U.S. patent application number 11/971482 was filed with the patent office on 2009-07-09 for soluble hydrophobic core carrier compositions for delivery of therapeutic agents, methods of making and using the same.
This patent application is currently assigned to PharmaIN Corporation. Invention is credited to Elijah M. Bolotin, Gerardo M. Castillo.
Application Number | 20090176892 11/971482 |
Document ID | / |
Family ID | 40845092 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176892 |
Kind Code |
A1 |
Castillo; Gerardo M. ; et
al. |
July 9, 2009 |
Soluble Hydrophobic Core Carrier Compositions for Delivery of
Therapeutic Agents, Methods of Making and Using the Same
Abstract
The present invention relates to a soluble hydrophobic-core
carrier composition comprising (i) a linear polymeric backbone;
(ii) a plurality of hydrophilic polymeric protective chains
covalently linked and pendant to the polymeric backbone and (iii)
at least one hydrophobic moiety covalently linked and pendant to
the polymeric backbone. In certain embodiments, the weight ratio of
hydrophilic protective chains to hydrophobic moieties in the
carrier is at least 15:1. In other embodiments, at least 90% of the
residues of the polymeric backbone are coupled to a hydrophilic
polymeric protective chain or a hydrophobic moiety. In other
embodiments, the composition further comprises (iv) a hydrophobic
load molecule dissociably linked to the hydrophobic moiety of the
carrier.
Inventors: |
Castillo; Gerardo M.;
(Bothell, WA) ; Bolotin; Elijah M.; (Bothell,
WA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH AND ROSATI / PHARMAIN LTD
650 PAGE MILL ROAD
PALO ALTO
CA
94304
US
|
Assignee: |
PharmaIN Corporation
Seattle
WA
|
Family ID: |
40845092 |
Appl. No.: |
11/971482 |
Filed: |
January 9, 2008 |
Current U.S.
Class: |
514/773 |
Current CPC
Class: |
A61K 47/60 20170801;
A61K 47/42 20130101; A61K 47/645 20170801; A61P 3/04 20180101; A61K
47/542 20170801; A61K 38/00 20130101; C07K 17/02 20130101; A61P
3/10 20180101 |
Class at
Publication: |
514/773 |
International
Class: |
A61K 47/00 20060101
A61K047/00 |
Goverment Interests
GOVERNMENTAL LICENSE RIGHTS
[0001] Work described herein was made in part with government
support under 5 R43 DK069727 awarded by the National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK). The U.S.
Government may have certain rights in subject matter provided
herein.
Claims
1. A soluble hydrophobic-core carrier composition comprising: (i) a
linear polymeric backbone; (ii) a plurality of hydrophilic
polymeric protective chains covalently linked and pendant to the
polymeric backbone, wherein each protective side chain has a
molecular weight between about 400 and about 20,000 Daltons; and
(iii) at least one hydrophobic moiety covalently linked and pendant
to the polymeric backbone; wherein the weight ratio of the
hydrophilic polymeric protective side chains and the hydrophobic
moieties is selected so that the composition is soluble in
water.
2. The composition of claim 1 wherein the weight ratio of the
hydrophilic polymeric protective side chains and the hydrophobic
moieties is at least 15:1, at least 17:1, at least 20:1, at least
50:1 or at least 100:1.
3. The composition of claim 1 wherein at least 90% of the residues
of the polymeric backbone are derivatized with either hydrophilic
protective chains or hydrophobic moieties.
4. The composition of claim 1, wherein the protective side chains
comprise polyethylene glycol, polypropylene glycol, a co-polymer of
polyethylene glycol, a co-polymer of polypropylene glycol,
polysaccharide, or alkoxy derivatives thereof.
5. The composition in claim 4, wherein the alkoxy derivative is
methoxypolyethylene glycol, methoxypolypropylene glycol, a
methoxylated co-polymer polyethylene glycol and
polypropyleneglycol, or ethoxylated polysaccharide.
6. The composition of claim 1, wherein the linear polymeric
backbone is selected from a group consisting of polylysine,
polyaspartic acid, polyglutamic acid, polyserine, polythreonine,
polycysteine, polyglycerol, natural saccharides, aminated
polysaccharides, aminated oligosaccharides, polyamidoamine,
polyacrylic acids, polyalcohols, sulfonated polysaccharides,
sulfonated oligosaccharides, carboxylated polysaccharides,
carboxylated oligosaccharides, aminocarboxylated polysaccharides,
aminocarboxylated oligosaccharides, carboxymethylated
polysaccharides, and carboxymethylated oligosaccharides.
7. The composition of claim 1 further comprising: (iv) a load
molecule dissociably linked to the hydrophobic moiety of the
backbone.
8. The composition of claim 7, wherein the hydrophilic protective
side chains comprise methoxypolyethylene glycol.
9. The composition of claim 8, wherein the polymeric backbone
comprises polylysine.
10. The composition of claim 9, wherein the hydrophobic moieties
comprise a fatty acid.
11. The composition of claim 10, wherein the load molecule is a
therapeutic agent.
12. The composition of claim 11, wherein the therapeutic agent is a
hydrophobic peptide, hydrophobic protein, or a hydrophobic
drug.
13. The composition of claim 11, wherein the therapeutic agent is
GLP-1.
14. The composition of claim 11, wherein the therapeutic agent is
selected from GLP-2, leptin, islet amyloid polypeptide and
vasoactive intestinal peptide.
15. The composition of claim 7, wherein the linear polymeric
backbone is polylysine.
16. The composition of claim 7, wherein the hydrophobic moiety(ies)
comprises a fatty acid selected from the range of 6-carbon fatty
acids to 36-carbon fatty acids.
17. The composition of claim 7, wherein the hydrophobic moiety(ies)
comprise a fatty acid with at least one double bond.
18. The composition of claim 7, wherein the hydrophobic moiety(ies)
comprises a multi-fatty acid-containing moiety.
19. The composition of claim 7, wherein the hydrophobic moiety(ies)
comprises an aromatic ring containing moiety.
20. The composition of claim 7, wherein the therapeutic agent is
hydrophobic peptide, hydrophobic protein, and hydrophobic
drugs.
21. The composition of claim 7, wherein the therapeutic agent is
selected from GLP-1, GLP-2, leptin, islet amyloid polypeptide and
vasoactive intestinal peptide.
22. The composition of claim 7, further comprising a targeting
molecule covalently linked to the protective side chains.
23. The composition of claim 22, wherein the targeting molecule is
selected from a group consisting of an antibody, fragment of an
antibody, chimeric antibody, lectins, receptor ligands, proteins,
enzymes, peptides, saccharides, quasi substrates of enzymes,
cell-surface-binding compounds, and extracellular-matrix-binding
compounds.
24. The composition of claim 7, further comprising a second set of
protective chain covalently linked to the hydrophobic moiety.
25. A pharmaceutical composition comprising a composition selected
from compositions in claims 7-24, wherein the load molecule is a
therapeutic agent.
26. A method of making a composition comprising: (a) dissolving a
polymeric backbone containing residues comprising free amino groups
in an aqueous buffer of pH 7-8 to obtain solution A; (b) activating
a carboxyl group or alkyl carboxyl group of a protective chain by
mixing it with a carbodiimide reagent in acidic buffer between pH
3-7 to obtain solution B; and (c) adding solution B to solution A
resulting in a solution C containing the polymeric backbone with
covalently linked protective chains, wherein the pH of solution C
is 7 or above.
27. A method of making a composition, comprising: (a) dissolving in
non-aqueous solvent with a tertiary amine, a component comprising a
polymeric backbone covalently linked to protective chains, wherein
the polymeric backbone comprises residues comprising free amino
groups, thereby obtaining solution E; (b) dissolving in a
non-aqueous solvent hydrophobic molecules containing free carboxyl
groups and activating the carboxyl groups by adding carbodiimide
reagent to obtain solution F; and (c) adding solution F to solution
E to obtain solution G to form covalent linkage between the
activated carboxyl groups and the free amine groups; wherein
solution E is added to solution G until at least 90% of the
residues are linked to protective chains or hydrophobic groups.
28. A method of making a composition, comprising: (a) dissolving in
partially-aqueous solvent at pH of 7 to 9 a component comprising a
polymeric backbone covalently linked to protective chains, wherein
the polymeric backbone comprises residues comprising free amino
groups, thereby obtaining solution E, (b) dissolving in
partially-aqueous solvent with pH of 3 to 7, hydrophobic molecules
containing free carboxyl groups and activating the carboxyl groups
by adding carbodiimide reagent resulting in solution F, (c) adding
solution F to solution E while maintaining the pH of the mixture
between 7-8 to obtain solution G, to form covalent linkage between
the activated carboxyl groups and the free amine groups; wherein
solution E is added to solution G until at least 90% of the
residues are linked to protective chains or hydrophobic groups.
29. A method of loading a composition, comprising: (a) dissolving
in aqueous or partially-aqueous solvent A soluble hydrophobic-core
carrier composition comprising: (i) a linear polymeric backbone;
(ii) a plurality of hydrophilic polymeric protective chains
covalently linked and pendant to the polymeric backbone, wherein
each protective side chain has a molecular weight between about 400
and about 20,000 Daltons; and (iii) at least one hydrophobic moiety
covalently linked and pendant to the polymeric backbone; wherein
the weight ratio of the hydrophilic polymeric protective side
chains and the hydrophobic moieties is selected so that the
composition is soluble in water, thereby obtaining solution A. (b)
dissolving the load molecule in aqueous or partially-aqueous
solvent to obtain solution B, (c) mixing solution A with solution B
to obtain solution C, incubating solution C for 30 minutes or
longer followed by lyophilization or solvent evaporation to obtain
a loaded carrier ready to be dissolved into appropriate
solvent.
30. A method of administering a therapeutic molecule to a subject
comprising: administering to the subject a composition comprising:
(i) a linear polymeric backbone; (ii) a plurality of hydrophilic
polymeric protective chains covalently linked and pendant to the
polymeric backbone, wherein each protective side chain has a
molecular weight between about 400 and about 20,000 Daltons; (iii)
at least one hydrophobic moiety covalently linked and pendant to
the polymeric backbone; (iv) a therapeutic molecule dissociably
linked to the hydrophobic moiety of the backbone; wherein the
weight ratio of the hydrophilic polymeric protective side chains
and the hydrophobic moieties is selected so that the composition is
soluble in water.
31. The method of claim 30 wherein the composition is administered
subcutaneously or intramuscularly.
32. A pharmaceutical composition comprising: (a) a composition
comprising: (i) a linear polymeric backbone; (ii) a plurality of
hydrophilic polymeric protective chains covalently linked and
pendant to the polymeric backbone, wherein each protective side
chain has a molecular weight between about 400 and about 20,000
Daltons; (iii) at least one hydrophobic moiety covalently linked
and pendant to the polymeric backbone; (iv) a therapeutic molecule
dissociably linked to the hydrophobic moiety of the backbone;
wherein the weight ratio of the hydrophilic polymeric protective
side chains and the hydrophobic moieties is selected so that the
composition is soluble in water; and (b) a pharmaceutically
acceptable carrier; wherein the composition is in unit dose form.
Description
BACKGROUND OF THE INVENTION
[0002] The development of new drugs, formulations and other systems
for administration of physiologically active peptides and proteins,
and other hydrophobic drugs or therapeutics is driven by the need
to achieve the desirable physiological effects. Peptides and
proteins have been observed to be unstable in blood and the
gastro-intestinal tract and therefore may need to be stabilized or
protected prior to delivery and remain protected once in either the
gastrointestinal tract or the circulation. Once the active
pharmaceutical gets into the systemic circulation, those that have
low molecular masses tend to have short biological half-lives due
to their efficient removal from systemic circulation via kidneys.
Furthermore, a fraction of them can also be removed via
reticuloendothelial uptake due to recognition by
monocyte/macrophages or as a result of opsonization by complement
components. They can also lose their activity in vivo due to
proteases and other enzymes.
[0003] Existing drug delivery systems can, in part, circumvent
these undesirable effects and can be useful for peptide and protein
delivery in vivo, with certain shortcomings, as noted. First, a
continuous systemic infusion of drug via a pump can be employed.
This strategy is proven efficient in clinical practice but may be
impractical for outpatients requiring high levels of mobility,
associated disadvantages of quality of life and potential
intravenous (I.V.) line infections.
[0004] Second, peptides and proteins can be included in an
implantable pump comprised of a capsule with a membrane allowing
diffusion of the drug, for example, at a desirable release rate.
Due to limited volume of these capsules, peptides and proteins are
often used in a concentrated formulation which leads to a loss of
solubility due to aggregation and potential loss of specific
activity. In most cases, the drug is usually released into the
extracellular space and distributed in lymphatics. Overall
concentration of peptide or protein may be affected by local lymph
node activity and the efficacy of lymph node drainage of the
implantation site. There is also a potential of host reaction to
capsule material but in general, this side effect is
infrequent.
[0005] Third, the drug release system can be made biodegradable as
a result of encapsulation or inclusion into degradable drug
delivery vehicles or carriers, e.g. polymeric matrices, particles
or membrane vesicles (liposomes). These delivery systems are
usually either implantable or injectable. Implantable drug delivery
systems are often placed under the epidermis where the components
of the system are usually slowly degraded as a result of biological
activity of surrounding cells (i.e. as a result of the release of
enzymes degrading chemical bonds that hold these implants
together).
