-based delivery systems encapsulating water-soluble
bioactive agents to reduce the burst release and degradation of the
bioactive agent, wherein the organic ion is selected to neutralize
the overall charge of a particular bioactive agent.
[0005] In each of these examples, however, and in the prior art in
general, the primary focus of such strategies is on manipulation of
the polymer-based delivery system to suit the requirements of a
particular bioactive agent, as opposed to manipulation or
adaptation of the bioactive agent itself.
SUMMARY OF THE INVENTION
[0006] In contravention of the conventional formulation methodology
of manipulating the polymer-based delivery system to suit the
encapsulated agent, the present invention provides methods and
compositions for modulating the release and/or drug-loading
characteristics of an encapsulated bioactive agent through direct
modification of the bioactive agents themselves. As demonstrated
herein, modification of the isoelectric point of a bioactive agent
such as a peptide molecule, e.g., alteration of the overall charge
of the peptide, can predictably modify the release and/or loading
characteristics of polymer-based delivery systems in clinically
meaningful ways including, e.g., reducing or enhancing the initial
diffusional release of the peptide from the polymer-based delivery
system, modulating the erosional release rate from biodegradable
systems, and/or increasing the encapsulation efficiency of such
peptides.
[0007] In one aspect, methods for increasing bioactive agent
loading efficiency in polymer-based delivery systems are provided,
comprising modifying the isoelectric point of the agent prior to
encapsulation in a polymer-based delivery system. In one
embodiment, the isoelectric point of the agent is modified such
that it carries a more positive charge compared to the parent
molecule in the environment of the desired polymer-based delivery
system.
[0008] In one embodiment, methods for increasing bioactive agent
loading efficiency in polymer-based delivery systems are provided,
comprising adding additional positive charge to a parent
molecule.
[0009] In one aspect, methods for modulating the erosional release
rate of a bioactive agent from a biodegradable polymer-based
delivery system are provided, comprising changing the isoelectric
point of the agent prior to encapsulation in the polymer-based
delivery system. In one embodiment, the isoelectric point of an
agent is quantitatively increased or decreased such that it carries
a greater net positive or negative charge, respectively, compared
to the parent molecule in the environment of the desired
polymer-based delivery system.
[0010] In one embodiment, methods for increasing the erosional
release rate of a bioactive agent from a biodegradable
polymer-based delivery system are provided, comprising adding
additional positive or negative charge to a parent molecule to
produce a stoichiometric increase or decrease, respectively, in net
charge relative to the parent molecule. In one embodiment,
additional positive charge is added to a neutral or cationic parent
molecule to increase the erosional release rate. In another
embodiment, additional negative charge is added to a neutral or
anionic parent molecule to increase the erosional release rate. In
a preferred embodiment, acid-terminated polymer-based delivery
systems are employed for enhanced effect.
[0011] Surprisingly, the present inventors have determined that an
increase in the net positive charge of a bioactive agent relative
to a cationic parent molecule can work as well as, and in some
cases even better than, a reduction in or neutralization of the net
charge to increase the erosional release rate of such an agent from
a biodegradable polymer-based delivery system. Significantly, as
also demonstrated for the first time herein, creating a greater
charge density in a charged bioactive agent relative to a parent
molecule provides for greater effect.
[0012] In one aspect, methods for modulating the initial
diffusional release of a bioactive agent from a polymer-based
delivery system are also provided, comprising changing the
isoelectric point of the agent prior to encapsulation in the
polymer-based delivery system. In one embodiment, the isoelectric
point of the agent is increased or decreased such that it carries a
greater net positive or negative charge, respectively, relative to
the parent molecule in the environment of the desired polymer-based
delivery system.
[0013] In one embodiment, methods for increasing the initial
diffusional release of a bioactive agent from a polymer-based
delivery system are provided, comprising adding additional positive
charge to the parent molecule to produce a stoichiometric increase
in net charge relative to the parent molecule. In a preferred
embodiment, ester-terminated polymer-based delivery systems are
employed for enhanced effect.
[0014] In one embodiment, methods for decreasing the initial
diffusional release of a bioactive agent from a polymer-based
delivery system are provided, comprising adding additional negative
charge to the parent molecule to produce a stoichiometric decrease
in net charge relative to the parent molecule. In a preferred
embodiment, ester-terminated polymer-based delivery systems are
employed for enhanced effect.
[0015] Any suitable means for modifying the isoelectric point of a
bioactive agent can be employed in conjunction with the subject
methods including, e.g., chemical modification, amino acid
substitution, conjugation of positively or negatively-charged
accessory molecules, and more preferably cleavable accessory
molecules, and the like. The additional positive or negative charge
may be distributed uniformly or non-uniformly across the bioactive
agent, and is preferably implemented at a location distal to the
known site(s) of action of the parent molecule, e.g. binding site,
etc. In one embodiment, the additional charge distribution is
clustered at the amino or carboxy terminus. In another embodiment,
the additional charge is conjugated to an amino acid side
chain.
[0016] Modification of the isoelectric point as disclosed herein
may also be employed in conjunction with more conventional
techniques such as conversion to water insoluble addition salts
(e.g., U.S. Pat. No. 5,776,886), pegylation (U.S. Pat. No.
6,706,289), and the like, to further modulate release kinetics
and/or loading efficiency. In a further aspect, improved
controlled-release pharmaceutical compositions are provided
comprising bioactive agents modified according to the above methods
and encapsulated in polymer-based delivery systems.
[0017] In one embodiment, the controlled-release pharmaceutical
composition comprises a modified bioactive agent encapsulated by a
polymer, wherein the isoelectric point of the modified bioactive
agent has been increased relative to the parent molecule to
increase drug loading efficiency, and/or to increase the erosional
release rate of the modified bioactive agent, preferably from an
acid-terminated polymer system, and/or to increase the diffusional
release of the modified bioactive agent, preferably from an
ester-terminated polymer system. In one such embodiment, the parent
molecule is neutral or cationic.