[0006] Polylysine and other polyamino acids have previously been
modified by the attachment of phospholipid groups and used in DNA
transfection (Zhou, X H et al (1991) Biochem. Biophys. Acta 1065:
8-14 and Zhou, X h, Huang L (1994) Biochem. Biophys Acta 1189:
195-203). Polylysine has also been modified by the attachment of
hydrophilic groups such as polyethylene glycol, a protective group
(Dash P R, et al (1997) J. Contr. Rel. 48: 269-276), and various
sugars (Kollen W J W, et al, (1996) Human Gene Ther. 13: 1577-1586
and Erbacher P, et al (1997) Biochimica Biophysica Acta 1324:
27-36) but no distinct hydrophobic moiety. Additionally, various
drugs (Hudecz F. et al (1993) Bioconjugate chemistry 4: 25-33) and
targeting residues such as transferrin (Wagner, E (1994) Adv. Drug
Delivery Rev. 14: 113-135), asialoglycoprotein (Chowdhury, N R et
al (1993) J. Biol. Chem. 268: 11265-11271) and monoclonal
antibodies (Chen, J B et al (1994) Febs Lett 338: 167-169) have
been conjugated to polylysine.
[0007] U.S. Pat. No. 5,871,710 to Bogdanov et al. discloses a
biocompatible graft co-polymer adduct including a polymeric
carrier, a protective chain linked to the polymeric carrier, a
reporter group linked to the carrier or to the carrier and
protective chain, and a reversibly linked Pt(II) compound for
diagnosis.
[0008] U.S. Pat. No. 7,138,105 to Bolotin discloses a biocompatible
graft co-polymer comprising of a metal bridge flanked by two metal
binding molecules wherein one of the metal binding molecules is
part of, or covalently linked to, the therapeutic agent. The bridge
links the carrier and therapeutic agent capable of binding
[0009] U.S. Pat. No. 6,576,254 to Uchegbu discloses polyamino acid
vesicles comprising of polylysine grafted with MPEG, fatty acids
and cholesterol.
SUMMARY OF THE INVENTION
[0010] The present invention is directed towards novel drug
delivery systems and methods of making and using the same. It is an
object of the present invention to provide a delivery system for a
therapeutic agent that has a sustained release capability, is safe,
biocompatible, readily prepared from known chemistries and
compounds, whose release rate can be readily adjusted by simple
mechanisms, and is amenable to a wide variety of therapeutic agents
such as peptides, proteins and other hydrophobic drugs. The
invention provides a novel protected graft co-polymeric carrier
with a linear polymeric backbone made up of repeating units (called
residues), preferably between 30 to 500 residues, with modifiable
functional groups (such as amino, carboxyl, hydroxyl, sulfur, and
phosphate), modified in such a way to contain at least one
hydrophobic moiety and a plurality of hydrophilic protective groups
pendant to the polymeric backbone in a weight ratio that renders
the composition soluble in water and size that allows for
subcutaneous delivery. The large number of protective groups acts
as a shield to protect load molecules from being exposed to the
surface of the carrier prior to release by dissociation.
[0011] The invention provides means to deliver hydrophobic
peptides, hydrophobic proteins and hydrophobic drugs in patients in
a controlled manner without the use of vesicles. Controlled manner
means that the level of the active therapeutic molecules in the
circulation will neither exceed a toxic level nor drop below the
therapeutically effective level for the desired period of time (see
FIG. 7). The ability of the carrier of the present invention to
release free and active therapeutic agent, or in a broader sense, a
"load molecule", when the level of free load molecule in the
circulation goes below the therapeutically effective level may be
readily adjusted. The carriers of the present invention may be
prepared to have both high loading capacity and adjustable release
rates by controlling the length, number and density of hydrophobic
moieties in which alkyl chains are more commonly used. Release rate
can also be controlled by the size of the carrier. The carriers of
the present invention are safe and non-immunogenic due to the
presence of multiple non-immunogenic protective chains that shield
the more immunogenic core of the carrier.
[0012] In one aspect this invention provides a soluble
hydrophobic-core carrier composition comprising: (i) a linear
polymeric backbone; (ii) a plurality of hydrophilic polymeric
protective chains covalently linked and pendant to the polymeric
backbone, wherein each protective side chain has a molecular weight
between about 400 and about 20,000 Daltons; and (iii) at least one
hydrophobic moiety covalently linked and pendant to the polymeric
backbone; wherein the weight ratio of the hydrophilic polymeric
protective side chains and the hydrophobic moieties is selected so
that the composition is soluble in water. In one embodiment the
weight ratio of the hydrophilic polymeric protective side chains
and the hydrophobic moieties is at least 15:1, at least 17:1, at
least 20:1, at least 50:1 or at least 100:1. (See Table 2). In
another embodiment at least 90% of the residues of the polymeric
backbone are derivatized with either hydrophilic protective chains
or hydrophobic moieties. In another embodiment the protective side
chains comprise polyethylene glycol, polypropylene glycol, a
co-polymer of polyethylene glycol, a co-polymer of polypropylene
glycol, polysaccharide, or alkoxy derivatives thereof. In another
embodiment the alkoxy derivative is methoxypolyethylene glycol,
methoxypolypropylene glycol, a methoxylated co-polymer polyethylene
glycol and polypropyleneglycol, or ethoxylated polysaccharide. In
another embodiment the linear polymeric backbone is selected from a
group consisting of polylysine, polyaspartic acid, polyglutamic
acid, polyserine, polythreonine, polycysteine, polyglycerol,
natural saccharides, aminated polysaccharides, aminated
oligosaccharides, polyamidoamine, polyacrylic acids, polyalcohols,
sulfonated polysaccharides, sulfonated oligosaccharides,
carboxylated polysaccharides, carboxylated oligosaccharides,
aminocarboxylated polysaccharides, aminocarboxylated
oligosaccharides, carboxymethylated polysaccharides, and
carboxymethylated oligosaccharides.
[0013] In another aspect, this invention provides the
aforementioned soluble hydrophobic-core carrier composition and
further comprises a load molecule dissociably linked to the
hydrophobic moiety of the backbone. In one embodiment the
hydrophilic protective side chains comprise methoxypolyethylene
glycol. In another embodiment the hydrophilic protective side
chains comprise methoxypolyethylene glycol and the polymeric
backbone comprises polylysine. In another embodiment the
hydrophilic protective side chains comprise methoxypolyethylene
glycol, the polymeric backbone comprises polylysine and the
hydrophobic moieties comprise a fatty acid. In another embodiment
the hydrophilic protective side chains comprise methoxypolyethylene
glycol, the polymeric backbone comprises polylysine, the
hydrophobic moieties comprise a fatty acid and the load molecule is
a therapeutic agent. In one embodiment, the therapeutic agent is a
hydrophobic peptide, hydrophobic protein, or a hydrophobic drug. In
another embodiment the therapeutic agent is GLP-1. In another
embodiment, the therapeutic agent is selected from GLP-2, leptin,
islet amyloid polypeptide and vasoactive intestinal peptide.
[0014] In another aspect, this invention provides the
aforementioned soluble hydrophobic-core carrier composition
comprising a load molecule dissociably linked to the hydrophobic
moiety of the backbone where the linear polymeric backbone is
polylysine. In one embodiment the hydrophobic moiety(ies) comprises
a fatty acid selected from the range of 6-carbon fatty acids to
36-carbon fatty acids. In another embodiment the hydrophobic
moiety(ies) comprise a fatty acid with at least one double bond. In
another embodiment the hydrophobic moiety(ies) comprises a
multi-fatty acid-containing moiety. In another embodiment the
hydrophobic moiety(ies) comprises an aromatic ring containing
moiety.
[0015] In some embodiments the carrier may optionally include a
second protective chains covalently linked to the hydrophobic
moiety for enhancing solubility or maintaining the hydrophilic
protective chains to hydrophobic moieties weight ratio above 15:1,
thus preventing vesicle formation and precipitation.
[0016] In various embodiments, the load molecule may be a
therapeutic agent or an imaging agent. In one embodiment the
therapeutic agent is a hydrophobic peptide, hydrophobic protein, or
a hydrophobic drug. In another embodiment, the therapeutic agent
can be GLP-2, leptin, islet amyloid polypeptide (IAPP, also known
as amylin) and vasoactive intestinal peptide (VIP). In one
embodiment, the therapeutic agent may be hydrophobic
polynucleotide, hydrophobic peptide, hydrophobic protein, or
hydrophobic drugs. The hydrophobic peptide/protein may be peptide
aptamer, glucagon-like-peptide, glucagon-like-peptide derivative,
exenatide, leptin, leptin fragment, Peptide YY, alpha-melanocyte
stimulating hormone, adiponectin, obestatin, Gastric inhibitory
polypeptide(GIP), Epidermal Growth Factor (EGF) receptor ligand,
EGF, Transforming Growth Factor alpha (TGF-alpha), Betacellulin,
Gastrin/Cholecystokinin receptor ligand, Gastrin, Cholecystokinin,
interferon, interferon gamma, interferon beta, interferon alpha,
interleukin-1, interleukin-2, interleukin-4, interleukin-6,
interleukin-8, interleukin-10, interleukin-12, tumor necrosis
factor, tumor necrosis factor alpha, tumor necrosis factor beta,
insulin, insulin-like growth factor, growth hormone, nerve growth
factor, brain-derived neurotrophic factor, enzymes, endostatin,
angiostatin, trombospondin, urokinase, streptokinase, blood
clotting factor VII, blood clotting factor VIII,
granulucyte-macrophage colony-stimulating factor (GM-CSF),
granulucyte colony-stimulating factor (G-CSF), thrombopoetin,
calcitonin, parathyroid hormone (PTH) and its fragments,
erythropoietin, atrial natriuretic factor, monoclonal antibodies,
monoclonal antibody fragments, somatostatin, protease inhibitors,
adrenocorticotropin, gonadotropin releasing hormone, oxytocin,
leutinizing-hormone-releasing-hormone, follicle stimulating
hormone, glucocerebrosidase, thrombopoietin, filgrastin,
terlipressin, and vasoactive intestinal peptide (VIP).
[0017] In further embodiment the present invention relates to all
the aforementioned compositions further comprising of targeting
moiety covalently linked to the distal end of the protective group.
The targeting moiety may be an antibody, fragment of an antibody,
chimeric antibody, lectins, receptor ligands, proteins, enzymes,
peptides, saccharides, quasi substrates of enzymes,
cell-surface-binding compounds, and extracellular-matrix-binding
compounds.
[0018] In another aspect, this invention provides a pharmaceutical
composition comprising any one composition selected from the
compositions above wherein the load molecule is a therapeutic
agent.
[0019] In another aspect, this invention provides a method of
making a composition comprising: (a) dissolving a polymeric
backbone containing residues comprising free amino groups in an
aqueous buffer of pH 7-8 to obtain solution A; (b) activating a
carboxyl group or alkyl carboxyl group of a protective chain by
mixing it with a carbodiimide reagent in acidic buffer between pH
3-7 to obtain solution B; and (c) adding solution B to solution A
resulting in a solution C with a pH of 7 or above, containing a
polymeric backbone with covalently linked protective chains.
[0020] In another aspect, this invention provides a method of
making a composition comprising: (a) dissolving in non-aqueous
solvent with a tertiary amine, a component comprising a polymeric
backbone covalently linked to protective chains, wherein the
polymeric backbone comprises residues comprising free amino groups,
thereby obtaining solution E; (b) dissolving in a non-aqueous
solvent hydrophobic molecules containing free carboxyl groups and
activating the carboxyl groups by adding carbodiimide reagent to
obtain solution F; and (c) adding solution F to solution E to
obtain solution G to form covalent linkage between the activated
carboxyl groups and the free amine groups; wherein solution E is
added to solution G until at least 90% of the residues are linked
to protective chains or hydrophobic groups.
[0021] In another aspect, this invention provides a method of
making a composition comprising: (a) dissolving in
partially-aqueous solvent at pH of 7 to 9 a component comprising a
polymeric backbone covalently linked to protective chains, wherein
the polymeric backbone comprises residues comprising free amino
groups, thereby obtaining solution E, (b) dissolving in
partially-aqueous solvent with pH of 3 to 7, hydrophobic molecules
containing free carboxyl groups and activating the carboxyl groups
by adding carbodiimide reagent resulting in solution F, (c) adding
solution F to solution E while maintaining the pH of the mixture
between 7-8 to obtain solution G, to form covalent linkage between
the activated carboxyl groups and the free amine groups; wherein
solution E is added to solution G until at least 90% of the
residues are linked to protective chains or hydrophobic groups.
[0022] In another aspect, this invention provides a method of
loading a composition comprising: a) dissolving in aqueous or
partially-aqueous solvent A soluble hydrophobic-core carrier
composition comprising: (i) a linear polymeric backbone; (ii) a
plurality of hydrophilic polymeric protective chains covalently
linked and pendant to the polymeric backbone, wherein each
protective side chain has a molecular weight between about 400 and
about 20,000 Daltons; and (iii) at least one hydrophobic moiety
covalently linked and pendant to the polymeric backbone; wherein
the weight ratio of the hydrophilic polymeric protective side
chains and the hydrophobic moieties is selected so that the
composition is soluble in water, thereby obtaining solution A. (b)
dissolving the load molecule in aqueous or partially-aqueous
solvent to obtain solution B, (c) mixing solution A with solution B
to obtain solution C, incubating solution C for 30 minutes or
longer followed by lyophilization or solvent evaporation to obtain
a loaded carrier ready to be dissolved into appropriate
solvent.
[0023] In another aspect, this invention provides a method of
administering a therapeutic molecule to a subject comprising
administering to the subject a composition comprising: (i) a linear
polymeric backbone; (ii) a plurality of hydrophilic polymeric
protective chains covalently linked and pendant to the polymeric
backbone, wherein each protective side chain has a molecular weight
between about 400 and about 20,000 Daltons; (iii) at least one
hydrophobic moiety covalently linked and pendant to the polymeric
backbone; (iv) a therapeutic molecule dissociably linked to the
hydrophobic moiety of the backbone; wherein the weight ratio of the
hydrophilic polymeric protective side chains and the hydrophobic
moieties is selected so that the composition is soluble in water.
In one embodiment, the composition is administered subcutaneously
or intramuscularly.
[0024] In another aspect this invention provides a pharmaceutical
composition comprising: (a) a composition comprising: (i) a linear
polymeric backbone; (ii) a plurality of hydrophilic polymeric
protective chains covalently linked and pendant to the polymeric
backbone, wherein each protective side chain has a molecular weight
between about 400 and about 20,000 Daltons; (iii) at least one
hydrophobic moiety covalently linked and pendant to the polymeric
backbone; (iv) a therapeutic molecule dissociably linked to the
hydrophobic moiety of the backbone; wherein the weight ratio of the
hydrophilic polymeric protective side chains and the hydrophobic
moieties is selected so that the composition is soluble in water;
and (b) a pharmaceutically acceptable carrier; wherein the
composition is in unit dose form.
BRIEF DESCRIPTION OF DRAWINGS
[0025] FIG. 1 depicts a schematic representation of one embodiment
of the hydrophobic-core composition of the invention: a linear
polymeric backbone; protective side chains covalently linked to
polymeric backbone; hydrophobic moieties covalently linked to
polymeric core, and hydrophobic load molecule with diameter of 3
nm. The dimension of the carrier is also shown to emphasize that it
is greater than the 4 nm glomerular filtration cut off, whereas
carrier and hydrophobic load molecules together are below this cut
off. The followings are example of proteins and their diameter:
siRNA (diameter<3 nm), albumin hydrated (diameter=7.2 nm);
growth hormone hydrated (diameter=3 nm); glomerular filtration
diameter<4 nm; beta-2 macroglobulin (diameter=3.2 nm); myoglobin
(diameter=3.9 nm); hemoglobin (diameter=6.5 nm); gamma globulin
(diameter=11.1 nm); and Bence-Jones protein (diameter=5.5 nm).
[0026] FIG. 2 depicts a diagram of various chemical reactions for
making amide bonds that are useful in making the composition of the
invention; R.sub.1 can be hydrophobic molecule and R.sub.2 can be
polylysine, or polylysine-PEG; or R.sub.1 can be PEG-carboxyl and
R.sub.2 can be polylysine, hydrophobic molecule-polylysine; or
R.sub.1 can be polyglutamate or polyaspartate and R.sub.2 can be
PEG-amine, hydrophobic molecule (such as alkyl amine from C6 to
C36); or R.sub.1 can be polyglutamate-PEG or polyaspartate-PEG and
R.sub.2 can be alkyl amine. EDC is a water soluble version of DCC;
both can be used to carry out the reactions.
[0027] FIG. 3 depicts a diagram of various chemical reactions for
attaching hydrophobic amine (R.sup.2) to carrier (R.sup.1)
containing functional groups such as isothiocyanate, succinimidyl
ester, or sulfonyl chloride. The carrier R.sup.1 can be any
backbone polymers. Polymer R.sup.1 can be polyglutamate,
polyaspartate, polyglutamate-PEG or polyaspartate-PEG.
[0028] FIG. 4 depicts some of the chemical reactions that may be
used to add PEG protective groups, analogs or derivatives thereof,
to amino group containing polymeric backbone.
[0029] FIG. 5 depicts some of the chemical reactions that may be
used to add aldehyde PEG derivatives to amino group containing
polymeric backbone. These are two step condensation-reduction
reactions (a & b).
[0030] FIG. 6 depicts some of the chemical reactions that may be
used to add PEG protective groups, analogs or derivatives thereof,
to hydroxyl containing polymeric backbone.
[0031] FIG. 7 is the hypothetical free hydrophobic load molecule
(peptide, proteins or hydrophobic drugs) in the blood with a
natural half-life of 20 minutes. There is significant fluctuation
in the concentration of hydrophobic load molecule without the
carrier. With the carrier, the hydrophobic load molecule will be
maintained at therapeutic concentration. The nM concentration of
carrier decreases with a half-life of 20 hrs. A) Hydrophobic load
molecule level resulting from injection 5 mg/kg, 3 times a day
without the carrier of the instant invention, this load molecule
has a blood half-life of 20 minutes; B) Carrier along with load
molecule has a half-life of 20 hours; C) therapeutic level of free
load molecule maintained by the carrier.
[0032] FIG. 8 is a graph showing the theoretical and actual
relationship between the amount of amino-group/mg of PLPEG
(polylysine-polyethyleneglycol copolymer) and % amino-group
saturation of polylysine. This is very useful as secondary
confirmation of the composition of PLPEG. This PEGylation process
is quite reproducible and adjustable during synthesis by continuing
the reaction until the desired % PEGylation is achieved using TNBS
amino group assay as a feedback guide during the reaction. The
yield is about 50-80% (5-8 gr) of the starting materials. The
theoretical prediction was calculated using the following equation:
X=[100.times.(C-Y)]/5YC+C]; where X is the % saturation; Y is the
mmol NH.sub.2 per gram of PLPEG as determined by TNBS; C is the
mmol of NH.sub.2 per gram of PL (polylysine) as determined by TNBS.
The 5 in the term 5YC in the equation represent the size of PEG
used which in this case is 5 kDa, thus 5YC. If 10 kDa PEG is used,
this will be 10YC. This is useful because once PLPEG product is
formed, the percent saturation of the amino group of polylysine can
be further confirmed by a single TNBS assay of the final product to
determine Y from which X can be calculated.
[0033] FIG. 9. These are Gel Filtration Chromatograms of the
products of the reaction before and after clean up through 100 kDa
MWCO membrane (Amersham Biosciences, Needham, Mass.) showing that
all unreacted PEG had been removed. The column used was
Ultrahydrogel linear (0.78.times.30 cm, Waters) eluted at flow rate
of 0.6 ml/min PBS with 15% Acetonitrile. The materials were
detected using refractive index detector. Panel A is 20PLPEG555 (20
kDa polylysine where 55% of the amino groups were reacted with PEG
succinate of 5 kDa molecular weight) prior to clean-up from
unreacted 5 kDa PEG. Panel B is 5 kDa PEG alone. Panel C is
20PLPEG555 after clean up.
[0034] FIG. 10. These are the Stoke's radii of various carriers
along with proteins of known stokes radii. These were analyzed on
the Ultrahydrogel Linear column (0.78 cm diameter.times.30 cm
length) using PBS with 15% Acetonitrile at a flow rate of 0.6
ml/min as mobile phase. The 20PL-PEG555 (20 kDa polylysine where
55% of the amino groups were reacted with PEG succinate of 5 kDa
molecular weight), 40PL-PEG530 (40 kDa polylysine where 30% of the
amino groups were reacted with PEG succinate of 5 kDa molecular
weight), 40PL-PEG551 (40 kDa polylysine where 51% of the amino
groups were reacted with PEG succinate of 5 kDa molecular weight),
and 40PL-PEG527 (40 kDa polylysine where 27% of the amino groups
were reacted with PEG succinate of 5 kDa molecular weight) are
larger than the glomerular filtration cut off that is above 4 nm
(40 Angstrom) in diameter (or 20 Angstrom in radius). Proteins with
known stokes radii were used as reference including Thyroglobulin
(669 kDa; 85.5 Angstroms stokes radius), Catalase (248 kDa; 52.2
Angstrom stokes radius), and BSA (67kDa; 35.5 Angstroms stokes
radius), Catalase (248 kDa; 52.2 Angstrom stokes radius), and BSA
(67 kDa; 35.5 Angstrom stokes radius).
[0035] FIG. 11. Binding of Doxorubicin-HCl to a carrier comprising
20 kDa polylysine backbone, 55% of the amino residues were
covalently linked to 5 kDa MPEGsuccinate, and the remainder
saturated with stearic acid. Ten mg/ml (30 uM) of Carrier is loaded
with various concentration of doxorubicin. This specific carrier
binds doxorubicin but did not bind with strong affinity. When
Scatchard plot is used and has apparent Kd of 315 uM. This is not
considered strong binding since to be considered strong for the
purpose of the present invention Kd has to be less than 100 uM. The
relative hydrophilicity of doxorubicin is evident from its
retention time on reverse phase HPLC column (Table 3, below)
compared to other load molecules tested.
[0036] FIG. 12. Binding of Nociceptin to a carrier comprising 20
kDa polylysine backbone, 55% of the amino residues were covalently
linked to 5 kDa MPEGsuccinate, and the remainder saturated with
stearic acid. Ten mg/ml (30 uM) of Carrier is loaded with 0.2 mg/ml
(111 uM) Nociceptin. This specific carrier binds Nociceptin but did
not bind with strong affinity. Very little binding is observed as
consistent with its hydrophilicity and the early retention time
(1.43 minutes; see table 3 below) on HPLC compared with the
retention time of GLP-1 (2.5 minutes; see table 3 below) in
identical reversed phase HPLC column (SynergiMaxRP 4.times.20 mm;
Phenomenex). The column was eluted at a flow rate of 1.5 ml/min
using a gradient of solvent A to B (25-50% B from 1-5 minutes)
where A is water with 0.1% Trifluoroacetic acid (TFA)/5%
Acetonitrile and solvent B is Acetonitrile with 0.1% TFA.
[0037] FIG. 13. Binding of Islet Amyloid Polypeptide or IAPP to a
carrier comprising 20 kDa polylysine backbone, 55% of the amino
residues were covalently linked to 5 kDa MPEGsuccinate, and the
remainder saturated with stearic acid. Ten mg/ml (30 uM) of Carrier
is loaded with various amount IAPP (for methods see example 9).
This carrier binds IAPP with strong affinity and will be useful in
prolonging the blood circulation half-life of IAPP or its
derivative when administered with the carrier. When analyzed by
Scatchard plot, the binding has Kd of 900 nM. The relative
hydrophobicity of IAPP based on reverse phase HPLC retention time
is shown in Table 3 below.
[0038] FIG. 14. Binding of Terlipressin to a carrier comprising 20
kDa polylysine backbone, 55% of the amino residues were covalently
linked to 5 kDa MPEGsuccinate, and the remainder saturated with
stearic acid. Ten mg/ml (30 uM) of Carrier is loaded with various
amount Terlipressin (for methods see example 9). This carrier does
not bind Terlipressin with strong affinity. The relative
hydrophobicity of terlipressin based on reverse phase HPLC
retention time is shown in Table 3 below.
[0039] FIG. 15. Binding of Vasoactive Intestinal Peptide (VIP) to a
carrier comprising 20 kDa polylysine backbone, 55% of the amino
residues were covalently linked to 5 kDa MPEGsuccinate, and the
remainder saturated with stearic acid. Ten mg/ml (30 uM) of Carrier
is loaded with various amount VIP (for methods see example 9). This
carrier binds VIP with strong affinity and will be useful in
prolonging the blood circulation half-life of VIP when administered
with the carrier. The relative hydrophobicity of VIP based on
reverse phase HPLC retention time is not greater than human growth
hormone as shown in Table 3 below. It is surprising that growth
hormone did not bind to the carrier with strong affinity while VIP
which is less hydrophobic did. Further investigation indicated that
at neutral pH in the presence of lipid (or perhaps carrier of the
present invention), VIP assumes more hydrophobic alpha helix
structure. Polypeptides such as this run under acidic HPLC
conditions will not follow the general trend and must be run under
neutral non-denaturing HPLC conditions to be more predictive.
[0040] FIG. 16. Binding of recombinant human growth hormone (hGH)
to a carrier comprising 20 kDa polylysine backbone, 55% of the
amino residues were covalently linked to 5 kDa MPEGsuccinate, and
the remainder saturated with stearic acid. Ten mg/ml (30 uM) of
Carrier is loaded with various amount hGH (for methods see example
9). This carrier does not bind hGH with strong affinity.
[0041] FIG. 17. Binding of Leptin to a carrier comprising 20 kDa
polylysine backbone, 55% of the amino residues were covalently
linked to 5 kDa MPEGsuccinate, and the remainder saturated with
stearic acid. 33 mg/ml (10 uM) of Carrier is loaded with various
amount Leptin (for methods see example 9). The carrier binds Leptin
with strong affinity and when analyzed by Scatchard plot, the
binding has Kd of 700 nM. This carrier will be useful in prolonging
the blood circulation half-life of Leptin when administered with
the carrier.
[0042] FIG. 18. Thin Layer chromatographic analysis of PEG-thapsic
acid product and starting amino PEG. TLC mobile phase is 5:1
dichloromethane/methanol. Solid phase is Silica Gel 60 F254 on
aluminum sheets. TLC was stained by Bromocresol Blue and Ninhydrin.
Bromocresol blue stains blue for PEG bearing materials, Rf of 5K
Amino PEG (s.m.)=0.76 and product thapsic acid-amino PEG conjugate
(Fatty PEG, Product)=0.63. Starting material and product both stain
bromocresol blue and 5K amino PEG stains positive for ninhydrin,
the product does not.