[0018] In another embodiment, the controlled-release pharmaceutical
composition comprises a modified bioactive agent encapsulated by a
biodegradable polymer, wherein the isoelectric point of the
modified bioactive agent has been decreased relative to the parent
molecule to increase the erosional release rate of the modified
bioactive agent, preferably from an acid-terminated polymer system,
and/or to decrease the diffusional release rate of the modified
bioactive agent, preferably from an ester-terminated polymer
system, relative to the parent molecule. In one such embodiment,
the parent molecule is neutral or anionic.
[0019] Unless otherwise specified, the compositions described
herein may comprise a non-biodegradable polymer-based delivery
system, e.g., a polymer system comprising a non-biodegradable
polymer. In one aspect, the non-biodegradable polymer is selected
from the group consisting of polyacrylates, polymers of
ethylene-vinyl acetates and other acyl substituted cellulose
acetates, non-degradable polyurethanes, polystyrenes, polyvinyl
chloride, polyvinyl fluoride, poly(vinyl imidazole),
chlorosulphonate polyolefins, polyethylene oxide, blends and
copolymers thereof.
[0020] In another aspect, the compositions described herein may
comprise a biodegradable polymer-based delivery system, e.g., a
polymer system comprising a biodegradable polymer. In another
aspect, the biodegradable polymer is selected from the group
consisting of homopolymers of poly(lactic acid) (PLA), polylactide
(PL) or poly(glycolic acid) (PGA), polyglycolide (PG), poly(lactic
acid)-co-(glycolic acid) (PLGA), poly(actide-co-glycolide (PLG),
poly(ortho esters), and polyanhydrides. Due to the biocompatibility
and the long history of their clinical applications, PLGA and PLA
are most generally used. Other biodegradable polymers that may be
used include polycaprolactone, polycarbonates, polyesteramides,
poly(amino acids), poly(dioxanones), poly(alkylene alkylate)s,
polyacetals, polycyanoacrylates, biodegradable polyurethanes,
blends and copolymers thereof.
[0021] The subject compositions and methods find advantageous use
with a variety of bioactive agents, including therapeutic proteins,
nucleic acids, peptides, polypeptides, oligonucleotides, and the
like.
[0022] In another aspect, the invention provides methods of
treating a patient in need of treatment, comprising administering a
therapeutically effective amount of a pharmaceutical composition of
the invention to the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is an exemplary schematic of a hypothesized triphasic
drug release from a degradable matrix.
[0024] FIG. 2 shows the actual loading (y-axis) of five peptides
(DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) or
negative (-) charge overall (x-axis). Unless otherwise noted, each
peptide molecule was loaded into the polymer as an acetate
salt.
[0025] FIG. 3 shows the percent theoretical loading efficiency
(y-axis) of five peptides (DP1, DP2, DP3, DP4, and DP5) with a
neutral, positive (+) or negative (-) charge overall (x-axis).
Unless otherwise noted, each peptide molecule was loaded into the
polymer as an acetate salt.
[0026] FIG. 4 shows the mean particle size (.mu.M) of five peptides
(DP1, DP2, DP3, DP4, and DP5) with a neutral, positive (+) or
negative (-) charge overall (x-axis). Unless otherwise noted, each
peptide molecule was loaded into the polymer as an acetate
salt.
[0027] FIG. 5 shows the release rate as percent release (y-axis) of
five peptides (DP1, DP2, DP3, DP4, and DP5) with a neutral,
positive (+) or negative (-) charge overall over 35 days (x-axis)
from a microparticle, polymer-based formulation containing an
acid-terminated, 50:50 poly(lactide-co-glycolide) with an
approximate i.v. of 0.2 dL/g as measured in chloroform at a
concentration of 0.5 g/dL at 30.degree. C. Unless otherwise noted,
each peptide molecule was loaded into the polymer as an acetate
salt.
[0028] FIG. 6 shows the release rate as percent release (y-axis) of
five peptides (DP1, DP2, DP3, DP4, and DP5) with a neutral,
positive (+) or negative (-) charge overall over 14 days (x-axis)
from a microparticle, polymer-based formulation containing an
acid-terminated, 50:50 poly(lactide-co-glycolide) with an
approximate intrinsic viscosity (i.v.) of 0.2 dL/g as measured in
chloroform at a concentration of 0.5 g/dL at 30.degree. C. Unless
otherwise noted, each peptide molecule was loaded into the polymer
as an acetate salt.
[0029] FIG. 7 shows the release rate as percent release (y-axis) of
four peptides (DP1, DP2, DP3, and DP5) with a neutral, positive (+)
or negative (-) charge overall over 17 days (x-axis) from a
microparticle, polymer-based formulation containing an
ester-terminated, 50:50 poly(lactide-co-glycolide) with an
approximate i.v. of 0.2 dL/g as measured in chloroform at a
concentration of 0.5 g/dL at 30.degree. C. Unless otherwise noted,
each peptide molecule was loaded into the polymer as an acetate
salt.
[0030] FIG. 8 shows the release rate as percent release (y-axis) of
five peptides (DP1, DP2, DP3, DP4, and DP5) with a neutral,
positive (+) or negative (-) charge overall over 29 days (x-axis)
from a microparticle, polymer-based formulation containing an acid
terminated 85:15 poly(lactide-co-glycolide) with an approximate
i.v. of 0.25 dL/g as measured in chloroform at a concentration of
0.5 g/dL at 30.degree. C. Unless otherwise noted, each peptide
molecule was loaded into the polymer as an acetate salt.