[0043] FIG. 19. Insoluble carrier causes skin necrosis whereas the
soluble carrier does not. Shown are photographs of rat skins taken
3 weeks after subcutaneous injection of insoluble carrier (A) with
PEG to fatty acid weight ratio of less than 10 and soluble carrier
(B) with PEG to fatty acid weight ratio of greater than 10. The
arrow in A shows that 15 mg of insoluble composition of U.S. Pat.
No. 6,576,254 injected subcutaneously causes necrosis after 3 weeks
(A, arrow) whereas no necrosis is observed in animals that received
similar amount of soluble carrier of the present invention (B,
arrow).
[0044] FIG. 20. Total VIP in the blood after subcutaneous
administration of formulated VIP (20PLPEG555C18 containing 2% by
weight of VIP). The 20PLPEG555C18 used in the formulation is a 20
kDa polylysine where 55% of the amino groups were reacted with PEG
succinate of 5 kDa molecular weight and the remaining aminogroups
were reacted with stearic acid or C18. The elimination half-life of
VIP administered alone is just a few minutes (not visible in the
graph) while the formulated VIP has half-life of more than 20
hours. Male Wistar rats were injected subcutaneous with 1 mg VIP
alone or 1 mg VIP in 20PLPEG555C18 formulation (N=7) in phosphate
buffered saline. Blood draws were done at given time points from
the tail and protease inhibitor cocktail was added (Calbiochem,
Cat#539131, La Jolla, Calif.). The total VIP (20PLPEG555C18 bound
and unbound) are measured by Elisa kit from Peninsula (San Carlos,
Calif.) and the background signal of rat serum were subtracted from
all data points.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0045] For convenience, before further description of the present
invention, certain terms employed in the specification, examples
and appended claims are collected here. These definitions should be
read in light of the remainder of the disclosure and understood as
by a person of ordinary skill in the art. Unless defined otherwise,
all technical and scientific terms used herein have the same
meaning as commonly understood by a person of ordinary skill in the
art.
[0046] The articles "a" and "an" are used to refer to one or to
more than one (i.e., to at least one) of the grammatical object of
the article. By way of example, "a protective chain" means one
protective chain or more than one protective chain.
[0047] The term "aptamer" means oligonucleic acid or peptide
molecules that bind a specific target molecule through specific
folding. One of the embodiments of the present invention is to
deliver hydrophobic peptide aptamers by providing carrier with
hydrophobic moiety. Aptamers are usually created by selecting them
from a large random sequence pool, but natural aptamers also exist
in riboswitches. Aptamers can be used for both basic research and
clinical purposes as macromolecular drugs. Aptamers can be combined
with ribozymes to self-cleave in the presence of their target
molecule. These compound molecules have additional research,
industrial and clinical applications. Aptamers offer the utility
for biotechnological and therapeutic applications as they offer
molecular recognition properties that rival that of the commonly
used biomolecule, antibodies. This is possible through specific
folding to create recognition sites. Although this folding can be
interrupted by binding to the carrier, upon release from the
carrier re-folding will occur to provide aptamers that has the
right folding to be biologically or therapeutically active. In
addition to their discriminate recognition, aptamers offer
advantages over antibodies as they can be engineered completely in
a test tube, are readily produced by chemical synthesis, possess
desirable storage properties, and elicit little or no
immunogenicity in therapeutic applications. Aptamers are cleared
rapidly from the bloodstream, with a half-life of minutes to hours,
mainly due to degradation and clearance from the body by the
kidneys, a result of the aptamer's inherently low molecular weight.
Peptide aptamers are proteins that are designed to interfere with
other protein interactions inside cells. They consist of a variable
peptide loop attached at both ends to a protein scaffold. This
double structural constraint greatly increases the binding affinity
of the peptide aptamer to levels comparable to an antibody's
(nanomolar range). The variable loop length is typically comprised
of 10 to 20 amino acids, and the scaffold may be any protein which
has good solubility (which for the purpose of the present invention
will preferably hydrophobic) and compact properties. These peptide
aptamers can be made to contain fatty acids to increase
hydrophobicity to be able to load into the present invention by
hydrophobic interaction. While the aptamer is loaded into the
carrier of the present invention it is protected from degradation
due to high density of protective chain shielding
[0048] The term "derivative" or "analog" as used herein refers to a
compound whose core structure is the same or closely resembles that
of, a parent compound, but which has a chemical or physical
modification, such as different or additional groups. The term also
includes a peptide with at least 50% sequence identity (i.e. amino
acid substitution is less than 50%) with the parent peptide. The
term also includes a peptide with additional groups attached to it
compared to the parent peptide, such as fatty acids and/or
additional amino acids that do not exceed the mass of the original
parent peptide. The term also includes a polymer with additional
group attached to it, such as, in the case of a protective group,
an alkoxy group, compared to the parent polymer. The term also
includes methoxylated or ethoxylated protective chains with
additional methoxy- or ethoxy-group(s) attached to it compared to
the parent protective chains.
[0049] The term "hydrophobic moiety" as used herein refers to a
molecule or molecular moiety attached to the backbone that is
non-polar and provides a hydrophobic environment for the load
molecule to interact in order to avoid the surrounding water
environment. Hydrophobic moieties may be aliphatic hydrocarbon
chains and/or ring compounds that do not have positive or negative
charge and are capable of binding to molecules by hydrophobic
interaction. The hydrophobic moieties are the portions of the
molecule that are typically made up of hydrogen and carbon with
minimal amount of oxygen and nitrogen. The hydrophobic moiety can
be a single continuous portion of a molecule having six or more
carbons linked together where the total number of nitrogen plus
oxygen bonded to this portion is one third or less than of the
number of carbon atoms. It is also understood that the hydrophobic
moiety counts as a separate entity from the polymeric backbone,
such that, for example, when the polymeric backbone is a polyamino
acid, the natural R group on the polyamino acid is not counted as a
hydrophobic moiety in the present invention. For example, a
hydrophobic moiety may be added to a polylysine backbone through
amide formation from amine group.
[0050] The term "load molecule" as used herein encompasses any
molecule that binds with high affinity (those with affinity
constant (Ka) of greater than 0.01 million/molar or dissociation
constant (Kd) of less than 100 micromolar) to the carrier, allowing
it to be loaded into the carrier. The affinity constant or
dissociation constant can easily be ascertained by those skilled in
the art. For the purpose of the present invention, these load
molecules include hydrophobic peptides (50 or less amino acids),
hydrophobic proteins (greater than 50 amino acids), polynucleotide
(RNA, DNA or their analogs) with additional hydrophobic moieties or
with additional agent that neutralize the negatively charged
polyphosphate backbone, and other hydrophobic molecules.
[0051] The term "non-proteinaceous polyamino acid" as used herein
refers to a polyaminoacid that is not naturally made by a living
organism unless recombinantly engineered by human. Non-limiting
examples of these are poly-(L and/or D)-lysine, poly-(L and/or
D)-glutamate, poly-(L and/or D)-glutamate, poly-(L and/or
D)-aspartate, poly-(L and/or D)-serine, poly-(L and/or
D)-threonine, poly-(L and/or D)-tyrosine, and poly-(L and/or
D)-arginine. The non-proteinaceous polyamino acid also includes
polyamino acids with R-groups that are not naturally occurring but
contains carboxyl, amino, hydroxyl, or thiol groups that can
provide repeating functional groups (from 30 to 1000 functional
groups) that are modifiable for the attachment of protective groups
and/or oligonucleotides. The non-proteinaceous polyaminoacids are
among the possible backbone component of the invention.
[0052] The term "hydrophilic protective side chain" as used herein
refers to a molecule(s) which protects the carrier-core and the
load molecule from contact with other macromolecules due to
extensive linking or binding of water to the chains. Because of
this extensive binding with water molecules the protective chain
also increases water solubility of the composition. The protective
group does not have significant amount of charge but is water
soluble. Generally, the groups are non-immunogenic. This also means
that protective chain provides hydrophilic property to the
composition. The term "protective side chain" is used
interchangeably with the terms "protective group" and "protective
chain". The protective chains of the present composition include
polyoxyethylene glycol also referred to as polyethylene glycol and
their derivatives. The protective chains of the present composition
also include uncharged polysaccharides and their derivatives such
as ethoxylated or methoxylated polysaccharides. In this context,
uncharged means that the main body of the chain does not have
positive or negative charge.
[0053] The term "targeting moiety," "targeting molecules," or
"targeting group" refers to any molecular structure which assists
the construct of the composition in localizing at a particular
target area, entering a target cell(s), and/or binding to a target
receptor. For example, lipids (including hydrophobic, neutral, and
steroidal lipids), antibodies, antibody fragments, chimeric
antibodies, lectins, ligands, receptor ligands, sugars,
saccharides, steroids, hormones, nutrients, peptides, proteins,
enzymes, quasi substrates of enzymes, cell-surface-binding
compounds, and extracellular-matrix-binding compounds may serve as
targeting moieties. Targeting moieties are preferably attached to
the distal portion of protective chains of the carrier.
[0054] The term "therapeutic agents" as used herein refer to any
chemical that is a biologically, physiologically, or
pharmacologically active and act locally or systemically in a
subject. For the purpose of the present invention, therapeutic
agent loaded into the carrier is understood to be hydrophobic such
as hydrophobic peptides and proteins or to have hydrophobic moiety
modification such as fatty acids or phenyl ring. Examples of
therapeutic agents (also referred to as "drugs") which has
significant hydrophobic moieties or may be enhanced to have
significant hydrophobic moieties by covalently linking it into
fatty acids includes glucagon-like-peptide, glucagon-like-peptide
derivatives, exenatide, glucagon-like-peptide-1,
glucagon-like-peptide-2, leptin fragment, Gastric inhibitory
polypeptide(GIP), Epidermal Growth Factor (EGF) receptor ligand,
EGF, Transforming Growth Factor alpha (TGF-alpha), Betacellulin,
Gastrin/Cholecystokinin receptor ligand, Gastrin, Cholecystokinin,
lysostaphin, interferon, interferon gamma, interferon beta,
interferon alpha, interleukin-1, interleukin-2, interleukin-4,
interleukin-6, interleukin-8, interleukin-10, interleukin-12,
auristatin, nisin, insulin, insulin-like growth factor 1, growth
hormone, growth hormone releasing hormone (GHRH), nerve growth
factor, brain-derived neurotrophic factor, enzymes, endostatin,
angiostatin, trombospondin, urokinase, streptokinase, blood
clotting factor VII, blood clotting factor VIII,
granulucyte-macrophage colony-stimulating factor (GM-CSF),
granulucyte colony-stimulating factor (G-CSF), thrombopoetin,
calcitonin, parathyroid hormone (PTH) and its fragments,
erythropoietin, atrial natriuretic factor, monoclonal antibodies,
monoclonal antibody fragments, somatostatin, protease inhibitors,
adrenocorticotropin, gonadotropin releasing hormone, oxytocin,
leutinizing-hormone-releasing-hormone, follicle stimulating
hormone, glucocerebrosidase, thrombopoietin, filgrastin,
prostaglandins, epoprostenol, prostacyclin, cyclosporine,
vasopressin, terlipressin, desmopressin, cromolyn sodium (sodium or
disodium chromoglycate), and vasoactive intestinal peptide (VIP).
Any hydrophobic molecules may be attached to the above therapeutic
agents to facilitate loading or improve their affinity to the
carrier. The hydrophobic moieties attached to the therapeutic agent
can be any fatty acids or their analogs. The therapeutic agents may
be natural hydrophobic peptides that are non-immunogenic but are
susceptible to breakdown and elimination without the protection of
the carrier.
[0055] The term "therapeutically effective amount" as used herein
refers to the amount of composition that will provide a therapeutic
benefit to the patient. In certain embodiments, the term refers to
an amount of the therapeutic agent that, when loaded to the
hydrophobic core carrier composition of the present invention and
administered to the patient, produces some desired effect at a
reasonable benefit/risk ratio applicable to any medical treatment.
The effective amount may vary depending on such factors as the
disease or condition being treated, the particular constructs being
administered, the size of the subject and/or the severity of the
disease or condition. One of ordinary skill in the art may
empirically determine the therapeutically effective amount of a
particular compound without necessitating undue experimentation. In
certain embodiments, the term refers to that amount necessary or
sufficient for a use of the subject compositions described herein.
In the treatment of obesity, the therapeutically effective amount
is the amount of composition of the present invention with
corresponding load molecule(s) such as, but not limited to, leptin
that will reduce appetite and/or weight. In the treatment of
obesity, the therapeutically effective amount is the amount of
composition of the present invention with corresponding load
molecule(s) such as, but not limited to, PYY, hat will reduce
appetite and/or weight. In the treatment of insulin-insufficient
diabetes, the therapeutically effective amount is the amount of
composition of the present invention with corresponding load
molecule(s) that will improve glucose homeostasis or normalize
blood glucose level of the patient and/or regenerate the beta-islet
cells in the pancreas. The regeneration of the beta-islet cells the
can be indirectly measured by monitoring blood glucose level,
Hemoglobin A1c level, C-peptide level, or insulin level in the
blood.
Descriptions of the Components of the Invention
[0056] This invention provides soluble compositions comprising a
linear polymeric backbone or carrier with hydrophilic side chains
and hydrophobic side chains appended thereto. Such compositions are
useful, among other things, as carriers of hydrophobic molecules,
such as therapeutic molecules. The hydrophobic molecules are
affinity-bound to the hydrophobic side chains. Upon administration
to a subject the composition is soluble and it releases the
hydrophobic load molecule as a function of the kinetics of binding
and local concentration. The compositions of the present invention
have a weight ratio of hydrophilic side chains to hydrophobic side
chains that renders the composition soluble in water, i.e., the
composition is soluble in water at a concentration of 50 mg/ml and
does not precipitate or render the solution cloudy. Generally, the
weight ratio of hydrophilic side chains to hydrophobic side chains
is at least 15:1. In various embodiments the weight ratio is above
17:1, above 20:1, above 25:1, above 50:1 or above 100:1. In other
embodiments, the weight ratio is between 15:1 and 60:1; between
20:1 and 45:1 or between 25:1 and 40:1.
[0057] Example 8 shows solubility of compositions of varying length
of polylysine and different ratios of MPEG to fatty acid. At
MPEG:fatty acid ratios above 14:1, the compositions are soluble at
a concentration of 50 mg/ml. At ratios between 10.5:1 and 13.7:1
the compositions are "partially soluble."
[0058] In certain embodiments, the composition of the present
invention is made up of at least four components; a) a linear
polymeric backbone, b) several hydrophilic protective chains
covalently linked to the polymeric backbone and/or to the
hydrophobic moiety c) several hydrophobic moieties covalently
linked and pendant (linked to side of the backbone) to the linear
polymeric back bone,, and d) hydrophobic load molecules such as
hydrophobic peptides (50 amino acids and less), hydrophobic
proteins (over 50 amino acids), or hydrophobic drugs dissociably
linked to the hydrophobic moieties. The preferable hydrophobic
peptides, proteins and hydrophobic molecules have retention times
greater than two minutes in one of their conformational states
under the following HPLC conditions; A reversed phase HPLC column
(SynergiMaxRP; 2.5 um, 4.times.20 mm; Phenomenex) eluted at a flow
rate of 1.5 ml/min using a gradient of solvent A to B (25-50% B
from 1-5 minutes) where A is water with 0.1% Trifluoroacetic acid
(TFA)/5% Acetonitrile and solvent B is Acetonitrile with 0.1% TFA.
The retention time of GLP-1 under this condition is 2.56 minute,
VIP is 1.63 minute but small amount in the alpha conformation is
2.56 minutes, and leptin is 4.9 minutes. VIP is believed to assume
more hydrophobic alpha conformation when exposed to the carrier
resulting in more binding than expected (see Table 2, below). Other
peptides and proteins that are not highly hydrophobic can be made
hydrophobic by attaching fatty acids and the process of such
modification is very well known in the art. The protective chain in
the present invention is essential to hide the load molecules from
degradation by enzymes and cells.
Polymeric Backbone
[0059] The polymeric backbone is a non-proteinaceous homo- or
heteropolymer with repeating amino or carboxyl groups and may be of
natural or synthetic origin. Preferably the polymeric backbone is
polyamino acid which may have D- or L-chirality or both and more
preferably a straight chain homopolymer. Preferred straight chain
homopolymers include polylysine and polyornithine, polyarginine,
polyglutamate, polyaspartate, polyserine, polythreonine,
polytyrosine or any other amide linked hemoropolymer made from
amino acids. A linear polyethylenimine may also be used a polymeric
backbone. If the polymeric backbone is a polyamino acid,
non-proteinaceous is preferable, meaning that it is not a protein
made by living organism to have a three dimensional conformation
associated activity. The polymeric backbone may have a molecular
weight of about 600-1,000,000, preferably 3,000-100,000. It is also
preferable to have 30 to 1000 modifiable functional groups. Other
polymeric backbone with repeating modifiable functional groups may
also be used such as those with repeating sulfhyryl(thiol),
phosphate, and hydroxyl groups. Carbohydrate polymers and other
synthetic polymers where monomers are non-biological may also be
used as polymeric backbone. The polymeric backbone provides the
multiple sites from where the hydrophobic chains and hydrophilic
protective chains can be attached. In certain embodiments, at least
80%, at least 90%, at least 95% or at least 99% of the residues of
the polymeric backbone are derivatized with hydrophilic or
hydrophobic side chains.
Hydrophobic Moiety
[0060] The hydrophobic moieties may comprise hydrophobic alkyl
groups, which have a general formula [CxHyOz] where x is 6-36; y is
2-71; z is 1-4. It is preferable that z=1, which is the minimum
required for amide bond formation with the amino group of the
polymeric backbone. The starting molecules (prior to attachment to
polymeric backbone) however, may have z greater than 1 prior to
amide bond formation. The hydrophobic moieties may also comprise
two ended hydrophobic alkyl groups (one end attached to polymeric
backbone) which have a general formula [--OC(CH.sub.2).sub.xCO--]
or [--OC(CH.sub.2).sub.xCN--] where x is 6-36, and may further
comprise protective groups covalently attached to the first end of
the hydrophobic moieties and the second end covalently attached to
the polymeric backbone.
[0061] In one embodiment, the chemical link of hydrophobic moieties
to the polymeric backbone comprises amide bond. In another
embodiment, the chemical link of hydrophobic moieties to the
polymeric backbone comprises ether bond. In another embodiment, the
chemical link of hydrophobic moieties to the polymeric backbone
comprises ester bond. In another embodiment, the chemical link of
hydrophobic moieties to the polymeric backbone comprises disulfide
bond. In one embodiment, the hydrophobic moiety attached to
polymeric backbone comprises an alkyl acyl derived from fatty
acids, or aromatic alkyl acyl derived from aromatic alkyl acids,
which has a general formula [CxHyOz] where x is 6-36; y is 2-71; z
is 1-4. It is preferable that z=1, the starting molecules however
may have z greater than 1 prior to bond formation with polymeric
backbone.
Protective Hydrophilic Group or Protective Chains
[0062] Preferably the "protective hydrophilic group" or "protective
chain" is non-ionic with a molecular weight of about 2000-20,000,
preferably 5,000-10,000. The protective chain of the composition is
preferably a polymer of ethylene oxide (or polyethyleneglycol also
called polyoxyethyleneglycol), i.e. PEG or a mono-methoxy ether of
polyethyleneglycol (i.e. MPEG). A protective chain is useful
because: 1) it ensures the solubility the composition while
maintaining a high drug payload, 2) a protective chain assists in
the formation of a stearic barrier which can prevent load molecules
(hydrophobic peptides, hydrophobic proteins and other hydrophobic
therapeutic agents) from binding or interacting with other
macromolecules, enzymes (nucleases and proteases) and cells in the
body; 3) a protective chain provides load molecules (hydrophobic
peptides, hydrophobic proteins and hydrophobic drugs) with long
circulation times or biological half-lives in vivo (e.g. for
decreasing glomerular filtration in kidneys, decreasing kidney and
liver uptake, decreasing macrophage uptake) and creates a
circulating depot; 4) a protective chain decreases undesirable
immunogenicity of the carrier or its load molecules such as
hydrophobic peptides and hydrophobic proteins and hydrophobic
drugs; and 5) the protective chains increases the size of the
carrier to take advantage of abnormal permeability of tumor vessels
and assists accumulation of the carrier with load molecules in a
tumor or inflammation site and delivering the load molecules or
anti tumor compounds to the tumor which is especially useful for
treating tumors and other highly vascularized areas of the
body.
[0063] The protective chain of the hydrophobic core carrier
composition may be polyethylene glycol, polypropylene glycol,
methoxypolyethylene glycol, methoxypolypropylene glycol, a
co-polymer of polyethylene glycol and polypropylene glycol; or a
alkoxy derivative thereof. It is preferable that the protective
chain is one of methoxypolyethylene glycol, methoxypolypropylene
glycol, or a co-polymer of methoxypolyethylene glycol and
methoxypolypropyleneglycol. The protective chain may also be
polyethylene glycol monoamine, methoxypolyethylene glycol
monoamine, methoxy polyethylene glycol hydrazine, methoxy
polyethylene glycol imidazolide or a polyethylene glycol diacid.
Protective chains are linked to the polymeric backbone and/or
hydrophobic moieties pendant to the polymeric backbone preferably
by a single linkage. Methoxylated or ethoxylated polysaccharides
can also be used as protective chains in the present invention
since alkoxylation will reduce or eliminate their immunogenicity
and will thus act as improved protective chains.
Hydrophobic Load Molecules
[0064] The hydrophobic load molecules are preferably hydrophobic
peptides and proteins; lipids, and hydrophobic drugs. Among these
includes hydrophobic imaging agent and hydrophobic therapeutic
agent. Hydrophobic peptides and proteins include cytokine,
lymphokine, hormone, and enzymes.
[0065] The load molecules of the present invention also include
therapeutic agents derivatized to contain hydrophobic moieties or
naturally hydrophobic therapeutic agent. These includes siRNA,
antisense-DNA, antisense-RNA, glucagon-like-peptide,
glucagon-like-peptide derivatives, exenatide,
glucagon-like-peptide-1, glucagon-like-peptide-2, leptin, leptin
fragment, Gastric inhibitory polypeptide(GIP), Epidermal Growth
Factor (EGF) receptor ligand, EGF, Transforming Growth Factor alpha
(TGF-alpha), Betacellulin, Gastrin/Cholecystokinin receptor ligand,
Gastrin, Cholecystokinin, lysostaphin, interferon, interferon
gamma, interferon beta, interferon alpha, interleukin-1,
interleukin-2, interleukin-4, interleukin-6, interleukin-8,
interleukin-10, interleukin-12, tumor necrosis factor, tumor
necrosis factor alpha, tumor necrosis factor beta, auristatin,
nisin, insulin, insulin-like growth factor, growth hormone, growth
hormone releasing hormone (GHRH), nerve growth factor,
brain-derived neurotrophic factor, enzymes, endostatin,
angiostatin, trombospondin, urokinase, streptokinase, blood
clotting factor VII, blood clotting factor VIII,
granulucyte-macrophage colony-stimulating factor (GM-CSF),
granulucyte colony-stimulating factor (G-CSF), thrombopoetin,
calcitonin, parathyroid hormone (PTH) and its fragments,
erythropoietin, atrial natriuretic factor, monoclonal antibodies,
monoclonal antibody fragments, somatostatin, protease inhibitors,
adrenocorticotropin, gonadotropin releasing hormone, oxytocin,
leutinizing-hormone-releasing-hormone, follicle stimulating
hormone, glucocerebrosidase, thrombopoietin, filgrastin,
prostaglandins, epoprostenol, prostacyclin, cyclosporine,
vasopressin, terlipressin, desmopressin, cromolyn sodium (sodium or
disodium chromoglycate), vasoactive intestinal peptide (VIP),
vancomycin, antimicrobials, polymyxin b, anti-fungal agents,
anti-viral agents, enfuvirtide, doxorubicin, etoposide, fentanyl,
ketamine, and vitamins. The preferred hydrophobic moieties to
attached therapeutic peptides and proteins to increase their
hydrophobicity are fatty acids from 6 to 36 carbon units, with the
intention of facilitating loading into the carrier during the
formulation.
Chemical Assembly of the Carrier from Individual Components
[0066] Attaching Protective chains to Amino groups of the Polymeric
Backbone: The present invention relates to a polymeric backbone
further comprising a protective chains and hydrophobic moiety. The
modification of the polymeric backbone containing amino groups is
the amide covalent attachment of protective chains comprising
methoxypolyethyleneglycol. A non-limiting example of an amino group
modification along the polymeric backbone is attachment of
protective chains comprising acyl methoxypolyoxyethyleneglycol
(MPEG). An example of a protective chain with or without
hydrophobic chain attached which is not intended to limit the scope
of this invention is an acyl PEG, analog or derivative thereof
which can be represented by formula:
--CO(CH.sub.2).sub.nCOOCH.sub.2CH.sub.2-A-OR.sub.3 or
--COCH.sub.2-A-OR.sub.3, where n is 2-22 (representing hydrophobic
moiety); A (representing protective chain) is [OCH2CH2].sub.x or
[OCH2CH2].sub.x or [OCHCH.sub.3CH.sub.2].sub.x, where x is 17-500,
or various combinations of [OCH2CH2], [OCH2CH2], and/or
[OCHCH.sub.3CH.sub.2] with total of 17-500 units, R.sub.3 is H,
(CH.sub.2).sub.pCH.sub.3 or (CH.sub.2).sub.pCOOH, and p is 0-7.
[0067] Another object of the present invention is to provide a
method of attaching protective chains to the amino group containing
polymeric backbone. The modifications can be done by amide bond
formation. As an example that is not intended to limit the scope of
this invention, the carboxyl containing protective chain can be
attached to the amino group of the polymeric backbone using
carbodiimide containing reagent such a
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide (see FIG. 2). A carbodiimide reagent
contains a functional group consisting of the formula
N.dbd.C.dbd.N. During the process of coupling reaction, the
activated carboxyl group O-acylisourea-intermediate can be
stabilized by forming N-hydroxysuccinimide ester using
N-hydroxysuccinimide. This relatively stable intermediate can react
with the amino group of carrier such as polylysine or chitosan to
form amino-acyl bond or amide bond. Similar result can also be
accomplished by reacting aldehyde containing protective group to
the amino group along the carrier. The aldehyde can react with the
amino group of carrier such as polylysine or chitosan to form
amino-acyl bond or amide bond.