[0031] FIG. 9 shows the release rate as percent release (y-axis) of
five peptides (DP1, DP2, DP3, DP4, and DP5) with a neutral,
positive (+) or negative (-) charge overall over 65 days (x-axis)
from a microparticle, polymer-based formulation containing an
ester-terminated 85:15 poly(lactide-co-glycolide) with an
approximate i.v. of 0.25 dL/g as measured in chloroform at a
concentration of 0.5 g/dL at 30.degree. C. Unless otherwise noted,
each peptide molecule was loaded into the polymer as an acetate
salt.
[0032] FIG. 10 shows the release rate as percent release (y-axis)
of five peptides (DP1, DP2, DP3, DP4, and DP5) with a neutral,
positive (+) or negative (-) charge overall over 15 days (x-axis)
from a microparticle, polymer-based formulation containing an
ester-terminated 85:15 poly(lactide-co-glycolide) with an
approximate i.v. of 0.25 dL/g as measured in chloroform at a
concentration of 0.5 g/dL at 30.degree. C. Unless otherwise noted,
each peptide molecule was loaded into the polymer as an acetate
salt.
DETAILED DESCRIPTION
[0033] Methods and formulations are provided for the
controlled-release of bioactive agents from polymer-based delivery
systems through direct modification of the bioactive agent. As
described herein, the isoelectric point of the modified bioactive
agent may be changed relative to a parent molecule, and/or the net
charge of the modified bioactive agent may be changed relative to a
parent molecule, etc. Encapsulated formulations comprising such
modified bioactive agents have enhanced controlled-release
properties, e.g., a lower initial diffusional or burst release, an
increased erosional release rate, increased encapsulation
efficiency, etc., compared to formulations comprising similarly
encapsulated formulations of the parent molecule.
[0034] Release of a bioactive agent from a polymer, e.g., a
biodegradable polymer such as a PLG microparticle, generally
follows a triphasic release profile as exemplified in FIG. 1. Phase
1 may generally be characterized as a diffusional release or
"burst" effect, during which the initial release rate of the
modified peptide molecule may be rapid, and may be dependent on
hydration of the polymer (occurring within minutes), swelling of
the matrix (hours), dissolution of the modified peptide molecule
(minutes) and diffusion from the matrix (hours).
[0035] The second phase of release (Phase 2, FIG. 1) may be
referred to as the induction or lag phase, and may be characterized
by a period of slower or no release. Phase 2 may be associated with
the time required for pores or channels to form or time for water
to fill such pores or channels in the polymer matrix thereby
allowing access to the modified peptide molecule entrapped within
the polymer matrix.
[0036] When a biodegradable polymer-based delivery system is used,
and as biodegradable polymer degradation continues, diffusional
paths may be formed through the eroding matrix, which may allow the
modified peptide molecule to travel down a concentration gradient
and escape the matrix. This erosional release corresponds to the
third phase of release as demonstrated in FIG. 1.
[0037] Release of a bioactive agent from a non-biodegradable
polymer generally follows a biphasic release profile, in which
phase 1 corresponds to a diffusional release of the bioactive
peptide and phase 2 corresponds to a lag phase. Accordingly,
skilled artisans are generally familiar with typical release rates
of bioactive agents from such polymer-based delivery systems.
[0038] In one embodiment, improved controlled-release compositions
and methods are provided wherein the isoelectric point of a parent
molecule is increased to produce a modified bioactive agent having
a more positive net charge and/or charge density, which as
demonstrated herein can increase drug-loading efficiency, increase
the erosional release rate of the modified bioactive agent, and
particularly from acid-terminated polymer-based systems, and/or
increase the diffusional release of the modified bioactive agent,
and particularly from ester-terminated polymer-based systems,
relative to the parent molecule. In one such embodiment, the parent
molecule is neutral or cationic.
[0039] In another embodiment, improved controlled-release
compositions and methods are provided wherein the isoelectric point
of a parent molecule is decreased to produce a modified bioactive
agent having a more negative net charge and/or charge density,
which as demonstrated herein can increase the erosional release
rate of the modified bioactive agent, and particularly from
acid-terminated polymer-based systems, and/or decrease the
diffusional release rate of the modified bioactive agent, and
particularly from ester-terminated polymer-based systems, relative
to the parent molecule. In one such embodiment, the parent molecule
is neutral or anionic.
[0040] "Bioactive agent" as used herein refers to any therapeutic
protein, therapeutic antibody, nucleic acid, peptide, polypeptide,
oligonucleotide, aptamer or other biologically active compound for
administration to a subject.
[0041] By "peptide molecule" as used herein is meant a polymeric
molecule comprising at least two amino acids covalently linked by a
peptide bond, and includes a protein, a polypeptide, an
oligopeptide and a peptide. A peptide molecule may be made up of
naturally occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures, i.e., "analogs", such as peptoids (see
Simon et al., 1992, Proc Natl Acad Sci USA 89(20):9367,
incorporated by reference).
[0042] By "amino acid" and "amino acid identity" as used herein is
meant one of the twenty naturally occurring amino acids or any
non-natural analogues that may be present at a specific, defined
position. Thus "amino acid", or "peptide residue", as used herein
means both naturally occurring and synthetic amino acids (including
analogues of naturally occurring amino acids). For example,
homophenylalanine, citrulline, 2-amino-3-guanidinoproprionic acid,
2-amino-3-ureidoproprionic acid, Lys(Me), Lys(Me).sub.2,
Lys(Me).sub.3, Ornitine, Omega-nitro-arginine, Arg(Me)2,
.alpha.-methyl Arg, .alpha.-methyl Lys, homolysine, homoarginine,
noreleucine, and the like are considered amino acids for the
purposes of the invention. "Amino acid" also includes imino acid
residues such as proline and hydroxyproline. The side chain may be
in either the (R) or the (S) configuration, and may be either D- or
L-isomers. In the preferred embodiment, the amino acids are in the
(S) or L-configuration, although D-isomers may be used to improve
serum stability. If non-naturally occurring side chains are used,
non-amino acid substituents may be used, for example to prevent or
retard in vivo degradation.