[0068] Attaching Protective chains to Carboxyl groups of Polymeric
backbone: The present invention relates to a polymeric backbone
further comprising a protective chains and hydrophobic moiety. The
modification of the polymeric backbone containing carboxyl groups
is the amide covalent attachment of an amino group containing
protective chains comprising amino methoxypolyoxyethyleneglycol
(MPEG). As an example that is not intended to limit the scope of
this invention, the protective chain with or without hydrophobic
moiety can be an amino PEG which can be represented by formula
--NH(CH.sub.2).sub.nNHCOCH.sub.2-A-OR.sub.3,
--NH(CH.sub.2).sub.nNHCO(CH.sub.2).sub.nCOOCH.sub.2CH.sub.2-A-OR.sub.3,
where n is 2-22 (representing the hydrophobic moiety); A
(representing protective group) is [OCH.sub.2CH2].sub.x or
[OCH.sub.2CH.sub.2].sub.x or [OCHCH.sub.3CH.sub.2].sub.x, where x
is 17-500, or various combinations of [OCH.sub.2CH.sub.2],
[OCH.sub.2CH.sub.2], and/or [OCHCH.sub.3CH.sub.2] with total of
17-500 units, R.sub.3 is H, (CH.sub.2).sub.pCH.sub.3 or
(CH.sub.2).sub.pCOOH, and p is 0-7.
[0069] Another object of the present invention is to provide
methods of attaching protective chains to the polymeric backbone.
These modifications can be done by amide bond formation. As an
example that is not intended to limit the scope of this invention,
the carboxyl group of the polymeric backbone can be activated to
react with amino functional group of the protective chains (see
FIG. 2). The activation can be accomplished using carbodiimide
containing reagent such a
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide. A carbodiimide reagent contains a
functional group consisting of the formula N.dbd.C.dbd.N. During
the process of activation the carboxyl group forms
O-acylisourea-intermediate that can be stabilized by
N-hydroxysuccinimide to form N-hydroxysuccinimide ester. This
relatively stable intermediate can react with the amino group of
protective molecules. If the protective group or molecule that
needs to be introduced into the carrier does not have amino group,
the amino group can be introduced to this molecule very easily and
this process is well known to those skilled in the art.
[0070] Attaching Protective chains to Hydroxyl groups of Polymeric
backbone: The present invention relates to a polymeric backbone
further comprising protective chains and hydrophobic moiety. The
modification of the polymeric backbone containing hydroxyl groups
is the ester or ether bond formation with protective chains
comprising methoxypolyethyleneglycol. The modification of hydroxyl
groups of polymeric backbone is by ester bond formation with
protective groups comprising acyl methoxypolyethyleneglycol. As an
example that is not intended to limit the scope of this invention,
the protective group can be a PEG with acyl or carbonyl represented
by --CO and attached to O of hydroxyl group of carrier to form
ester. The acyl PEG with or without hydrophobic group or its
derivative can be represented by formula
--CO(CH.sub.2).sub.nNHCOCH.sub.2-A-OR.sub.3,
--COCH.sub.2CH.sub.2-A-OR.sub.3, or --COCH.sub.2-A-OR.sub.3, where
n is 2-22 (representing the hydrophobic moiety); A (representing
protective group) is [OCH.sub.2CH.sub.2].sub.x or
[OCH.sub.2CH.sub.2].sub.x or [OCHCH.sub.3CH.sub.2] where x is
17-500, or various combinations of [OCH.sub.2CH.sub.2],
[OCH.sub.2CH.sub.2], and/or [OCHCH.sub.3CH.sub.2] with total of
17-500 units, R.sub.3 is H, (CH.sub.2).sub.pCH.sub.3 or
(CH.sub.2).sub.pCOOH, and p is 0-7.
[0071] The modification of hydroxyl group can be facilitated by
synthesis of acyl halides of protective chains. Synthesis of acyl
halides can be done by reaction of the carboxylic acid moiety of
protective chains with dichlorosufoxide (SOCl.sub.2) or other
reagent known to those skilled in the art. The resulting acyl
halides are reactive to alcohols including serine, threonine, and
tyrosine residue of poly amino acids. The reaction will result in
an ester bond formation essentially attaching the protective groups
or molecules into the carrier. PEG-epoxide, PEG-isocyanate, PEG-PNC
(PEG-nitrophenylcarboxyester) are the PEG analogs that may be used
to modify the hydroxyl groups forming ether, ester, and urethane
linkage respectively between protective group and the carrier.
[0072] Attaching Hydrophobic moiety to Amino-groups of Polymeric
backbone: The present invention relates to a polymeric backbone
further comprising a protective chains and hydrophobic moiety. Once
the polymeric backbone contains protective chains, the hydrophobic
moiety can be attached by activating carboxyl groups in hydrophobic
molecule (such as fatty acids) to react with the remaining amino
groups of polymeric backbone (see FIG. 3). Another object of the
present invention is to provide a method of attaching hydrophobic
moiety to the polymeric backbone with amino groups along its
length. As an example that is not intended to limit the scope of
this invention, the modifications can also be done by amide bond
formation with fatty acid anhydride. Another method is to activate
the carboxyl containing hydrophobic molecule using a carbodiimide
containing reagent such a
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide. A carbodiimide reagent contains a
functional group consisting of the formula N.dbd.C.dbd.N. During
the process of coupling reaction, the activated carboxyl group
(O-acylisourea-intermediate) can optionally be stabilized by
forming N-hydroxysuccinimide ester using N-hydroxysuccinimide. This
relatively stable intermediate can react with the amino group of
the polymeric backbone to form amino-acyl bond or amide bond.
Alternatively, hydrophobic moieties can be introduced to amino
groups containing polymeric backbone using fatty acyl halide.
Synthesis of acyl halides can be done by reaction of the carboxylic
acid moiety with dichlorosulfoxide (SOCl.sub.2) or other reagent
known to those skilled in the art. The resulting acyl halides are
reactive to amino functional groups present in the polymeric
backbone. The reaction will result in amide bond formation
attaching the hydrophobic moieties or molecules to the polymeric
backbone.
[0073] Attaching Hydrophobic moiety to Carboxyl-groups of Polymeric
backbone: The present invention relates to a polymeric backbone
with repeating carboxyl groups derivatized to contain protective
chains and hydrophobic moieties. Once the polymeric backbone
contains protective chains (see above), the hydrophobic moiety can
be attached to the remaining carboxyl groups of the polymeric
backbone by activating the carboxyl groups to react with amino
containing hydrophobic molecules such as alkyl amine containing 6
to 36 carbon units or derivatives thereof. Alternatively, the
activated polymeric backbone can be reacted with di-alkyl
hydrophobic molecule such as phosphatidyl amine or phosphatidyl
ethanolamine. Another object of the present invention is to provide
a method of attaching hydrophobic moiety to the polymeric backbone
with carboxyl groups along its length. As an example that is not
intended to limit the scope of this invention, the modifications
can be done by amide bond formation with hydrophobic molecule. The
carboxyl containing polymeric backbone can be attached to the amino
group of the hydrophobic moiety using a carbodiimide containing
reagent such a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide. A carbodiimide reagent contains a
functional group consisting of the formula N.dbd.C.dbd.N. During
the process of coupling reaction, the activated carboxyl group
(O-acylisourea-intermediate) can optionally be stabilized by
forming N-hydroxysuccinimide ester using N-hydroxysuccinimide. This
relatively stable intermediate can react with the amino group of
the hydrophobic molecule to form amino-acyl bond or amide bond.
[0074] Attaching Hydrophobic Moieties to Hydroxyl-groups of
Polymeric Backbone: The present invention relates to a polymeric
backbone further comprising a protective chains and hydrophobic
moiety. Once the polymeric backbone contains protective chains (see
above), the hydrophobic moieties can be attached by first modifying
the remaining hydroxyl groups of the polymeric backbone into a
carboxyl containing group such as but not limited to reaction with
succinic-anhydride or other anhydride containing molecules. Once
the hydroxyl groups have been converted to carboxyl groups, the
carboxyl groups can be activated to react with hydrophobic
molecules containing amino groups such as alkyl amine containing 6
to 36 carbon units or derivatives thereof. Alternatively, the
activated polymeric backbone can be reacted with di-alkyl
hydrophobic molecule such as phosphatidyl amine or phosphatidyl
ethanolamine. Another object of the present invention is to provide
a method of attaching hydrophobic moiety to the polymeric backbone
with hydroxyl groups along its length. As an example that is not
intended to limit the scope of this invention, the modifications
can be done by ester bond formation with cyclic anhydride molecule
followed by amide bond formation with amino containing hydrophobic
molecule. After modification of hydroxyl groups to carboxyl groups,
the new carboxyl group polymeric backbone can be attached to the
amino group of the hydrophobic moiety using a carbodiimide
containing reagent such a
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide. A carbodiimide reagent contains a
functional group consisting of the formula N.dbd.C.dbd.N. During
the process of coupling reaction, the activated carboxyl group
(O-acylisourea-intermediate) can optionally be stabilized by
forming N-hydroxysuccinimide ester using N-hydroxysuccinimide. This
relatively stable intermediate can react with the amino group of
the hydrophobic moiety to form amino-acyl bond or amide bond.
[0075] Attaching Protective Group-Containing Hydrophobic Molecules
to polymeric backbone: The present invention relates to a polymeric
backbone further comprising a protective chains pendant to the
backbone; hydrophobic moiety pendant to the backbone, and second
protective chains covalently linked to the hydrophobic moieties. A
two ended hydrophobic molecule such as thapsic acid can easily be
attached to the amino group containing protective chains. Limiting
activation can be performed on thapsic acid such that most of the
molecules are singly activated. This can be done by using one half
molar equivalent (compared to thapsic acid) of a carbodiimide
containing reagent such a
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or
dicyclohexylcarbodiimide compared to thapsic acid. A carbodiimide
reagent contains a functional group consisting of the formula
N.dbd.C.dbd.N. During the process of coupling reaction, the
activated carboxyl group (O-acylisourea-intermediate) can
optionally be stabilized by forming N-hydroxysuccinimide ester
using N-hydroxysuccinimide (one molar equivalent to thapsic acid).
Once activated and stabilized, the thapsic acid will have mostly
singly activated end which can then be reacted to amino containing
protective chains. If the protective chains or molecule does not
have an amino group, the amino group can be introduced to this
molecule very easily and the chemistry is well known to those
skilled in the art. More details in the synthesis of various
embodiment of the composition can also be seen in U.S. patent
application Ser. No. 11/613,183 which hereby incorporated by
reference and to which this application claims priority.
[0076] The type of chemical link to use in attaching the
hydrophobic moiety and protective groups will depend on the desired
biological half-life of the complex and the therapeutic agent
associated with the complex. If longer half-life is desired amide
bonds will be preferred, while ester bonds will be used for carrier
that need shorter half-lives or stabilities in biological fluid or
tissue. Mixtures of both chemical bonds can be used to achieve the
desired stability for a specific therapeutic agent to be delivered.
The S--S bond may be used to achieve a desired property of the
carrier that would be beneficial for its intended therapeutic and
diagnostic purpose.
[0077] These embodiments of the present invention, other
embodiments, and their features and characteristics, will be
apparent from the description, drawings and claims that follow.
Exemplification
Synthetic Method Overview
[0078] Hydrophobic core carriers of the present invention include a
central polymeric backbone, a hydrophobic moiety, a protecting
group, and, optionally hydrophobic moiety and/or a targeting group.
Each group is linked together covalently and the hydrophobic moiety
group is capable of forming reversible binding (hydrophobic
interaction) with hydrophobic load molecule (therapeutic or
diagnostic agent) such as hydrophobic peptides/proteins,
hydrophobic drugs and derivatives thereof. The reversible linkage
between the carrier and a load molecule includes hydrophobic
interactions between the hydrophobic load molecule and the
hydrophobic moiety of the carrier.
[0079] The synthesis of hydrophobic-core carrier from a polymeric
backbone containing amino, carboxyl, hydroxyl groups, or thiol
groups generally involves three synthetic stages: 1) covalent
modification of a back bone carrier with protective chains; 2)
modification of the product from step 1 with a hydrophobic moiety;
and 3) incubating the product from step 2 with a load molecule, for
example with leptin, to achieve formation of a hydrophobic core
carrier-leptin complex without vesicle formation.
EXAMPLE 1
[0080] Synthesis of MPEG-poly-L-lysine (5000; 40,000; 27%;
40PLPEG527): The reagents, MPEG-succinimidyl-succinate and
polylysine, are commercially available and their syntheses are well
known in the art. Poly-L-lysine (200 mg; Polylysine Hydrobromide;
Sigma chemical Co.; DPvis:264; MWvis: 55,200; DPmalls:190;
MWmalls:39,800; 0.7 mmoles amino groups by TNBS assay by Sparado et
al. Anal Biochem 96:317, 1979) was dissolved in 200 ml of 0.1 M
carbonate buffer pH 8.35 and 1150 mg of MPEG-succinimidyl-succinate
(pre-activated 5 kDa PEG from NOF, Tokyo, Japan) was added,
vortexed followed by overnight incubation at room temperature. The
next day, aliquots were taken and the amount of amino groups
remaining was quantified using trinitrobenzenesulfonic acid
(Sparado et al. Anal Biochem 96:317, 1979). The result indicated
that 73% of amino group remains. The solution (200 ml) was washed
by ultra-filtration through 100 kDa cut-off membrane (UFP-100-E-3A,
GE-Amersham Biosciences Corp, Westborough, Mass.) with ten changes
of water. The resulting PLPEG complex was lyophilized and weighed
giving a yield of 860 mg. The resulting product has an estimated Mw
of 310 kDa based on the number of amino groups that had been
derivatized by MPEG. The number of free amino groups per mg of
final product is 0.43 umole/mg (FIG. 9).
EXAMPLE 2
[0081] Synthesis of MPEG-poly-L-lysine (5kDa PEG; 40 kDa PL; 55%
saturation of amino groups; 40PLPEG555): The reagents,
MPEG-succinimidyl-succinate and polylysine, are commercially
available and their syntheses are well known in the art. One gm of
40PL (Polylysine Hydrobromide; Sigma chemical Co.; DPvis: 264;
MWvis: 55,200; DPmalls: 190; MWmalls:39,800; one gram contains 3.0
mmol amino groups) was dissolved in 190 ml of 200 mM HEPES. Five
grams (1 mmol) of MPEG-succinimidyl-succinate (pre-activated 5 kDa
PEG from NOF, Tokyo, Japan) was added to 40PL solution and allowed
to react. After 2 hrs, additional 5 g of
MPEG-succinimidyl-succinate was added as above and allowed to react
over the weekend. Amino group content was measured by TNBS (Sparado
et al. Anal Biochem 96:317, 1979) and found to be 1.4 mmol total
indicating 53% saturation of amino group. The solution (200 ml) was
washed by filtration through 100 kDa cut-off membrane
(UFP-100-E-5A, GE-Amersham Biosciences Corp, Westborough, Mass.)
with ten changes of water. The resulting PLPEG complex was
lyophilized and weighed giving a yield of 8.6 g. The resulting
product has an estimated Mw of 570 kDa based on the number of amino
groups that had been derivatized by MPEG. The number of free amino
groups per mg of final product is 0.20 umole/mg (FIG. 9).
EXAMPLE 3
[0082] Synthesis of MPEG-poly-1-lysine (5 kDa PEG; 40 kDa PL; 22%
saturation of amino groups; 40PLPEG522): The reagents,
MPEG-succinimidyl-succinate and polylysine, are commercially
available and their syntheses are well known in the art.
Poly-L-lysine (200 mg; Polylysine Hydrobromide; Sigma chemical Co.;
DPvis:264; MWvis: 55,200; DPmalls:190; MWmalls:39,800; 0.7 mmoles
amino group by TNBS assay Sparado et al. Anal Biochem 96:317, 1979)
was dissolved in 10 ml of 0.2 M HEPES buffer pH 7.35 and 500 mg of
MPEG-succinimidyl-succinate (pre-activated 5 kDa PEG from NOF,
Tokyo, Japan) was added, vortexed, repeated (another 500 mg) and
incubated overnight at room temperature. The next day, aliquots
were taken and the amount of amino groups remaining was quantified
using trinitrobenzenesulfonic acid (Sparado et al. Anal Biochem
96:317, 1979). The result indicated that 22% of amino groups had
been conjugated to MPEG. The solution was washed by
ultra-filtration through 100 kDa cut-off membrane (UFP-100-E-3A,
GE-Amersham Biosciences Corp, Westborough, Mass.) with ten changes
of water. The resulting PLPEG complex was lyophilized and weighed
giving a yield of 820 mg. The resulting product has an estimated Mw
of 260 kDa based on the number of amino groups that had been
derivatized by MPEG. The number of free amino groups per mg of
final product is 0.40 umole/mg (FIG. 9).
EXAMPLE 4
[0083] Synthesis of 20PLPEG555 DA (5 kDa MPEGcarboxyl; 20 kDa PL;
55% saturation of amino groups; 20PLPEG555DA). The MPEG used in
this synthesis is carboxyl MPEG without ester bond resulting in
direct amide (DA) connection to polymeric backbone without ester
bond which is different than succinate ester MPEG above where the
linkage between the MPEG and succinate is ester bond. One gm of
20PL (P7890 Sigma lot# 065K5101; one gram contains 2.4 mmol amino
groups) was dissolved in 50 ml of 200 mM HEPES. Five grams (1 mmol)
of MPEGCarboxyl (pre-activated but lost some activation; NOF,
Tokyo, Japan) in 15 ml of 10 mM MES pH=4.7 was re-activated by
adding 250 mg of NHSS (mw=217.14; 1.15 mmol, followed by 500 mg EDC
(mw=191.71; 2.6 mmol). Activation is allowed to proceed for 20
minutes. The activated MPEGCarboxyl was added to 20PL solution and
allowed to react. After 2 hrs, additional 5 grams of MPEGCarboxyl
was activated and added as above and allowed to react over the
weekend. Amino group was measured by TNBS (Sparado et al. Anal
Biochem 96:317, 1979) and found to be 1.05 mmol total indicating
56% saturation of amino group. This was confirmed by Size Exclusion
chromatography using TosohG4000WXL with Retention time of 12.35 min
(17 nm). The solution was washed by ultra-filtration through 100
kDa cut-off membrane (UFP-100-E-5A, GE-Amersham Biosciences Corp,
Westborough, Mass.) and lyophilized (9.9 g). One gm was saturated
with FITC for determination of carrier Cmax (maximum concentration
in blood) and Tmax (Time for maximum concentration in blood) in
animals and washed with ethanol, water and lyophilized (960 mg).
The remaining 8.9 g of 20PLPEG555DA was divided into 2 (in 53 ml
DCM each) and one was saturated with activated C22 (2.5 mmol in 30
ml in 2:1 DMF:DCM) and the other was saturated with C18 (2.5 mmol
in 30 ml in 2:1 DMF:DCM) below.
EXAMPLE 5
[0084] Synthesis of 20PLPEG555DAC18 and 20PLPEG555DAC22. The
remaining 8.9 g of 20PLPEG555DA was divided into 2 (in 53 ml DCM
each) and one was saturated with activated behenic acid and the
other is saturated with activated stearic acid.
[0085] Behenic acid or C22 (0.851 gm or 2.5 mmol in 30 ml in 2:1
DMF:DCM) was activated by adding 290 mg NHS(Mw=115; 2.5 mmol) and 1
ml DCC (Mw=206; 1 ml=1.3 g=6.3 mmol) and incubating for an hour.
The urea precipitate was removed by centrifugation and the
supernatant was added to the 20PLPEG555DA solution along with 200
ul TEA and allowed to react. This was repeated twice and allowed to
react overnight. The product was rotary evaporated at 37.degree.
C., dissolved in 80% ethanol and precipitate was removed by
centrifugation. The supernatant (200 ml) was washed with 3 liter of
80% ethanol followed by 1 liter of water. This was
filter-sterilized using 200 nm filter and lyophilized giving 2.9 g.
Note that insoluble compositions will not go through this
filter-sterilization: compositions with PEG/Fatty acid weight ratio
of less than 14 (see table 2) and those that form vesicles. The
diameter of the composition is 19.8 nm with retention time of 12
minutes in gel permeation chromatography using G4000PWXL column
(0.78.times.30 cm; TSK Gel; Tosoh Biosep; Montgomeryville, Pa.)
eluted with PBS containing 15% acetonitrile at flow rate of 0.6
ml/min. Amino group content is 18 nmol/mg (very small). The
composition is water soluble at 100 mg/ml forming a yellowish true
solution without cloudiness. The composition does not form vesicles
upon addition of cholesterol.
[0086] Stearic acid or C18 (0.7 gm or 2.5 mmol in 30 ml in 2:1
DMF:DCM) was activated by adding 290 mg NHS(Mw=115; 2.5 mmol) and 1
ml DCC (Mw=206; 1 ml=1.3 g=6.3 mmol) and incubating for an hour.
The urea precipitate was removed by centrifugation and the
supernatant was added to the 20PLPEG555DA solution along with 200
ul TEA and allowed to react. This was repeated twice and allowed to
react overnight. The product was rotary evaporated at 37 C,
dissolved in 80% ethanol, precipitate was removed by
centrifugation, and supernatant was washed with 2 liter of 80%
ethanol after followed by water. This was filter sterilized using
200 nm filter and lyophilized giving 3.35 g. Note that insoluble
compositions will not go through this filter sterilization
especially those that form vesicles. Diameter is 19.4 nm with
retention time of 12.05 minutes. Amino group content is 6 nmol/mg
(very small). The composition is water soluble at 100 mg/ml forming
a yellowish true solution without cloudiness. The composition does
not form vesicles upon addition of cholesterol.
EXAMPLE 6
[0087] Reproducibility of the synthesis of 20PLPEG555C18: The
carrier 20PLPEG555C18 which is made up of 20 kDa polylysine (from
Sigma Chemical Co. with degree of polydispersity of 1.2), wherein
55 percent of the TNBS reactive amino groups was reacted with 5 kDa
MPEG-ester-succinate and the remaining 44-45% the TNBS reactive
amino groups was conjugated with stearic acids, was made. To make
this, one gm of 20PL (P7890 Sigma lot# 065K5101; 1 gm has 2.4 mmol
NH2) was dissolved in 50 ml of 200 mM HEPES. Five grams of
MPEG-succinate (1 mmol) in 15 ml of 10 mM MES pH=4.7 was activated
by adding 250 mg of NHSS (mw=217.14; 1.15 mmol, followed by 500 mg
EDC (mw=191.71; 2.6 mmol). Activation is allowed to proceed for 20
minutes. The activated MPEG-succinate was added to 20PL solution
and allowed to react. After 2 hrs, additional five grams of
MPEG-succinate was activated and added as above and allowed to
react over the weekend. Amino group was measured and found to be
1.017 mmol indicating 57% saturation of amino group. This was
confirmed by Size Exclusion chromatography using TosohG400WXL (flow
rate of 0.6 ml/min; in PBS with 15% acetonitrile) with retention
time of 12.9 min (14 nm) also showing 90% incorporation of total
PEG added. The reaction mixture containing the 20PLPEG555 was
lyophilized and dissolved in 50 ml DCM, precipitates were removed
by centrifugation, precipitates were washed with DCM, and the
pooled supernatant (total 200 ml) was saturated with activated C18
after addition of 400 ul TEA. To do this, stearic acid (C18; 0.7 gm
or 2.5 mmol in 30 ml in 1:2 DMF:DCM) was activated by adding 290 mg
NHS (Mw=115; 2.5 mmol) and 0.5 ml DCC (Mw=206; 0.5 ml=0.65 g=3.2
mmol ) and incubating for an hour. The urea precipitate was removed
by filtration and the activated fatty acid was added to the
20PLPEG555 solution and allowed to react. This was repeated twice
and allowed to react overnight, followed by rotary evaporation of
the reaction mixture, dissolution in 50% ethanol water and removal
of the precipitate and top fatty layer by centrifugation. The
precipitate was washed twice with 50% ethanol and all supernatants
were pooled together and made up to 80% ethanol to clarify the
solution before ultra-filtration. The pooled supernatant was
concentrated to 200 ml using ultra-filtration apparatus (100,000
MWCO Ultra-filtration cartridge; UFP-100-E-5A, GE-Amersham), washed
with 10 volumes of 80% ethanol followed by 10 volumes of water in
the same ultra-filtration apparatus, concentrated 150 ml and
collected. The remaining material in cartridge was washed with
2.times.50 ml water to and pooled with the sample. Sample
(20PLPEG555C18) was filtered sterilized using 0.2 um filter
(polysulfone membrane; Nalgene) and lyophilized (total 8.9 grams).
It should be noted that the insoluble materials will not go through
this filter as well as materials greater than 0.2 um. The molecular
diameter of the carrier was determined by Exclusion chromatography
using TosohG4000WXL (flow rate of 0.6 ml/min; in PBS with 15%
acetonitrile) and found to have 12 minute retention time
corresponding to 19 nm using globular protein standards. One mg/ml
of 20PLPEG555C18 was analyzed by TNBS and found contain 6+/-5 uM
NH2 or 6 nmol/mg indicating greater than 90% saturation.
[0088] The above synthesis was repeated two more times to determine
reproducibility. The resulting carrier is water soluble and has
diameter of 19 nm and retains load molecule by affinity. Acid
digestion and amino acid analysis indicated that the overall lysine
content of the composition is 4.8 to 5% by weight. The acid
digestion used is similar to the analysis done in with protein and
is know to those skilled in the art. The following table shows the
results of the triplicate synthesis of the carrier showing the
reproducibility of the synthesis.
TABLE-US-00001 TABLE 1 20PLPEG555C18 Lot # A B C Polylysine degree
of polymerization 115 115 115 (Polydispersity Mn/Mw) (1.2) (1.2)
(1.2) % PEG Sat 54% 54% 55% Retention Time Before Fatty acid 12.9
12.9 12.9 Addition (min) Size Diameter of PLPEG 14 nm 14 nm 14 nm
Carrier Retention Time After Fatty acid 12.16 12.22 12.03 Addition
(min) Carrier Diameter 19 nm 18 nm 19 nm Yield 79% 81% 81% (based
on starting PEG plus Polylysine weight as 100%) Amount 8.9 g 9.2 g
4.5 g NH2 left in Carrier 6 nmol/mg 8 nmol/mg 10 nmol/mg (started
at 3 umol/mg PL; % +/- STD) (4 +/- 2%) (5.2 +/- 2%) (6.6 +/- 2%)
GLP1 loading test (% loaded +/- STD 95.8 +/- 0.05 96.1 +/- 0.43
95.9 +/- 0.09 when GLP1 at 2% of carrier weight was loaded in 10
mg/ml Carrier) Lysine content by weight after complete 4.96% 4.87%
4.82% acid digestion
EXAMPLE 7
[0089] The following experiments were performed to determine the
solubility properties of various compositions of Polylysine/Fatty
acids/MPEG conjugates. The transition to become water soluble at 50
mg/ml started when the MPEG:fatty acid weight ratio is somewhere
between 12:1 and 14:1. All compositions in which the MPEG:fatty
acid weight ratio is at least 14:1 are water soluble. This provides
a composition that is soluble in the absence of cholesterol and
non-vesicle forming even in the presence of cholesterol when mixed
in aqueous solvent or partially aqueous solvent. The methods to
synthesize the following compositions shown in table 2 are as in
example 6. To vary the percent of MPEG in the composition the
amount of MPEG in the synthesis was varied proportionately and
added in small portion with amino group measurement in between
until the desired amino groups saturation was achieved. With the
exception of C8 and C12, the fatty acids were activated with NHS
and DCC. For C8, C12 and C16, the caprylic, lauric, and palmitic
anhydride reagents were used instead of activating the caprylic,
lauric, and palmitic acids. For each composition the solubility was
noted at 50 mg/ml and data are presented in Table 2.