[0043] "Parent molecule" as used herein refers to a bioactive agent
that is subsequently modified in accordance with the invention
teachings to generate a "modified bioactive agent." In some
embodiments, a parent molecule may be any bioactive agent molecule
requiring a controlled-release, polymer-based formulation for
therapeutic use. As described herein, encapsulation and release
from polymers can be manipulated by modification of the parent
molecule, e.g., modification of the isoelectric point, net charge,
solubility etc. of the parent molecule.
[0044] Similarly, by "parent peptide molecule," "parent
polypeptide," "parent protein," and the like as used herein is
meant a polypeptide, protein and the like that is subsequently
modified to generate a "modified peptide molecule." For example, a
parent peptide molecule, a parent polypeptide, a parent protein or
the like may serve as a template and/or basis for at least one
modification described herein, e.g., modification of the
isoelectric point, modification of the net charge, modification of
the solubility, etc. Said parent peptide molecule may be a
naturally occurring polypeptide, or a variant or engineered version
of a naturally occurring polypeptide. Parent polypeptide may refer
to the polypeptide itself, compositions that comprise the parent
polypeptide, or the amino acid sequence that encodes it.
[0045] By "isolectric point", "pI", or the like as used herein is
meant the pH at which a peptide molecule carries no net electrical
charge. The isoelectric point may be determined using well known
methods, e.g., by isoelectric focusing. In case of smaller peptide
molecules the approximate pI may be also calculated. In general,
the pI of a peptide molecule depends on the pKa values of its basic
and acidic groups, e.g., in case of a peptide formed with encoded
amino acids only, the primary amine of the N-terminus or the lysine
side chain, the guanidine group of the arginine side chain, the
imidazole ring system of histidine and the carboxylic acid groups
of the peptide C-terminus, the aspartic acid side chain and
glutamic acid side chain.
[0046] Modification of the pI of a parent peptide molecule may
occur by, e.g., chemical modification. Non-limiting examples of
such modifications include acylation, alkylation or other chemical
modification of the N-terminal amine group; amidation,
esterification or other chemical modification of the C-terminal
carboxylic acid group; substitution of a non-ionizable amino acid
residue for a lysine, histidine, arginine, glutamic acid, aspartic
acid or other non-encoded amino acids with acidic or basic chain
groups; acylation, alkylation or other chemical modification of
side chain groups of lysine, histidine, arginine or basic functions
of other, non-encoded amino acids; amidation, esterification or
other chemical modification of side chain carboxylic acid groups,
conjugation with pI shifting accessory molecules. etc. In case of
ionized peptides the pI of the salt form also depends on the
pK.sub.a of the counter ion.
[0047] As used herein, the "net charge" of a parent peptide
molecule depends on its pI and the pH of the peptide solution. The
net charge of a parent peptide molecule may be modified by any of
the following non-limiting examples: acylation, alkylation or other
chemical modification of the N-terminal amine group; amidation,
esterification or other chemical modification of the C-terminal
carboxylic acid group; substitution of a non-ionizable amino acid
residue for a lysine, histidine, arginine, glutamic acid, aspartic
acid or other non-encoded amino acid with acidic or basic chain
groups; modification of the isoelectric point of the parent
peptide; conjugation with positively or negatively charged
accessory molecules, and the like.
[0048] As disclosed herein, modification of charge distribution
and/or density can also be considered for modulation of polymer
encapsulation and release properties of the parent peptide. Added
charge density may be distributed uniformly or non-uniformly across
the modified peptide molecule. In one embodiment, a non-uniform
distribution of additional negative or positive charge comprises
clustering the additional negative or positive charge,
respectively, at one or more positions within the peptide chain.
The cluster(s) of additional negative or positive charge may be
anywhere along the peptide, e.g., near or at the end of the
peptide, near or at the center of the peptide, etc., but are
preferably positioned distal to the active site of the peptide,
which can be readily determined by reference to the known
characteristics of the parent molecule. Alternatively, the
additional negative or positive charge may be distributed evenly
along the polymer.
[0049] In one embodiment, a modified peptide molecule may comprise
an additional negative or positive charge relative to its parent
peptide molecule, e.g., via substitution of appropriate amino
acids. In one embodiment, the addition of positive charge is
accomplished by substitution of negative and/or non-ionizable amino
acids in the parent peptide molecule with lysine, arginine,
histidine, or analogues thereof. In another embodiment, the
addition of negative charge is accomplished by substitution of
positive and/or non-ionizable amino acids in the parent peptide
molecule with glycine, aspartic acid, glutamic acid, or analogues
thereof (e.g., any alpha or beta amino alkanedioic acid (e.g.,
amino suberic acid).
[0050] In one embodiment, a modified peptide molecule may comprise
an additional negative or positive charge relative to its parent
peptide molecule, e.g., via conjugation with one or more
negatively-charged accessory molecule(s) or positively-charged
accessory molecule(s), respectively. "Conjugation" as used herein
refers to covalent linkage of two molecules as opposed to mere
complexation or other close physical association. Exemplary
negatively-charged accessory molecules include conjugations of in
general any chemical functionality of a peptide such as the
hydroxyl group of tyrosine, threonine and serine side chains, the
thiol group of the cysteine side chain or the N-terminal amino
group or amino groups of the arginine and lysine side chains with
phospholipids (phosphoamid bound), mono-, di-, and/or
tri-phosphates, sulphates, citrates, tartaric acids, polyaspartic,
polyglutamic and diacids (e.g. oxalic acids, malonic acids,
succinic acids, etc.). Exemplary negatively-charged structures also
include, but are not limited to, poly(glutamic acid), anionic
lipids, poly(aspartic acid), and alginates. In some cases the
chemical functionality of the peptide may also have to be induced
or introduced in order to facilitate conjugation (e.g. formation of
reactive thioesters, aldhydes, hydroxylamines, alkylbromides,
maleimides, etc). Exemplary positively-charged accessory molecules
include, polylysine, polyarginine, polyhistidine, poly(allyl
amine), poly(ethyl imine), chitosan or positively charged lipid
structures.
[0051] Accessory molecules may also comprise a "tail" of positive
or negative amino acids, and may be conjugated to the bioactive
agent by a more neutral linker moiety, e.g., polyethylene glycol
(PEG), poly --CH.sub.2--, and the like.
[0052] Modified peptide molecules may further include
pharmaceutically acceptable counterions, representative examples of
which are set forth in Table 1 below.
TABLE-US-00001 TABLE 1 Potential counterions Salt Class Examples
inorganic acids hydrochloride, sulfate, nitrate, phosphate sulfonic
acids tosylate, mesylate, esylate, isethionate carboxylic acids
acetate, proprionate, maleate, benzoate, salicylate, fumarate
hydroxyacids citrate, lactate, succinate, tartrate, glycollate
anionic amino acids glutamate, aspartate fatty acids stearate,
hexanoate, octanoate, decanoate, oleate
[0053] Modification of solubility in water and/or organic solvents
as well as alteration of the hydrophilicity of a parent peptide
molecule may also be employed in conjunction with the subject
methods to further modulate the encapsulation and release
characteristics of a peptide in a polymer-based delivery system.
The solubility and/or hydrophilicity of a peptide therapeutic may
be modified by changing its salt form or by pegylation as described
in, e.g., U.S. Pat. No. 5,776,885 and U.S. Pat. No. 6,706,289, the
disclosures of which are both expressly incorporated by reference
herein.
[0054] As used herein "relative to a parent peptide molecule"
refers to a change (e.g., an increase or decrease) in a
quantifiable characteristic, e.g., isoelectric point, net charge,
etc., by a modified peptide compared to the parent peptide molecule
(e.g., the peptide molecule that was subsequently modified to
generate the modified peptide molecule) when the amounts of
modified peptide molecule and parent peptide molecule are
essentially the same in the same assay.
[0055] Described herein are methods and compositions for modulating
the release and/or drug loading characteristics of encapsulated
peptide molecules in polymer-based delivery systems through direct
modification of the peptide molecules themselves. Polymer-based
delivery systems described herein are generally biocompatible
polymer-based delivery system. The polymer-based delivery systems
described herein may comprise a biodegradable or non-biodegradable
polymer, blends or copolymers thereof. A polymer-based delivery
system, or a polymer, is biocompatible if the polymer, and any
degradation products of the polymer, are non-toxic to the recipient
and also present no significant deleterious or untoward effects on
the recipient's body.
[0056] Biocompatible, non-biodegradable polymers suitable for
encapsulating bioactive agents (e.g., peptide molecules) include,
but are not limited to, non-biodegradable polymers selected from
the group consisting of polyacrylates, polymers of ethylene-vinyl
acetates and other acyl substituted cellulose acetates,
non-degradable polyurethanes, polystyrenes, polyvinyl chloride,
polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonate
polyolefins, polyethylene oxide, blends and copolymers thereof.
[0057] Biodegradable polymers may also be used for encapsulating
bioactive agents (e.g., peptide molecules) e.g., for
controlled-release formulations. In one embodiment, biodegradable
polymers for which the degradation products are known to be
innocuous or biocompatible are used. Accordingly, such
biodegradable polymers need not be surgically removed at the end of
a treatment. Commonly used biodegradable polymers, which have been
investigated for the controlled-release of peptide molecules,
include homopolymers of poly(lactic acid) (PLA), polylactide (PL)
or poly(glycolic acid) (PGA), polyglycolide (PG), poly(lactic
acid)-co-(glycolic acid) (PLGA), poly(actide-co-glycolide (PLG),
poly(ortho esters), and polyanhydrides. Due to the biocompatibility
and the long history of clinical applications, PLG and PL are most
generally used. Other biodegradable polymers that may be used
include polycaprolactone, polycarbonates, polyesteramides,
poly(amino acids), poly(dioxanones), poly(alkylene alkylate)s,
polyacetals, polycyanoacrylates, biodegradable polyurethanes,
blends and copolymers thereof.
[0058] In one aspect, polymeric delivery systems can be
microparticles including, but not limited to microspheres,
microcapsules, nanospheres and nanoparticles comprising
biodegradable polymeric excipients, non-biodegradable polymeric
excipients, or mixtures of polymeric excipients thereof, or the
polymeric delivery systems can be, but not limited to rods or other
various shaped implants, wafers, fibers, films, in situ forming
boluses and the like comprising biodegradable polymeric excipients,
non-biodegradable polymeric excipients, or mixtures thereof. These
systems can be made from a single polymeric excipient or a mixture
or blend of two or more polymeric excipients.
[0059] The term "microparticle" is used herein to include
nanoparticles, microspheres, nanospheres, microcapsules,
nanocapsules, and particles, in general. As such, the term
microparticle refers to particles having a variety of internal
structure and organizations including homogeneous matrices such as
microspheres (and nanospheres) or heterogeneous core-shell matrices
(such as microcapsules (and nanocapsules), porous particles,
multi-layer particles, among others. Microparticles are particles
that have sizes in the range of about 10 nanometers to about 1000
micrometers (microns).
[0060] A variety of techniques known in the art can be used to form
microparticles including e.g., single or double emulsion steps
followed by solvent removal. Solvent removal may be accomplished by
extraction, evaporation or spray drying among other methods.
[0061] In the solvent extraction method, the polymer is dissolved
in an organic solvent that is at least partially soluble in the
extraction solvent such as water. The modified bioactive agent,
either in soluble form or dispersed as fine particles, is then
added to the polymer solution, and the mixture is dispersed into an
aqueous phase that contains a surface-active agent such as
poly(vinyl alcohol). The resulting emulsion is added to a larger
volume of water where the organic solvent is removed from the
polymer/bioactive agent to form hardened microparticles.
[0062] In the solvent-evaporation method, the polymer is dissolved
in a volatile organic solvent. The bioactive agent, either in
soluble form or dispersed as fine particles, is then added to the
polymer solution, and the mixture is suspended in an aqueous phase
that contains a surface-active agent such as poly(vinyl alcohol).
The resulting emulsion is stirred until most of the organic solvent
evaporates, leaving internally, solid microparticles.
[0063] In the spray drying method, the polymer is dissolved in a
suitable solvent, such as methylene chloride (e.g., 0.04 g/mL). A
known amount of the modified bioactive agent is then suspended (if
insoluble) or co-dissolved (if soluble) in the polymer solution.
The solution or the dispersion is then spray dried. Microparticles
ranging in diameter between one and ten microns can be obtained
with a morphology, which depends on the selection of polymer.
[0064] Other known methods, such as phase separation and
coacervation, and variations of the above, are known in the art and
also may be employed in the present invention.
[0065] A suitable polymeric excipient includes, but is not limited
to, a poly(diene) such as poly(butadiene) and the like; a
poly(alkene) such as polyethylene, polypropylene, and the like; a
poly(acrylic) such as poly(acrylic acid) and the like; a
poly(methacrylic) such as poly(methyl methacrylate), a
poly(hydroxyethyl methacrylate), and the like; a poly(vinyl ether);
a poly(vinyl alcohol); a poly(vinyl ketone); a poly(vinyl halide)
such as poly(vinyl chloride) and the like; a poly(vinyl nitrile), a
poly(vinyl ester) such as poly(vinyl acetate) and the like; a
poly(vinyl pyridine) such as poly(2-vinyl pyridine),
poly(5-methyl-2-vinyl pyridine) and the like; a poly(styrene); a
poly(carbonate); a poly(ester); a poly(orthoester) including a
copolymer; a poly(esteramide); a poly(anhydride); a poly(urethane);
a poly(amide); a cellulose ether such as methyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methyl cellulose, and the
like; a cellulose ester such as cellulose acetate, cellulose
acetate phthalate, cellulose acetate butyrate, and the like; a
poly(saccharide), a protein, gelatin, starch, gum, a resin, and the
like. These materials may be used alone, as physical mixtures
(blends), or as co-polymers. Derivatives of any of the polymers
listed above are also contemplated.
[0066] In one aspect, the polymeric excipient of the delivery
system includes a biocompatible, non-biodegradable polymer such as,
for example, a silicone, a polyacrylate; a polymer of
ethylene-vinyl acetate; an acyl substituted cellulose acetate; a
non-degradable polyurethane; a polystyrene; a polyvinyl chloride; a
polyvinyl fluoride; a poly(vinyl imidazole); a chlorosulphonate
polyolefin; a polyethylene oxide; or a blend or copolymer
thereof.
[0067] In another aspect, the polymeric excipient includes a
biocompatible, biodegradable polymer such as, for example, a
poly(lactide); a poly(glycolide); a poly(lactide-co-glycolide); a
poly(lactic acid); a poly(glycolic acid); a poly(lactic
acid-co-glycolic acid); a poly(caprolactone); a poly(orthoester); a
poly(phosphazene); a poly(hydroxybutyrate) or a copolymer
containing a poly(hydroxybutarate); a
poly(lactide-co-caprolactone); a polycarbonate; a polyesteramide; a
polyanhydride; a poly(dioxanone); a poly(alkylene alkylate); a
copolymer of polyethylene glycol and a polyorthoester; a
biodegradable polyurethane; a poly(amino acid); a polyetherester; a
polyacetal; a polycyanoacrylate; a
poly(oxyethylene)/poly(oxypropylene) copolymer, or a blend or
copolymer thereof.
[0068] In one aspect, the polymer-based delivery system comprises a
non-biodegradable polymer. In another aspect, the polymer-based
delivery system comprises a biodegradable polymer, wherein the
peptide is imbedded within the polymer of the delivery system. In
one aspect, the peptide is encapsulated in a delivery system
composed of poly(lactide-co-glycolide), poly(lactide),
poly(glycolide), polycaprolactone, poly(lactide-co-caprolactone),
poly(lactide-co-glycolide-co-caprolactone),
poly(glycolide-co-caprolactone), or a mixture thereof.
Lactide/glycolide polymers for drug-delivery formulations are
typically made by melt polymerization through the ring opening of
lactide and glycolide monomers. Some polymers are available with or
without carboxylic acid end groups. Some polymers are available
with a block of polyethylene glycol (PEG). When the end group of
the poly(lactide-co-glycolide), poly(lactide), or poly(glycolide)
is not a carboxylic acid, for example, an ester, then the resultant
polymer is referred to herein as blocked or capped. The unblocked
polymer, conversely, has a terminal carboxylic group.
[0069] In one aspect, linear lactide/glycolide polymers are used;
however branched polymers can be used as well. In certain aspects,
high-molecular-weight polymers (e.g., i.v. >1 dL/g as measured
in chloroform at a concentration of 0.5 g/dL at 30.degree. C.) can
be used for medical devices, for example, to meet strength
requirements. In other aspects, low-molecular weight polymers
(e.g., i.v. <1 dL/g as measured in chloroform at a concentration
of 0.5 g/dL at 30.degree. C.) can be used for drug-delivery and
vaccine delivery products where resorption time and not material
strength is more important. The lactide portion of the polymer has
an asymmetric carbon. Commercially racemic DL-, L-, and D-polymers
are available. The L-polymers are more crystalline and resorb
slower than DL-polymers. In addition to copolymers comprising
glycolide and DL-lactide or L-lactide, copolymers of L-lactide and
DL-lactide are available. Additionally, homopolymers of lactide or
glycolide are available. Also, the lactide monomer can also contain
alkyl groups.
[0070] In the case when the biodegradable polymer is
poly(lactide-co-glycolide), poly(lactide), or poly(glycolide), the
amount of lactide and glycolide in the polymer can vary. In one
aspect, the biodegradable polymer contains 0 to 100 mole %, 40 to
100 mole %, 50 to 100 mole %, 60 to 100 mole %, 70 to 100 mole %,
or 80 to 100 mole % lactide and from 0 to 100 mole %, 0 to 60 mole
%, 10 to 40 mole %, 20 to 40 mole %, or 30 to 40 mole % glycolide,
wherein the amount of lactide and glycolide is 100 mole %. In one
aspect, the biodegradable polymer can be poly(lactide), 85:15
poly(lactide-co-glycolide), 75:25 poly(lactide-co-glycolide), or
65:35 poly(lactide-co-glycolide) where the ratios are mole
ratios.
[0071] In one aspect, when the biodegradable polymer is
poly(lactide-co-glycolide), poly(lactide), or poly(glycolide), the
polymer has an intrinsic viscosity of from 0.15 to 1.5 dL/g, 0.25
to 1.5 dL/g, 0.25 to 1.0 dL/g, 0.25 to 0.8 dL/g, 0.25 to 0.6 dL/g,
or 0.25 to 0.4 dL/g as measured in chloroform at a concentration of
0.5 g/dL at 30.degree. C.
[0072] Pharmaceutical Compositions
[0073] In a further embodiment of the present invention, the
modified peptides and polymer-based delivery systems according to
the subject invention are admixed with an appropriate
pharmaceutical carrier suitable for administration in primates,
especially humans, to provide pharmaceutical compositions.
[0074] Pharmaceutically acceptable carriers which can be employed
in the present pharmaceutical compositions can be any and all
solvents, dispersion media, isotonic agents and the like. Except
insofar as any conventional media, agent, diluent or carrier is
detrimental to the recipient or to the therapeutic effectiveness of
the polymer-based delivery system or therapeutic peptide or other
bioactive agent encapsulated therein, its use in the pharmaceutical
compositions of the present invention is appropriate.
[0075] The carrier can be liquid, semi-solid, e.g., pastes, or
solid carriers. Examples of carriers include oils, water, saline
solutions, alcohol, sugar, gel, lipids, liposomes, resins, porous
matrices, binders, fillers, coatings, preservatives and the like,
or combinations thereof.
[0076] In a further embodiment of the present invention, the
modified peptides and polymer-based delivery systems according to
the subject invention can be administered as a powder without
carrier.
[0077] Methods of Treatment
[0078] In a further aspect of the present invention, methods are
provided for treating a disease in a vertebrate, preferably a
mammal, preferably a primate, with human subjects being an
especially preferred embodiment, by administering a peptide
formulation of the invention desirable for treating such
disease.
[0079] Experimental
EXAMPLE 1
Formulation of Model Peptides
[0080] Manufacturing Process:
[0081] The peptide-loaded microparticles were prepared using a
standard solvent extraction method. Approximately 200 mg of peptide
was dissolved in 2 grams of DMSO. Separately, 2 g of
poly(lactide-co-glycolide (PLG) was dissolved in 10 g of
dichloromethane. The peptide solution was then added to the polymer
solution while homogenizing at high revolutions per minute (rpm)
using an IKA homogenizer. The peptide/polymer phase was next
dispersed into a continuous phase consisting of 3 g of poly(vinyl
alcohol) (PVA) and 2.7 g of dichloromethane in 150 mL distilled
water by homogenizing at 700 rpm using a Silverson L4RT mixing
assembly. Once the droplets had been sufficiently formed (3
minutes) the emulsion was diluted with 1 L of distilled water and
stirred on a stirplate for 1 hour, allowing the extraction of the
dichloromethane and solidification of the PLG microparticles.
Thereafter, the microparticles were isolated by passing the
suspension through a 125 micron sieve and collecting microparticles
on a 20-micron sieve. The collected microparticles were then
lyophilized to remove residual water. The finished product was a
white to off-white free-flowing powder and was subsequently stored
at -20.degree. C.
[0082] Drug Content:
[0083] The drug content was determined by carefully extracting
peptide from the PLG microparticle formulations. Typically, PLG
formulations are dissolved in an appropriate organic solvent (or
the polymer is hydrolyzed) and the drug is extracted into a
suitable aqueous buffer. Drug in the extract is then quantified by
HPLC. The concentration value is used to determine the amount of
drug contained in the microparticle, which is reported as weight
percent (wt %).
[0084] In Vitro Release:
[0085] In-vitro release analysis consisted of placing samples of
the microparticle formulations into an appropriate receiving fluid
(PBS at pH 7.4) maintained at 37.degree. C. with mild agitation.
The pH of the receiving fluid was checked routinely to assure that
the PBS maintains a pH of 7.4. The receiving fluid was exchanged at
various time points and the amount of peptide released into the
receiving fluid was quantified by HPLC. Control studies were
performed to ensure the stability of the peptide in the receiving
fluid once released.
[0086] Microparticle Size
[0087] The mean size and size distribution of the microparticle
formulations was determined using a Beckman Coulter LS 13 320 Laser
Diffraction Particle Size Analyzer.
[0088] FIGS. 2 and 3 show the effect of charge on drug load and
loading efficiency. Within the variation of the data, the drug load
and loading efficiency were consistent and similar across the
neutral and positively charged peptides. The negatively charged
peptide (DP5) was incorporated (loaded) less efficiently when
compared to the other peptides studied.
[0089] FIG. 4 shows that, with the exception of DP2 in the
microparticle formulation containing an ester-terminated 50:50
poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g as
measured in chloroform at a concentration of 0.5 g/dL at 30.degree.
C., particle size was unaffected by the peptide incorporated.
[0090] FIGS. 5 and 6 demonstrate the effect of peptide charge on
release from a microparticle formulation containing an
acid-terminated 50:50 poly(lactide-co-glycolide) with an
approximate i.v. of 0.2 dL/g as measured in chloroform at a
concentration of 0.5 g/dL at 30.degree. C. The charged peptides
release at a consistently faster rate when compared to the neutral
peptide. It may also be noted that the higher charge-density
peptides (DP3 DP4, DP5 verses DP2) released more rapidly. It is
also of note that no "burst" was observed form this polymer-based
delivery system.
[0091] FIG. 7 shows the release profiles form a similar
microparticle, polymer-based formulation, an ester-terminated 50:50
poly(lactide-co-glycolide) with an approximate i.v. of 0.2 dL/g as
measured in chloroform at a concentration of 0.5 g/dL at 30.degree.
C. While the neutral and negatively charged peptide showed very
little release over the period studied, the positively charged
peptide exhibited a significant burst from the formulation.
Further, the greater the positive charge (higher charge density)
the more significant the effect on the release rate.
[0092] FIG. 8 shows the release of the peptides from a
microparticle formulation containing an acid-terminated 85:15
poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g as
measured in chloroform at a concentration of 0.5 g/dL at 30.degree.
C. Over the period studied, release of peptide was not observed
from any microparticle formulation.
[0093] FIG. 9 shows the release of the peptides from a
microparticle formulation containing an ester-terminated 85:15
poly(lactide-co-glycolide) with an approximate i.v. of 0.25 dL/g as
measured in chloroform at a concentration of 0.5 g/dL at 30.degree.
C. While the neutral and negatively charged peptide showed very
little release over the period studied, the positively charged
peptide exhibited a more significant release from the microparticle
formulation. The greater the positive charge (higher charge
density) the more significant the effect on the release rate.
EXAMPLE 2
Exemplary Modifications of Parent Peptides
[0094] Calcitonin
[0095] Calcitonin is a hormone used in the treatment of
osteoporosis. The amino acid sequence of human Calcitonin is
Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-P-
he-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro (set forth
as SEQ ID NO:1).
[0096] The pI of Calcitonin is modified to enhance its use in a
controlled released formulation by adding a tri-lysine moiety at
the N-terminus to increase its encapsulation efficacy (set forth as
SEQ ID NO:2). Alternatively, to increase its initial burst from an
ester-terminated polyester, the glycine residues are replaced with
lysines (set forth as SEQ ID NO:3). To increase erosional release
from an acid-terminated polyester, the glycine residues are
replaced with aspartic acids (set forth as SEQ ID NO:4).
[0097] Leuprolide
[0098] Leuprolide is a gonadotropin-releasing hormone agonist that
may be used in the treatment of prostate cancer or endometriosis.
The amino acid sequence of Leuprolide is
Glu-His-Trp-Ser-Tyr-DLeu-Leu-Arg-Pro-NHEt (set forth as SEQ ID
NO:5).
[0099] The pI of Leuprolide is modified to enhance its use in a
controlled released formulation by replacing the glutamic acid with
glutamine (set forth as SEQ ID NO:6) to increase its diffusional
release from an ester-terminated polyester and/or replacing the
arginine with aspartic acid (set forth as SEQ ID NO:7) to decrease
diffusional release from an ester-terminated polyester.
[0100] Octreotide
[0101] Octreotide is an octapeptide that may be used as an
inhibitor of growth hormone and/or the treatment of acromegaly. The
amino acid sequence of Octreotide is
DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr (set forth as SEQ ID NO:8).
[0102] The threonine is replaced with lysine (set forth as SEQ ID
NO:9) to modify the pI of Octreotide and to enhance its use in a
controlled-released formulation, e.g., to increase the
encapsulation efficiency and drug load of Octreotide.
[0103] All citations are expressly incorporated herein in their
entirety by reference.
Sequence CWU 1
1
9132PRTHomo sapiens 1Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr
Tyr Thr Gln Asp Phe 1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr
Ala Ile Gly Val Gly Ala Pro 20 25 30 235PRTHomo sapiens 2Lys Lys
Lys Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr 1 5 10 15
Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val 20
25 30 Gly Ala Pro 35 332PRTHomo sapiens 3Cys Lys Asn Leu Ser Thr
Cys Met Leu Lys Thr Tyr Thr Gln Asp Phe 1 5 10 15 Asn Lys Phe His
Thr Phe Pro Gln Thr Ala Ile Lys Val Lys Ala Pro 20 25 30 432PRTHomo
sapiens 4Cys Asp Asn Leu Ser Thr Cys Met Leu Asp Thr Tyr Thr Gln
Asp Phe 1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Asp
Val Asp Ala Pro 20 25 30 59PRTHomo sapiensMISC_FEATURE(6)..(6)D
amino acid 5Glu His Trp Ser Tyr Leu Leu Arg Pro 1 5 69PRTHomo
sapiensMISC_FEATURE(6)..(6)D amino acid 6Gln His Trp Ser Tyr Leu
Leu Arg Pro 1 5 79PRTHomo sapiensMISC_FEATURE(6)..(6)D amino acid
7Glu His Trp Ser Tyr Leu Leu Asp Pro 1 5 88PRTHomo
sapiensMISC_FEATURE(1)..(1)D amino acid 8Phe Cys Phe Trp Lys Thr
Cys Thr 1 5 98PRTHomo sapiensMISC_FEATURE(1)..(1)D amino acid 9Phe
Cys Phe Trp Lys Lys Cys Lys 1 5
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