TABLE-US-00002 TABLE2 Shown are the solubility properties of
various compositions of Polylysine/ Fatty acids/MPEG conjugates %
Amino groups in % Amino groups in MPEG:Fatty Water polylysine
linked to polylysine linked to acid solubility at MPEG (size in
kDa) Fatty acid (type) weight ratio 50 mg/ml 55 (5 kDa) 44(C8) 43
Soluble 35 (5 kDa) 64(C4) 31 Soluble 55 (5 kDa) 44(C16) 24 Soluble
55 (5 kDa) 44 (C18) 22 Soluble 37 (5 kDa) 62 (C8) 20 soluble 35 (5
kDa) 64(C8) 19 Soluble 55 (5 kDa) 44(C22) 18 Soluble 55 (5 kDa)
44(C24) 17 Soluble 37 (5 kDa) 62(C12) 15 Soluble 35 (5 kDa) 64(C12)
13.7 Partially soluble 26 (10 kDa) 73(C18) 12.5 Partially soluble
37 (5 kDa) 62(C16) 11.6 Partially soluble 37 (5 kDa) 62(C18) 10.5
Insoluble gel 27 (5 kDa) 72(C16) 7.3 Insoluble gel 27 (5 kDa) 72
(C18) 6.6 insoluble gel 27 (5 kDa) 72(C24) 5.0 Insoluble gel 9(5
kDa) 90(16) 1.95 Insoluble gel 9 90 (C18) 1.76 Insoluble gel
EXAMPLE 8
[0090] Synthesis of MPEG-thapsic acid: To make 5 kDa MPEG-thapsic
acid, thapsic acid (1.15 g; 4 mmol, Sigma-Aldrich, St Louis, Mo.)
was dissolved 50 mL of Dimethylformamide (DMF, Sigma-Aldrich) with
1.4 mL of triethylamine (TEA; 10 mmol, Fisher, Waltham, Mass.) and
1.65 g of N,N'-Dicyclohexylcarbodiimide (DCC, 8 mmol, Peirce,
Rockford, Ill.). The solution was stirred for 15 min and
N-hydroxysuccinimide (NHS; 920 mg; 8 mmol, Fisher) was added and
stirred for another 15 min. MPEG amine (5 grams, 1 mmol of 5 kDa,
Sunbio, South Korea) was dissolved in 45 mL of DMF by slight
heating and added slowly into the activated thapsic acid solution
over 15 minutes and stirred overnight. To determine if the reaction
was complete, a 250 uL aliquot was removed, precipitated the MPEG
with 5 mL of diethyl ether, dissolved in 1 mL of 1 N NaOH,
acidified with HCL, extracted with dichloromethane (.about.2 mL),
bottom layer collected, concentrated by a stream of air, added to
diethyl ether (5 mL) to precipitate. The precipitate was collected,
dissolved in 1 mL of dichloromethane, analyzed by thin layer
chromatography (TLC). TLC mobile phase is 5:1
dichloromethane/methanol. Solid phase is Silica Gel 60 F254 on
aluminum sheets. Visualized the following TLC stains by Bromocresol
Blue and Ninhydrin. Bromocresol blue stains blue for PEG bearing
materials, Rf of 5K Amino PEG (s.m.)=0.76 and product thapsic
acid-amino PEG conjugate (Fatty PEG, Product)=0.63. Starting
material and product both stain bromocresol blue and 5K amino PEG
stains positive for ninhydrin, the product does not (see FIG. 18).
Once reaction is complete, MPEG-thapsic was precipitated with ether
(800 mL contained in a 1 L beaker, equipped with a stir bar),
collected by vacuum filtration (Q8, filter paper, FisherBrand) and
the precipitate in the filter was washed with additional diethyl
ether (50 mL) The crude MPEG-thapsic acid was dissolved in 1N NaOH
(20 mL) to remove excess activated carboxylic acid and the pH was
restored back to acidic using HCl followed dilution with 50 ml
water and extraction of MPEG-thapsic acid with Dichlomethane (100
mL) three times. The combined dichloromethane solution was dried
with magnesium sulfate (50 g), filtered through a glass filter
funnel (medium frit), and concentrated on a rotary evaporation
under vacuum (bath temp .about.50.degree. C.). The residue was
added to a stirring solution of diethyl ether (800 mL contained in
a 1L beaker, equipped with a stir bar) and the MPEG-thapsic acid
solids were filtered through a Buchner funnel equipped with filter
paper (Q8, qualitative, Fisher brand, pre-washed with ether) under
house vacuum. The MPEG-thapsic acid product was dissolved in
ethanol (50 ml) and washed with 10 volumes of 80% ethanol by
ultra-filtration using 3K MWCU ultra-filtration cartridge
(GE-Amersham, UFP-3-E-3MA, Batch #3-1067), followed with 10 volumes
of water. The product was filter-sterilized using 0.2 um
polysulfone filter (Nalgene, Rochester, N.Y.), and lyophilized
giving 3.5 grams of materials.
EXAMPLE 9
[0091] Synthesis of 20PLC16PEGF: This carrier is made up of 20 kDa
polylysine (with degree of polydispersity of 1.2), wherein 90-99
percent of the TNBS reactive amino groups was reacted with 5 kDa
MPEG-thapsic acid (see example 8). A warm stirring polylysine
solution was prepared by dissolving 25 mg of Polylysine (Mw is 20
kDa with amine content=3.1 mmol/g as determined by TNBS assay, Cat
#P7890, DPVIS=115, batch#017k5101, Sigma) in 50 ml of 200 mM HEPES
and stirred in warmed oil bath at 40-50.degree. C. Separately,
MPEG-thapsic acid (320 mg, 0.060 mmol), N-hydroxysulfosuccinimide
sodium salt (NHSS, 26.4 mg, 0.12 mmol, Fluka) and
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC, 23.2 mg, 0.12
mmol) were placed in 8 ml of 10 mM MES. The mixture was vortexed
and incubated for 20 min. The activated MPEG-thapsic acid was added
to the polylysine solution and stirred for 60 minutes. The
activation of similar amount of MPEG-thapsic acid and addition to
polylysine solution was repeated four more times at approximately
60 minutes apart and the mixture was stirred overnight. The
reaction mixture concentrated to 50 ml and washed with 10 volumes
of 80% ethanol followed by 10 volumes of water using
ultra-filtration apparatus with 100 k MWCU ultra-filtration
cartridge (GE-Amersham, UFP-100-E-3MA). The washes product was
sterile filtered (0.2 micron, 115 mL, Nalgene) and lyophilized
giving 400-500 mg of 20PLC16PEGF. TNBS assay of final material (1
mg/mL) indicated an amine content of 7-10 nmol/mg (TNBS assay)
indicating very little amino groups left in polylysine. GPC
analysis using TosohG4000WXL eluted with PBS with 15% CAN at a flow
rate of 0.6 mL/min indicated that the retention time is 11.9 min,
indication a structure with diameter of 21 nm. This synthesis was
repeated three times determine the reproducibility of the process.
The following table shows the results of the triplicate synthesis
of the carrier showing the reproducibility of the synthesis.
TABLE-US-00003 TABLE 3 20PLC16PEGF.; Lot # A B C Polylysine Degree
of 115 115 115 polymerization Retention Time of 11.92 11.92 11.89
finished Carrier (min) Corresponding Carrier 20.6 20.6 20.6
Diameter (nm) Yield based on PL + 26% (430 mg) 26% (420 mg) 28%(450
mg) PEG-Thapsic acid % (Amount) NH2 left in Carrier 7.1 nmol/mg
10.3 nmol/mg 10.5 nmol/mg (started at 3 umol/ mg PL)
EXAMPLE 10
[0092] In order to fully characterized the type of load molecules
that can be loaded into these carriers, a table showing the
relative retention time of various molecules in reverse phase HPLC
columns which reflects their relative hydrophobicity in acidic
conditions (0.1% TFA). The results are a general guideline and
should not be taken as absolute as can be seen with VIP. Most
therapeutics has been run on reverse phase HPLC columns either
during purifications after synthesis (or biosynthesis) or for
analytical purpose. Therefore, this process of determining
retention time on HPLC column does not constitute undue
experimentation to enable the invention disclosed in the instant
application. For the purpose of demonstration, candidate load
molecules were eluted from reversed phase HPLC column (SynergiMaxRP
4.times.20 mm; Phenomenex, Torrance, Calif.) at a flow rate of 1.5
ml/min using a gradient of solvent A to B (25-50% B from 1-5
minutes) where A is water with 0.1% Trifluoroacetic acid (TFA)/5%
Acetonitrile and solvent B is Acetonitrile with 0.1% TFA. Based on
the binding studies in FIGS. 11-17, those with retention time of
greater than 2.5 minutes under the above HPLC condition will likely
demonstrate high affinity binding to the hydrophobic core carrier
(Table 4) below
TABLE-US-00004 TABLE 4 List of retention times of various load
molecules under the above chromatographic conditions and their
corresponding Kd to the 20PLPRG555C18. Retention time Dissociation
Load molecules (minutes) Constant (Kd) Doxorubicin 1.63 >100 uM
Nociceptin 1.42 >100 uM Terlipressin 1.45 >100 uM Glucagon
Like Peptide 1 (GLP-1; 3 kDa) 3.2 <500 nM* Glucagon Like Peptide
2 (GLP-2; 3 kDa) 2.46 <500 nM* Islet Amyloid Polypeptide (IAPP
or 2.36 <900 uM* Amylin; 3.6 kDa) Vasoactive Intestinal Peptide
1.64 <10 uM* Human Growth Hormone (26 kDa) 1.82 >100 uM
Leptin (16 kDa) 4.90 <800 nM* Insulin (5 kDa) 1.79 Not
determined *Those with Kd in bold binds with high affinity or has
Kd less than 100 uM.
[0093] To some extent, it is reasonable to extrapolate that it is
almost always the case that load molecules eluting with retention
time of greater than 2 minutes under this condition will have high
affinity (Kd less than 100 uM) to the carrier, making the carrier
sufficiently useful in prolonging the circulation half-lives of
these molecules. One exception to the rule of using HPLC to predict
affinity to the carrier is the vasoactive intestinal peptide. This
peptide is quite hydrophilic in acidic condition (0.1% TFA) as can
be seen from its retention time of 1.64 minutes. However in the
presence of lipid at neutral pH it is known to assume an alpha
helix conformation which is more compatible with hydrophobic lipid
environment. Its high affinity to the carrier (20PLPEG555C18) in
PBS may be due to the formation of more hydrophobic alpha helix.
Although HPLC can be performed at neutral pH to be more predictive
of the ability of the load molecule to bind to the hydrophobic core
carrier of the present invention, it is a little challenging due to
the broadening of the reverse phase HPLC peak under this condition.
It is, however, quite proper and will still allow accurate
prediction. This process is not an undue experimentation as the
reverse phase HPLC is a standard and universal practice in the art.
In addition to HPLC, assay for the binding of the carrier to the
present composition can easily be performed by those with ordinary
skill in the art as shown in example in FIGS. 11-17.
EXAMPLE 11
[0094] Binding of load molecules to 20PLPEG555C18: Polypropylene
micro-centrifuge tubes were prepared in triplicate. Aliquots (25
ul) of carrier stock (20PLPEG555C18; 100 mg/ml or 33 mg/ml) were
placed in polypropylene micro-centrifuge tubes and 25 ul of water
for the corresponding controls. Load molecules of various
concentrations were added to the tubes so that the corresponding
weight of load molecules will be between 2 to 30% of the weight of
the carrier. To each tube, 25 ul of 10.times. Phosphate buffered
saline was added and the total volume of the solution was made up
to 250 ul. The solutions were incubated for 2 hours, followed by
filtration through a 100 kDa molecular weight cut-off filter made
up of regenerated cellulose (Microcon Ultracell YM-100; Millipore,
Bedford, Mass.). The free unbound load molecules in the filtrate
were analyzed by reverse phase HPLC using the conditions described
above to generate Table 3. The total amount of load molecule loaded
or the control is represented by the filtrate of the solution
without the carrier. The amount of load molecules bound to the
carrier is represented by the difference in the amount of load
molecules between the filtrate without the carrier and the filtrate
with the carrier. Prior to this analysis, the background filter
binding was determined and accounted for whenever significant
filter binding was found. FIG. 11 to 17 were generated in this
manner.
[0095] Although the foregoing invention has been described in some
detail by way of illustration and example for the purposes of
clarity of understanding, one skilled in the art will easily
ascertain that certain changes and modifications may be practiced
without departing from the spirit and scope of the appended
claims.
Incorporation by Reference
[0096] All of the patents and publications cited herein are hereby
incorporated by reference.
Equivalents
[0097] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *