U.S. patent application number 10/176768 was filed with the patent office on 2002-11-14 for biodegradable sustained-release alginate gels.
Invention is credited to Goldenberg, Merrill Seymour, Gu, Jian Hua.
Application Number | 20020168406 10/176768 |
Document ID | / |
Family ID | 22159900 |
Filed Date | 2002-11-14 |
United States Patent
Application |
20020168406 |
Kind Code |
A1 |
Goldenberg, Merrill Seymour ;
et al. |
November 14, 2002 |
Biodegradable sustained-release alginate gels
Abstract
The present invention relates to sustained-release formulations
using biodegradable alginate delayed gels or particles and methods
thereof.
Inventors: |
Goldenberg, Merrill Seymour;
(Thousand Oaks, CA) ; Gu, Jian Hua; (Thousand
Oaks, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Family ID: |
22159900 |
Appl. No.: |
10/176768 |
Filed: |
June 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10176768 |
Jun 20, 2002 |
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09080832 |
May 18, 1998 |
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6432449 |
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Current U.S.
Class: |
424/468 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
3/10 20180101; Y10S 514/909 20130101; A61P 9/08 20180101; A61P
31/00 20180101; A61P 3/06 20180101; A61P 3/00 20180101; A61K 47/36
20130101; A61P 3/04 20180101; A61P 7/00 20180101; A61K 9/0019
20130101; Y10S 514/944 20130101; A61K 9/1652 20130101; A61P 1/16
20180101 |
Class at
Publication: |
424/468 |
International
Class: |
A61K 009/22 |
Claims
We claim:
1. A sustained-release delayed gel composition, comprising: a) a
hydrophilic polymer; b) a biologically active agent; and c) at
least one bound polyvalent metal ion, wherein said gel is
biodegradable.
2. The sustained-release composition of claim 1 wherein the bound
polyvalent metal ion is a mixture of bound and unbound polyvalent
metal ion.
3. The sustained-release delayed gel of claim 1 further comprising
excipients for stabilizing the biologically active agent or the
hydrophilic polymer.
4. The composition of claim 1 wherein the bound polyvalent metal
ion is a salt selected from the group consisting of acetates,
phosphates, lactates, tartrates, citrates, chlorides, carbonates or
hydroxides thereof.
5. The composition of claim 4 wherein the metal ion is selected
from the group consisting of manganese, strontium, iron, magnesium,
calcium, barium, copper, aluminum or zinc.
6. The composition of claim 5 wherein the metal ion is calcium.
7. The composition of claim 1 wherein the proton donor is from an
acid source.
8. The composition of claim 7 wherein the acid source is selected
from the group consisting of buffers, esters, slowly dissolving
acids or lactones.
9. The composition of claim 1 wherein the hydrophilic polymer is a
polyanion.
10. The composition of claim 1 wherein the hydrophilic polymer is a
polysaccharide.
11. The composition of claim 10 wherein the polysaccharide is an
acidic polysaccharide.
12. The composition of claim 11 wherein the polysaccharide is
alginate.
13. The composition of claim 12 wherein the alginate contains at
least 30% guluronic acid.
14. The composition of claim 12 wherein the alginate consist of at
least 0.05% by weight.
15. The composition of claim 1 wherein the biologically active
agent comprises a protein, and wherein the composition demonstrates
improved bioavailability.
16. The composition of claim 15 wherein the protein consist of at
least 0.001 mg/ml.
17. The composition of claim 15 wherein the protein is selected
from the group consisting of hematopoietic factors, colony
stimulating factors, anti-obesity factors, growth factors, trophic
factors, and antiinflammatory factors.
18. The composition of claim 15 wherein the protein is selected
from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3,
GM-CSF, IL-1ra, IL2, TNF-bp, MGDF, OPG, interferons,
erythropoietin, KGF, insulin and analogs or derivatives
thereof.
19. The composition of claim 1 wherein the biologically active
agent is a complexed biologically active agent.
20. The composition of claim 19 wherein the complexed biologically
active agent is a precipitated protein.
21. The composition of claim 20 wherein the precipitated protein is
a zinc leptin precipitate.
22. A method of producing a sustained-release delayed gel
composition, wherein said gel is biodegradable, comprising the
steps of: a) mixing a biologically active agent and a hydrophilic
polymer in a solvent to form a first mixture; b) mixing to the
first mixture at least one bound polyvalent metal ion to form a
second mixture.
23. A method of claim 22 further comprising the step of c) mixing
to the second mixture at least one proton donor capable of
releasing the bound polyvalent metal ion.
24. The method of claims 22 wherein the first mixture is
concentrated before mixing the proton donor or bound polyvalent
metal ion.
25. The method of claim 22 wherein the bound polyvalent metal ion
is a salt selected from the group consisting of acetates,
phosphates, lactates, citrates, tartrates, chlorides, carbonates or
hydroxides thereof.
26. The method of claim 22, wherein said method provides for a
substantially constant blood level of said biologically active
agent over time in the patient.
27. The composition of claim 25 wherein the metal ion is selected
from the group consisting of manganese, strontium, iron, magnesium,
calcium, barium, copper, aluminum or zinc.
28. The composition of claim 27 wherein the metal ion is
calcium.
29. The composition of claim 24 wherein the proton donor is from an
acid source.
30. The composition of claim 29 wherein the slow dissolving acid is
selected from the group consisting of buffers, esters, slowly
dissolving acids or lactones.
31. The composition of claim 30 wherein the acid source is
.delta.-gluconolactone.
32. The composition of claim 22 wherein the hydrophilic polymer is
a polyanion.
33. The composition of claim 22 wherein the hydrophilic polymer is
a polysaccharide.
34. The composition of claim 33 wherein the polysaccharide is an
acidic polysaccharide.
35. The composition of claim 34 wherein the polysaccharide is
alginate.
36. The composition of claim 35 wherein the alginate contains at
least 30% guluronic acid.
37. The composition of claim 35 wherein the alginate consist of at
least 0.05% by weight.
38. The composition of claim 22 wherein the biologically active
agent comprises a protein.
39. The composition of claim 38 wherein the protein consist of at
least 0.001 mg/ml.
40. The composition of claim 38 wherein the protein is selected
from the group consisting of hematopoetic factors, colony
stimulating factors, anti-obesity factors, growth factors, trophic
factors, and antiinflammatory factors.
41. The composition of claim 38 wherein the protein is selected
from the group consisting of leptin, G-CSF, SCF, BDNF, GDNF, NT3,
GM-CSF, IL-1ra, IL2, TNF-bp, MGDF, OPG, interferons,
erythropoietin, KGF and analogs or derivatives thereof.
42. The composition of claim 22 wherein the biologically active
agent is a complexed biologically active agent.
43. The composition of claim 42 wherein the complexed biologically
active agent is a precipitated protein.
44. The composition of claim 43 wherein the precipitated protein is
a zinc leptin precipitate.
45. The method of claim 22 further comprising the step of isolating
the sustained-release composition.
46. The sustained-release product produced by the method of claims
22 or 45.
47. A pharmaceutical formulation comprising the sustained-release
composition according to claims 1, 2, 3 or 46 in a pharmaceutically
acceptable carrier, diluent or adjuvant.
48. The pharmaceutical formulation of claim 47, wherein the
formulation is in a syringe.
49. A method of treating an indication with a sustained-release
composition according to claims 1, 2, 3 or 46 in a pharmaceutically
acceptable carrier, diluent or adjuvant.
50. A method of treatment of a disorder selected from the group
consisting of excess weight, diabetes, high blood lipid level,
artherial sclerosis, artherial plaque, the reduction or prevention
of gall stones formation, insufficient lean tissue mass,
insufficient sensitivity to insulin, and stroke, with a
sustained-release composition according to claims 1, 2, 3, or 46 in
a pharmaceutically acceptable carrier, diluent, or adjuvant wherein
the biologically active agent is leptin, an analog or derivative
thereof
51. A method of treating a disorder selected from the group
consisting of hematopoietic cell deficiencies, infection, and
neutropenia with a sustained-release composition according to
claims 1, 2, 3, or 46 in a pharmaceutically acceptable carrier,
diluent, or adjuvant wherein the biologically active agent is
G-CSF, an analog or derivative thereof.
52. A method of treating inflammation with a sustained-release
composition according to claims 1, 2, 3, or 46 in a
pharmaceutically acceptable carrier, diluent, or adjuvant, wherein
the biologically active agent is an IL-1ra, an analog or derivative
thereof.
53. A sustained-release composition, comprising: a) a hydrophilic
polymer; b) a biologically active agent; and c) at least one
precipitating agent; characterized in that the biologically active
agent is co-precipitated within the hydrophilic polymer, wherein
said composition is in the form of a gel particle, and wherein said
particle is biodegradable.
54. The composition of claim 53 wherein the precipitating agent is
selected from the group consisting of polyvalent metal ions or
salts, acetates, citrates, chlorides, carbonates or hydroxides
thereof.
55. The composition of claim 54 wherein the metal ion is selected
from the group consisting of manganese, strontium, iron, magnesium,
calcium, barium, aluminium or zinc.
56. The composition of claim 55 wherein the precipitating agent is
a polyvalent ion selected from the group consisting of zinc,
calcium or a combination thereof.
57. The composition of claim 53 wherein the hydrophilic polymer is
a polysaccharide.
58. The composition of claim 57 wherein the polysaccaharide is
alginate.
59. A method of producing a sustained-release composition,
comprising the steps of: a) dissolving a biologically active agent
and a hydrophilic polymer with a solvent to form a first mixture;
b) dissolving at least one precipitating agent in a solvent to form
a second mixture; c) adding the first mixture with the second
mixture; and d) co-precipitating the biologically active agent with
the hydrophilic polymer to form a co-precipitated gel particle,
wherein said particle is biodegradable.
60. The method of claim 59 further comprising the step of isolating
the co-precipitated particle.
61. The sustained-release product produced by the method of claim
60.
62. A pharmaceutical formulation according to claims 53 in a
pharmaceutically acceptable carrier, diluent or adjuvant.
63. A method of treating an indication with a sustained-release
composition according to claim 53 in a pharmaceutically acceptable
carrier, diluent or adjuvant.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to sustained-release
formulations using biodegradable alginate gel beads and/or delayed
gels and methods thereof.
BACKGROUND OF THE INVENTION
[0002] With the advances in genetic and cell engineering
technologies, the availability of recombinant proteins has
engendered advances in the use of proteins as medicaments for
therapeutic applications. Many illnesses or conditions treated with
pharmaceutical proteins require sustained protein levels to achieve
the most effective therapeutic result. However, as with most
protein pharmaceuticals, the generally short biological half-life
requires frequent administration. These repeated injections are
given at various intervals which result in fluctuating medication
levels at a significant physical and monetary burden on the
patients. Since many conditions respond better to controlled levels
of a pharmaceutical, a need exists for controlled release of a
medicament to provide longer periods of consistent release. Such
sustained-release medicaments would provide the patient with not
only enhanced prophylactic, therapeutic or diagnostic effects, but
also a decrease in the frequency of injections as well as in
overall costs.
[0003] Current attempts to sustain medication levels in humans or
animals between doses have included the use of biodegradable
polymers as matrices to control medicament release. For example,
Great Britain Patent No. 1,388,580 discloses the use of hydrogels
for sustained-release of insulin. U.S. Pat. No. 4,789,550 discloses
the use of polylysine coated alginate microcapsules for delivery of
protein by encapsulating living cells. Sustained-release attempts
have also utilized anionic or cationic polymer compositions
surrounded by ionic polymers of the opposite charge for
encapsulating cells capable of producing biologically active
compositions; U.S. Pat. No. 4,744,933. Likewise, multiple coats of
anionic or cationic cross-linking polymers have also been disclosed
as means for obtaining controlled release; U.S. Pat. Nos. 4,690,682
and 4,789,516. In addition, further attempts disclose the use of
alginates alone, or alginates coated with other biodegradable
polymers, for controlled release of polypeptide compositions or
cation precipitates thereof; PCT WO 96/00081, PCT WO 95/29664 and
PCT WO 96/03116.
[0004] These attempts, however, have provided insufficient means
for obtaining sustained-release delivery of desired protein
pharmaceuticals. It is generally known that the use of certain
biodegradable polymers, e.g., polylactide co-glycolide, under in
vivo conditions, exhibit high initial bursts of medicament release;
Johnson, O. et al., Nature Med., 2(7): 795 (1996). Furthermore, it
is generally known that proteins used with current forms of
sustained-release preparations can undergo denaturation and lose
their bioactivity upon exposure to the encapsulating agents. Such
preparations use organic solvents which can have deleterious
effects on the protein of choice. Finally, as discussed below, use
of alginate alone has not provided the desired controlled protein
release necessary for effective therapeutic results.
[0005] In general, alginates are well known, naturally occurring,
anionic, polysaccharides comprised of
1,4-linked-.beta.-D-mannuronic acid and .alpha.-L-guluronic acid;
Smidsrod, O. et al., Trends in Biotechnol., 8: 71-78 (1990);
Aslani, P. et al., J. Microencapsulation, 13(5): 601-614 (1996).
Alginates typically vary from 70% mannuronic acid and 30% guluronic
acid, to 30% mannuronic acid and 70% guluronic acid; Smidsrod,
supra. Alginic acid is water insoluble whereas salts formed with
monovalent ions like sodium, potassium and ammonium are water
soluble; McDowell, R. H., "Properties of Alginates" (London,
Alginate Industries Ltd, 4th edition 1977). Polyvalent cations are
known to react with alginates and to spontaneously form gels.
[0006] Alginates have a wide variety of applications such as food
additives, adhesives, pharmaceutical tablets and wound dressings.
Alginates have also been recommended for protein separation
techniques. For example, Gray, C. J. et al., in Biotechnology and
Bioengineering, 31: 607-612 (1988) entrapped insulin in
zinc/calcium alginate gels for separation of insulin from other
serum proteins.
[0007] Alginate matrices have also been well documented for drug
delivery systems; see for example, U.S. Pat. No. 4,695,463 which
discloses an alginate based chewing gum delivery system and
pharmaceutical preparations. Alginate beads have been used for
controlled release of various proteins such as: tumor necrosis
factor receptor in cation-alginate beads coated with polycations;
Wee, S. F, Proceed. Intern. Symp. Control. Rel. Bioact. Mater., 21:
730-31 (1994); transforming growth factor encapsulated in alginate
beads; Puolakkainen, P. A. et al., Gastroenterology, 107: 1319-1326
(1994); angiogenic factors entrapped in calcium-alginate beads;
Downs, E. C. et al., J. of Cellular Physiology, 152: 422-429
(1992); albumin entrapped in chitosan-alginate microcapsules; Polk,
A. et al., J. Pharmaceutical Sciences, 83(2): 178-185 (1994);
chitosan-calcium alginate beads coated with polymers; Okhamafe, A.
O. et al., J. Microencapsul., 13(5): 497-508 (1996); hemoglobulin
encapsulated with chitosan-calcium alginate beads; Huguet, M. L. et
al., J. Applied Polymer Science, 51: 1427-1432 (1994), Huguet, M.
L. et al., Process Biochemistry, 31: 745-751 (1996); and
interleukin-2 encapsulated in alginate-chitosan microspheres; Liu,
L. S. et al., Proceed. Intern. Symp. Control. Rel. Bioact. Mater,
22: 542-543 (1995).
[0008] Systems using alginate gel beads, or alginate/calcium gel
beads, to entrap proteins suffer from lack of any sustained-release
effect due to rapid release of the protein from the alginate beads;
Liu, L. et al., J. Control. Rel., 43: 65-74 (1997). To avoid such
rapid release, a number of the above systems attempt to use
polycation polymer coatings (e.g., polylysine, chitosan) to retard
the release of the protein alginate beads; See, e.g., Wheatley, M.
A. et al., J. Applied Polymer Science, 43: 2123-2135 (1991); Wee,
S. F. et al. supra; Liu, L. S. et al. supra; Wee, S. F. et al.,
Controlled Release Society, 22: 566-567 (1995) and Lim, et al.
supra.
[0009] Polycations, such as polylysine, are positively charged
polyelectrolytes which interact with the negatively charged
alginate molecules to form a polyelectrolyte complexes that act as
diffusion barriers on the bead surface. Problems can occur with the
use of polycations in that: (1) such formulations maybe cytotoxic
due to the polycations; Huguet, M. L. et al., supra; Zimmermann,
Ulrich, Electrophoresis, 13: 269 (1992); Bergmann, P. et al.,
Clincial Science, 67: 35 (1984); (2) polycations are prone to
oxidation; (3) beads with polycation coatings tend not to be
erodible and build up in the body; (4) such formulations are made
via laborious coating procedures which include multiple coatings of
the polycation polylysine; Padol, et al., Proceed. Intern. Symp.
Control. Rel. Bioact. Mater, 2: 216 (1986) and (5) ionic
interactions between the protein and the polycations can result in
loss of protein activity or cause protein instability.
[0010] Francesco et al., U.S. Pat. No. 5,336,668 (and references
cited therein) describe total and partial esters of alginic acid,
made by different processes, and possessing interesting
pharmaceutical qualities. It is described how the alginic esters
could be utilized as biodegradable plastic materials for
medical-surgical use; as additives for a wide range of polymeric
materials; or used in the preparation of various medicaments.
Potential use the esterified alginates in sustained release
formulations is not discussed nor are esterified alginate hydrogels
described.
[0011] Nightlinger et al., Proceed. Inter. Symp. Control. Rel.
Bioact. Mater., 22: 738-739 (1995) describe esterified hyaluronic
acid (HA) microspheres having controlled release capabilities. The
references generally addresses the different degradation rates for
their HA derivatives and describe how the ester "breaks off" to
liberate the alcohol and HA moieties. There is no discussion
relating how or whether the HA backbone itself breaks down into
lower molecular weight polymer units.
[0012] In order for a polysaccharide based sustained delivery
system to be useful, the polysaccharide must be biodegradable into
non-toxic products. It has been found that certain alginate gel
systems, while effective in providing sustained release of drug,
results in a "bump" (or nodule) at the site of injection due to the
very slow dissipation of the gel. In a therapeutic setting
involving low doses of drug and infrequent injections, this might
not be a major problem. However, in a therapeutic setting involving
high doses of drug and more frequent injections, this effect could
be prohibitive. A means for increasing the dissipation rate of the
alginate gel from the injection site must be developed.
[0013] There is thus still a need to develop pharmaceutical
formulations which achieve a more versatile and effective means of
sustained-release for clinical applications. Numerous recombinant
or natural proteins could benefit from constant long term release
and thereby provide more effective clinical results.
[0014] The present invention provides such advances. Pharmaceutical
compositions using the biodegradable alginate gel particles or gels
of the present invention are capable of providing increased
bioavailability, protein protection, decreased degradation and slow
release with increased protein stability and potency. Also,
pharmaceutical compositions of the present invention provide a
simple, rapid and inexpensive means of controlled recombinant
protein release for effective prophylactic, therapeutic or
diagnostic results.
SUMMARY OF THE INVENTION
[0015] The present invention grew out of studies using unmodified
alginate (a class of anionic polysaccharides) hydrogels for the
sustained release of proteins. These protein-containing unmodified
alginate hydrogels (see copending U.S. applications Ser. No.
08/857,913 and 08/912,902) are formed in a time-delayed manner
whereby the materials can be filled in a syringe and left to later
gel in the same syringe; these gels are found to be injectable.
After a single subcutaneous injection in rodent models evidence of
many days of sustained protein is observed; however, a perceptible
bump or nodule remains at the injection site for long periods of
time with little change in its size. This bump consists of the
water-filled alginate hydrogel and the size of the bump is a
function of the volume of gel that is injected. Gel beads also
remain at the injection site.
[0016] The present invention thus relates to a novel class of
biodegradable biocompatible polysaccharide hydrogels, e.g.,
alginate ester hydrogels, for the sustained release of therapeutic
proteins. Unexpectedly, the alginate ester hydrogels, in addition
to having the gelation, injectability and sustained release
properties of the unmodified alginates, did not leave a bump at the
injection site--that is, the alginate ester hydrogels are
biodegradable or erodible and are gradually resorbed into the
surrounding tissues with little injection site reaction.
[0017] The compositions of the present invention comprise alginate
esters or their derivatives ionically crosslinked in a hydrogel
(water-containing) matrix containing a therapeutic agent such as a
protein.
[0018] The present invention further relates to a method of
producing biodegradable sustained release compositions.
[0019] The present invention further relates to the use of the
ester alginate materials in liquid mixtures for time delay gelation
in the body.
[0020] The present invention further relates to compositions
wherein the alginate ester hydrogels are in the form of beads or
microspheres for the sustained release of active agents preferably
therapeutic proteins.
[0021] In one embodiment of the present invention the alginate
ester hydrogels provide compositions for application at target
sites in the body of a patient. These compositions are useful: for
preventing or inhibiting the formation of tissue adhesions
following surgery and traumatic injury; for supplementing tissues
especially for filling soft and hard tissues; to fill a confined
space with a resorbable material; as a scaffold for tissue growth;
and as a wound dressing.
[0022] In another embodiment the alginate ester hydrogels provide
active agent containing devices for implantation in the body
whereby the agent can be either bound or unbound to the alginate
polymer.
[0023] In another embodiment the alginate ester hydrogel
compositions of the present invention provide a method for
improving the bioavailability of the active agent in the
composition.
[0024] Finally, the alginate ester hydrogel compositions of the
present invention further provide a method for obtaining a
substantially constant blood level over time in the patient.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Compositions
[0026] Hydrophilic polymers including alginates and derivatives
thereof, can be obtained from various commercial, natural or
synthetic sources well known in the art. As used herein, the term
hydrophilic polymer refers to water soluble polymers or polymers
having affinity for absorbing water. Hydrophilic polymers are well
known to one skilled in the art. These include but are not limited
to polyanions, including anionic polysaccharides such as alginate,
carboxymethyl amylose, polyacrylic acid salts, polymethacrylic acid
salts, ethylene maleic anhydride copolymer (half ester),
carboxymethyl cellulose, dextran sulfate, heparin, carboxymethyl
dextran, carboxy cellulose, 2,3-dicarboxycellulose,
tricarboxycellulose, carboxy gum arabic, carboxy carrageenan,
pectin, carboxy pectin, carboxy tragacanth gum, carboxy xanthan
gum, pentosan polysulfate, carboxy starch, carboxymethyl
chitin/chitosan, curdlan, inositol hexasulfate, b-cyclodextrin
sulfate, hyaluronic acid, chondroitin-6-sulfate, dermatan sulfate,
heparin sulfate, carboxymethyl starch, carrageenan,
polygalacturonate, carboxy guar gum, polyphosphate,
polyaldehydo-carbonic acid, poly-1-hydroxy-1-sulfonate-propen-2,
copolystyrene maleic acid, agarose, imesoglycan, sulfopropylated
polyvinyl alcohols, cellulose sulfate, protamine sulfate, phospho
guar gum, polyglutamic acid, polyaspartic acid, polyamino acids,
derivatives or combinations thereof. One skilled in the art will
appreciate other various hydrophilic polymers that are within the
scope of the present invention.
[0027] Likewise, bound polyvalent metal ions can be obtained from
various commercial, natural or synthetic sources which are well
known in the art. In particular, the metal ions can include but are
not limited to aluminum, barium, calcium, iron, manganese
magnesium, strontium and zinc. Preferably the metal ions are
calcium and zinc or the salts thereof, like zinc acetate, calcium
acetate or chloride salts. Water soluble small molecules and salts
can also be used such as ammonium sulfate, acetone, ethanol and
glycerol.
[0028] Alcohols of the aliphatic series for use as esterifying
components of the carboxy groups of alginic acid according to the
present invention are, for example, those with a maximum of 34
carbon atoms, which may be saturated or unsaturated and which may
possibly also be substituted by other free or functionally modified
groups, such as amino, hydroxy, aldehydo, keto, mercapto, carboxy
groups or by groups deriving from the same, such as hydrocarbyl or
dihydrocarbylamino (hereafter the term "hydrocarbyl" should be
taken to mean not only monovalent radicals of hydrocarbons such as
the C.sub.nH.sub.2n+1 type but also bivalent or trivalent radicals,
such as "alkylenes" --C.sub.nH.sub.2- or
"alkylidenes"=C.sub.nH.sub.2n), ether or ester groups, acetal or
ketal groups, thio-ether or thioester groups and esterified carboxy
groups or carbamidic or carbamidic groups substituted by one or two
hydroxy groups, by nitrile groups or by halogens.
[0029] In the above groups containing hydrocarbyl radicals these
are preferably lower aliphatic radicals, such as heteroatoms, such
as oxygen, nitrogen and sulfur. Preference is given to alcohols
substituted with one or two of the aforesaid function groups.
[0030] Alcohols of the above group to be used preferentially within
the terms of the present invention are those with a maximum of 12
and especially with a maximum of 6 carbon atoms and in which the
hydrocarbyl radicals in the above mentioned amino, ether, ester,
thioether, thioester, acetal, ketal groups representing alkyl
groups with a maximum of 4 carbon atoms, and also in the esterified
carboxy or substituted carbamidic groups the hydrocarbyl groups are
alkyls with the same number of carbon atoms, and in which the amino
or carbamidic groups may be alkyleneamino or alkylenecarbamidic
groups with a maximum of 8 carbon atoms. Of these alcohols those to
be mentioned first and foremost are the saturated and unsubstituted
ones such as methyl, ethyl, propyl, isopropyl alcohols, n-butyl
alcohol, isobutyl, tertbutyl alcohols, amyl, pentyl, hexyl, octyl,
nonyl, and dodecyl alcohols and above all those with a linear chain
such as n-octyl or n-dodecyl alcohols. Of the substituted alcohols
of this group the bivalent alcohols should be listed, such as
ethyleneglycol, propylene glycol or butylene glycol, the trivalent
alcohols such as glycerin, aldehydo alcohols such as tartronic
alcohol, carboxy alcohols such as lactic acids, for example
alpha-oxypropionic acid, glycolic acid, malic acid, tartaric acids,
citric acid, aminoalcohols, such as aminoethanol, aminopropanol,
n-aminobutanol and their dimethyl and diethyl derivatives in the
aminic function, choline, pyrrolidinylethanol, piperidinylethanol,
piperazinylethanol and the corresponding derivatives of n-propyl or
n-butyl alcohols, monothioethyleneglycol or its alkyl derivatives,
for example the ethylderivative in the mercapto function.
[0031] Of the higher saturated aliphatic alcohols, those worthy of
special mention are for example cetyl alcohol and myristyl alcohol,
but especially important for the purposes of the present invention
are the higher unsaturated alcohols with one or two double bonds,
such as especially those contained in many essential oils and
having an affinity with terpenes such as citronellol, geraniol,
nerol, nerolidol, linalool, farnesol, phytol.
[0032] Of the lower unsaturated alcohols consideration should be
given to propargyl alcohol.
[0033] Of the aliphatic alcohols those to be mentioned above all
are those with only one benzene residue and in which the aliphatic
chain has a maximum of 4 carbon atoms, in which also the benzene
residue may be substituted by between 1 and 3 methyl or hydroxy
groups or by halogen atoms, especially chlorine, bromine or iodine
and in which the aliphatic chain may be substituted by one or more
functional groups selected from the group consisting of free amino
groups or mono- or dimethyl groups or by pyrrolidine or piperidine
groups. Of these alcohols, benzyl alcohol and phenethyl alcohol are
especially preferred. The alcohols of the cycloaliphatic or
aliphatic cycloaliphatic series may derive from mono or polycyclic
hydrocarbons and may have a maximum of 34 carbon atoms. Of the
alcohols derived from cyclic monoanular hydrocarbons special
mention should be given to those with a maximum of 12 carbon atoms,
with rings containing preferably between 5 and 7 carbon atoms,
possibly substituted for example by between one and three lower
alkyl groups, such as methyl, ethyl, propyl or isopropyl groups.
Specific alcohols of this group are cyclohexanol, cyclohexanediol,
1,2,3-cyclohexanetriol and 1,3,5-cyclohexanetriol (phloroglucitol),
inositol, the alcohols deriving from p-menthane such as
carvomenthol menthol, alpha and gamma-terpineol 1-terpineol
alcohols known as "terpineol", 1,4-and 1,8-terpin. Alcohols
deriving from hydrocarbons with condensed rings are, for example,
those of the thujane, pinane, campbane groups, particularly
thujanol, sabinol pinol hydrate, D and L-borneol and D and
L-isoborneol.
[0034] Also to be included are alcohols derived from the
esterification reaction of epoxy-containing compounds with
alginates (See e.g., U.S. Pat. No. 2,463,824 and U.S. Pat. No.
2,426,125).
[0035] The total and partial ester group containing polyanions of
the present invention are generally acidic polysaccharides where
the glycosidic oxygen is attached beta to the carbonyl carbon of
the ester. While not being bound to any specific mechanism, this
arrangement of the moieties allows the breakdown of the polymer
chain by a beta-elimination mechanism which can occur under
physiological conditions.
[0036] Alginic acid esters of the present invention are comprised
of mannuronic acid residues (m-COOH or m-COO anion) and guluronic
acid residues (g-COOH or g-COO anion) joined together by glycosidic
ether oxygen linkages of the following general formula I:
-(M)n1-(M')n2-(G)n3-(G')n4-(A)n5- I
[0037] where:
[0038] M is a mannuronic acid residue, m-COOH or m-COO anion;
[0039] M' is a mannuronic acid ester residue, m-COOR1;
[0040] G is a guluronic acid residue, g-COOH or g-COO anion;
[0041] G' is a guluronic acid ester residue, g-COOR2;
[0042] A represents non-g or non-m chain units, such as sugars,
sugar oxidation products, or aliphatic, aromatic, araliphatic,
alaromatic, cycloaliphatic radicals which can be substituted and
interrupted by heteroatoms linked within or at the chain ends;
[0043] n1, n2, n3, n4 and n5 are integers representing the average
relative number of incorporated units;
[0044] R1 and R2 are independently aliphatic, aromatic,
araliphatic, alaromatic, cycloaliphatic radicals which can be
substituted and interrupted by heteroatoms;
[0045] and derivatives (e.g. where the hydroxy groups are
acetylated and are reacted with isocyanates) and pharmaceutically
acceptable salts thereof.
[0046] In the esters of the present invention, it is desirable that
R1=R2=aliphatic or alaromatic and further that
100(n2+n4)/(n1+n2+n3+n4) is from 1-99 mole, preferably 5-50 mole,
more preferably 6-30 mole, still more preferably 6-15 mole and most
preferably 7-12 mol % and 100 n5/(n1+n2+n3+n4+n5) is preferably
less than 10 mol %.
[0047] In the partial esters of the invention the non-esterified
carboxy groups may be kept free or may be salified. The bases for
the formation of these salts are chosen according to the ultimate
end use of the product. Inorganic salts maybe formed from alkaline
metals, such as potassium and in particular sodium and ammonium, or
deriving from alkaline earth metals such as calcium or magnesium or
aluminum salts. of particular interest are the salts with organic
bases, especially azotized bases and, therefore, aliphatic,
araliphatic, cycloaliphatic or heterocyclic amines. These ammonium
salts may derive from therapeutically acceptable amines or nontoxic
but therapeutically inactive amines, or from amines with a
therapeutic action. Of the first type, preferred are aliphatic
amines, for example mono-, di- and tri-alkylamines with alkyl
groups with a maximum of 8 carbon atoms or arylalkylamines with the
same number of carbon atoms in the aliphaic part and where aryl
means a benzene group possibly substituted by between 1 and 3
methyl groups or halogen atoms or hydroxy groups. The biologically
inactive bases for the formation of the salts may also be cyclic,
such as monocyclic alkyleneamines with cycles of between 4 and 6
carbon atoms, possibly interrupted in their cycle by heteroatoms
chosen from the group formed by nitrogen, oxygen and sulphur, such
as piperazine or morpholine, or may be substituted, for example by
amino or hydroxy functions such as aminoethanol, ethylenediamol,
ethylenediamine, ephedrine or choline.
[0048] The degree of and type of esterification can be controlled
by synthetic methods known in the art. Preferably, the alginate
esters are prepared by treatment of the quaternary ammonium salts
of alginic acid with conventional alkylating agents in an aprotic
organic solvent such as dimethyl sulfoxide. The resultant esters
are preferably the esters of monovalent alcohols such as lower
alkyl such as ethyl or aralkyl such as benzyl or their mixtures.
One can also form esters by the reaction of alginic acid with
oxirane or epoxy containing compounds such as ethylene or propylene
oxide.
[0049] It is also possible to form quaternary ammonium salts of
partial esters, for example the salts of tetraalkylammonium with
the above said number of carbonatoms and-preferably salts of this
type in which the fourth alkyl group has between 1 and 4 carbon
atoms, for example a methyl group.
[0050] The degree of esterification (expressed in mol %) of the
alginate is related to the desired disappearance rate of the gel in
the patient tissue. This disappearance rate of the gel is generally
related to the desired release rate of the active agent from the
gel that is over a period of 5 years or less, usually 2 days to 270
days, more usually 2 days to 180 days, more usually 2 days to 90
days. The degree of esterification (DE) is from 1 mole to 99 mole,
preferably from 5 molt to 50 mol %, more preferably from 6 mol % to
30 mol %, more preferably from 6 mol % to 15 mol %, more preferably
from 7 mol % to 12 mol %.
[0051] As used herein, the term buffer or buffer solution refers to
use of inorganic or organic acids or a combination thereof to
prepare a buffer solution as known in the art. Inorganic acids
within the scope of the present invention include hydrogen halide
(e.g., hydrochloric acid), phosphoric, nitric or sulfuric. Other
inorganic acids would be well known to one skilled in the art and
are contemplated herein. Organic acids within the scope of the
invention include aliphatic carboxylic acids and aromatic acids
such as formic, carbonic, acetic, propionic, butyric, valeric,
caproic, acrylic, malonic, succinic, glutaric, adipic, maleic,
fumaric, glycine or phenol sulfonic. Other organic acids would be
well known to one skilled in the art.
[0052] As used herein, biologically active agents refers to
recombinant or naturally occurring proteins, whether human or
animal, useful for prophylactic, therapeutic or diagnostic
application, as well as non-protein based agents such as small
molecules. The biologically active agent can be natural, synthetic,
semi-synthetic or derivatives thereof. The biologically active
agents of the present invention must be precipitable. A wide range
of biologically active agents are contemplated. These include but
are not limited to hormones, cytokines, hematopoietic factors,
growth factors, antiobesity factors, trophic factors,
anti-inflammatcry factors, and enzymes (see also U.S. Pat. No.
4,635,463 for additional examples of useful biologically active
agents). One skilled in the art will readily be able to adapt a
desired biologically active agent to the compositions of present
invention.
[0053] Such proteins would include but are not limited to
interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293 4,897,471,
and 4,695,623 hereby incorporated by reference including drawings),
interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by
reference including drawings), erythropoietins (see, U.S. Pat. Nos.
4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby
incorporated by reference including drawings), granulocyte-colony
stimulating factors (see, U.S. Pat. Nos. 4,810,643, 4,999,291,
5,581,476, 5,582,823, and PCT Publication No. 94/17185, hereby
incorporated by reference including drawings), stem cell factor
(PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby
incorporated by reference including drawings), and the OB protein
(see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010
and 97/06816 hereby incorporated by reference including figures).
In addition, biologically active agents can also include but are
not limited to anti-obesity related products, insulin, gastrin,
prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating
hormone (TSH), luteinizing hormone (LH), follicle stimulating
hormone (FSH), human chorionic gonadotropin (HCG), motilin,
interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12),
tumor necrosis factor (TNF), tumor necrosis factor-binding protein
(TNF-bp), brain derived neurotrophic factor (BDNF), glial derived
neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast
growth factors (FGF), neurotrophic growth factor (NGF), bone growth
factors such as osteoprotegerin (OPG), insulin-like growth factors
(IGFs), macrophage colony stimulating factor (M-CSF), granulocyte
macrophage colony stimulating factor (GM-CSF), megakeratinocyte
derived growth factor (MGDF), thrombopoietin, platelet-derived
growth factor (PGDF), colony simulating growth factors (CSFs), bone
morphogenetic protein (BMP), superoxide dismutase (SOD), tissue
plasminogen activator (TPA), urokinase, streptokinase and
kallikrein. The term proteins, as used herein, includes peptides,
polypeptides, consensus molecules, analogs, derivatives or
combinations thereof.
[0054] Derivatives of biologically active agents may include the
attachment of one or more chemical moieties to the protein moiety.
Chemical modification of biologically active agents has been found
to provide additional advantages under certain circumstances, such
as increasing the stability and circulation time of the therapeutic
protein and decreasing immunogenicity. One skilled in the art will
be able to select the desired chemical modification based on the
desired dosage, circulation time, resistance to proteolysis,
therapeutic uses and other considerations.
[0055] As used herein, biodegradability refers to the breakdown of
the molecular weight of a particular polymer into a smaller number
of units in the chain, i.e., breakdown into lower molecular weight
units. Biodegradble gel refers to the dissipation of the gel in the
environment of use, where the idssipation is contingent on the
breakdown of the molecular weight of the constituent polymers,
resulting in fewer units in the polymer chain.
[0056] Complexes
[0057] The proteins, analog or derivative may be administered
complexed to a binding composition. Such binding composition may
have the effect of prolonging the circulation time of the protein,
analog or derivative or enhancing the activity of the biologically
active agent. Such composition may be a protein (or synonymously,
peptide), derivative, analog or combination. For example, a binding
protein for the OB protein is OB protein receptor or portion
thereof, such as a soluble portion thereof. Other binding proteins
may be ascertained by examining OB protein, or the protein of
choice, in serum, or be empirically screening for the presence of
binding. Such binding will typically not interfere with the ability
of OB protein or analog or derivative to bind to endogenous OB
protein receptor and/or effect signal transduction. In addition to
the OB protein, binding complexes will also be applicable to other
therapeutic proteins of the present invention as well. Those well
skilled in the art will be able to ascertain appropriate binding
proteins for use with the present invention.
[0058] Likewise, precipitating agents used to precipitate the
biologically active agent can be obtained from various commercial,
natural or synthetic sources which are well known in the art.
Precipitating agents include but are not limited to polyvalent
metal ions or their salts such as acetates, citrates, chlorides,
carbonates, hydroxides, oxalates, tartrates or hydroxides thereof,
acids or water soluble polymers. In particular, the metal ions can
include but are not limited to aluminum, barium, calcium, iron,
manganese magnesium, strontium and zinc. Preferably the metal ion
is zinc or the salts thereof, like acetate chloride salts. Water
soluble small molecules and salts can also be used such as ammonium
sulfate, acetone, ethanol and glycerol.
[0059] As for water soluble polymers these include but are not
limited to polyethylene glycol, ethylene glycol/propylene glycol
copolymers, carboxylmethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymers, polyaminoacids, dextran, poly
(n-vinyl pyrolidone) polyethylene glycol, propylene glycol
homopolymers, polypropylene oxide/ethylene oxide copolymers,
polyoxyethylated polyols, polyvinyl alcohol succinate, glycerine,
ethylene oxides, propylene oxides, poloxamers, alkoxylated
copolymers, water soluble polyanions, derivatives or combinations
thereof. The water soluble polymer may be of any molecular weight,
and may be branched or unbranched. For example, the preferred
molecular weight of polyethylene glycol is between about 700 Da and
about 100 kDa for ease in handling and efficiency of
precipitation.
[0060] Other sizes and types of precipitating agents, may be used,
depending on the desired therapeutic profile (e.g., the duration of
sustained-release desired, the effects, if any on biological
activity, the ease in handling, the degree or lack of antigenicity
and other known effects of a desired precipitating agent to a
therapeutic protein or analog). One skilled in the art will
appreciate other precipitating agents that are within the scope of
the invention.
[0061] In addition, the compositions of the present invention-may
also include extra excipients necessary to stabilize the
biologically active agent and/or the hydrophilic polymer. These can
be contained in the buffer and may include but are not limited to
preservatives.
[0062] Pharmaceutical Compositions
[0063] The sustained-release pharmaceutical compositions of the
present invention may be administered by oral (e.g., capsules such
as hard capsules and soft capsules, solid preparations such as
granules, tablets, pills, troches or lozenges, cachets, pellets,
powder and lyophilized forms, liquid preparations such as
suspensions) and non-oral preparations (e.g., intramuscular,
subcutaneous, transdermal, visceral, IV (intravenous), IP
(intraperitoneal), intraarterial, intrathecal, intracapsular,
intraorbital, injectable, pulmonary, nasal, rectal, and
uterine-transmucosal preparations)
[0064] In general, comprehended by the invention are
sustained-release pharmaceutical compositions comprising effective
amounts of protein, or derivative products, with the
sustained-release compositions of the invention together with
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers needed for administration.
See PCT 97/01331 hereby incorporated by reference. The optimal
pharmaceutical formulation for a desired biologically active agent
will be determined by one skilled in the art depending upon the
route of administration and desired dosage. Exemplary
pharmaceutical compositions are disclosed in Remington's
Pharmaceutical Sciences (Mack Publishing Co., 18th Ed., Easton,
Pa., pgs. 1435-1712 (1990)).
[0065] Due to the thixotropic nature of the delayed gel
formulation, syringes can be used to administer subcutaneously. The
composition may be gelled in a syringe for later injection. This
gelation can be performed in a time-delayed manner. The timing is
controlled by the judicious adjustment of the quantity of the
gelling agent and the proton donor in the mixture, if needed. Such
a preparation would be used for later re-gelation in the body after
injection. The term thixotropic as used herein refers to the
viscosity of the gel mixture which decreases under pressure, e.g.,
from the syringe plunger, at which point the mixture can flow,
e.g., through the syringe needle, and then reform a gel at the
injection site.
[0066] The concept of delayed gelation can also be applied to
filling a syringe where a sustained-release gel composition is
filled in a syringe and at a preset time gels in the syringe, e.g.,
from a few minutes to many hours after filling. This avoids the
problem of filling a syringe with material that has already gelled.
These prefilled syringes can be stored for later injection into
patients.
[0067] Components that may be needed for administration include
diluents of various buffer content (e.g., Tris-HCl, acetate), pH
and ionic strength; additives such as surfactants and solubilizing
agents, (e.g., Tween 80, HCO-60, Polysorbate 80), anti-oxidants
(e.g., ascorbic acid, glutathione, sodium metabisulfite),
additional polysaccharides (e.g., carboxymethylcellulose, sodium
alginate, sodium hyaluronate, protamine sulfate, polyethylene
glycol), preservatives (e.g., Thimersol, benzyl alcohol, methyl
paraben, propyl paraben) and building substances (e.g., lactose,
mannitol); incorporation of the material into particulate
preparations of polymeric compounds such as polylactic/polyglycolic
acid polymers or copolymers, etc. or combined with liposomes.
Hylauronic acid may also be used as an administration component and
this may have the effect of promoting even further the sustained
duration in the circulation. Additionally, sustained-release
compositions of the present invention may also be dispersed wIth
oils (e.g., sesame oil, corn oil, vegetable), or a mixture thereof
with a phospholipid (e.g., lecitin), or medium chain fatty acid
triglycerides (e.g., Miglyol 812) to provide an oily suspension.
The compositions of the present invention may also be dispersed
with dispersing agents such as water-soluble polysaccharides (e.g.,
mannitol, lactose, glucose, starches), hyaluronic acid, glycine,
fibrin, collagen and inorganic salts (e.g., sodium chloride).
[0068] In addition, also contemplated for use in the administration
of the sustained-release compositions of the present invention are
mechanical devices designed for pulmonary delivery of therapeutic
products, including but not limited to nebulizers, metered dose
inhalers, and powder inhalers, all of which are familiar to those
skilled in the art.
[0069] The administration components may influence the physical
state, stability, rate of in vivo release, and rate of in vivo
clearance of the present proteins and derivatives. One skilled in
the art will appreciate the appropriate administration components
and/or the appropriate mechanical devices to use depending on the
therapeutic use, route of administration, desired dosage,
circulation time, resistance to proteolysis, protein stability and
other considerations.
[0070] Methods of Use
[0071] Therapeutic. Therapeutic uses depend on the biologically
active agent used. One skilled in the art will readily be able to
adapt a desired biologically active agent to the present invention
for its intended therapeutic uses. Therapeutic uses for such agents
are set forth in greater derail in the following publications
hereby incorporated by reference including drawings. Therapeutic
uses include but are not limited to uses for proteins like
interferons (see, U.S. Pat. Nos. 5,372,808, 5,541,293 4,897,471,
and 4,695,623 hereby incorporated by reference including drawings),
interleukins (see, U.S. Pat. No. 5,075,222, hereby incorporated by
reference including drawings), erythropoietins (see, U.S. Pat. Nos.
4,703,008, 5,441,868, 5,618,698 5,547,933, and 5,621,080 hereby
incorporated by reference including drawings), granulocyte-colony
stimulating factors (see, U.S. Pat. Nos. 4,999,291, 5,581,476,
5,582,823, 4,810,643 and PCT Publication No. 94/17185, hereby
incorporated by reference including drawings), stem cell factor
(PCT Publication Nos. 91/05795, 92/17505 and 95/17206, hereby
incorporated by reference including drawings), and the OB protein
(see PCT publication Nos. 96/40912, 96/05309, 97/00128, 97/01010
and 97/06816 hereby incorporated by reference including
figures)
[0072] In addition, therapeutic uses of the present invention
include uses of biologically active agents including but not
limited to anti-obesity related products, insulin, gastrin,
prolactin, adrenocorticotropic hormone (ACTH), thyroid stimulating
hormone (TSH), luteinizing hormone (LH), follicle stimulating
hormone (FSH), human chorionic gonadotropin (HCG), motilin,
interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12),
tumor necrosis factor (TNF), tumor necrosis factor-binding protein
(TNF-bp), brain derived neurotrophic factor (BDNF), glial derived
neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast
growth factors (FGF), neurotrophic growth factor (NGF), bone growth
factors such as osteoprotegerin (OPG), insulin-like growth factors
(IGFs), macrophage colony stimulating factor (M-CSF), granulocyte
macrophage colony stimulating factor (GM-CSF), megakeratinocyte
derived growth factor (MGDF), thrombopoietin, platelet-derived
growth factor (PGDF), colony simulating growth factors (CSFs), bone
morphogenetic protein (BMP), superoxide dismutase (SOD), tissue
plasminogen activator (TPA), urokinase, streptokinase and
kallikrein. The term proteins, as used herein, includes peptides,
polypeptides, consensus molecules, analogs, derivatives or
combinations thereof. In addition, the present compositions may
also be used for manufacture of one or more medicaments for
treatment or amelioration of the conditions the biologically active
agent is intended to treat.
[0073] By way of example, therapeutic uses oxygenation in the
blood) and a decrease in bone resorption or osteoporosis may also
be achieved in the absence of weight loss.
[0074] Combination Therapies. The present compositions and methods
may be used in conjunction with other therapies, such as altered
diet and exercise. Other medicaments, such as those useful for the
treatment of diabetes (e.g., insulin, and possibly amylin),
cholesterol and blood pressure lowering medicaments (such as those
which reduce blood lipid levels or other cardiovascular
medicaments), activity increasing medicaments (e.g., amphetamines),
diuretics (for liquid elimination), and appetite suppressants. Such
administration may be simultaneous or may be in seriatim. In
addition, the present methods may be used in conjunction with
surgical procedures, such as cosmetic surgeries designed to alter
the overall, appearance of a body (e.g., liposuction or laser
surgeries designed to reduce body mass, or implant surgeries
designed to increase the appearance of body mass). The health
benefits of cardiac surgeries, such as bypass surgeries or other
surgeries designed to relieve a deleterious condition caused by
blockage of blood vessels by fatty deposits, such as arterial
plaque, may be increased with concomitant use of the present
compositions and methods. Methods to eliminate gall stones, such as
ultrasonic or laser methods, may also be used either prior to,
during or after a course of the present therapeutic methods.
Furthermore, the present methods may be used as an adjunct to
surgeries or therapies for broken bones, damaged muscle, or other
therapies which would be improved by an increase in lean tissue
mass.
[0075] Dosages
[0076] One skilled in the art will be able to ascertain effective
dosages by administration and observing the desired therapeutic
effect. The dosage of the sustained-release preparation is the
amount necessary to achieve the effective concentration of the
biologically active agent in vivo, for a given period of time. The
dosage and the preferred administration frequency of the
sustained-release preparations varies with the type of the
biologically active agent, the desired duration of the release, the
target disease, desired administration frequency, the subject
animal species and other factors. Preferable, the formulation of
the molecule will be such that between about 0.10 .mu.g/kg/day and
100 mg/kg/day will yield the desired therapeutic effect.
[0077] The effective dosages may be determined using diagnostic
tools over time. By way of example, the present invention provides
the dosages of OB protein. For example, a diagnostic for measuring
the amount of OB protein in the blood (or plasma or serum) may
first be used to determine endogenous levels of OB protein. Such
diagnostic tool may be in the form of an antibody assay, such as an
antibody sandwich assay. The amount of endogenous OB protein is
quantified initially, and a baseline is determined. The therapeutic
dosages are determined as the quantification of endogenous and
exogenous OB protein (that is, protein, analog or derivative found
within the body, either self-produced or administered) is continued
over the course of therapy. For example, a relatively high dosage
may be needed initially, until therapeutic benefit is seen, and
then lower dosages used to maintain the therapeutic benefits.
[0078] Materials and Methods
[0079] Materials. Alginate in the form of sodium alginate can be
found from sources well known in the art. OB protein and GCSF are
from Amgen Inc. Other chemicals are from sources well known in the
art.
[0080] Alginate Hydrogel Particle/Bead Preparation. The preparation
of the alginate hydrogel particles and beads, with and without
proteins, is described in detail in co-pending U.S. application
Ser. No. 08/842,756, hereby incorporated by reference.
[0081] Delayed Gel Preparation. The preparation of the delayed
alginate hydrogels, with and without proteins, is described in
detail in copending,U.S. applications Ser. No. 08/857,913 and
08/912,902, each of which is hereby incorporated by reference.
[0082] The following examples are offered to more fully illustrate
the invention, but are not to be construed as limiting the scope
thereof. In addition, with respects to the above disclosure or the
examples below, one skilled in the art will be able to make the
necessary changes to the disclosures for large scale
production.
EXAMPLE 1
[0083] The following example describes the preparation of alginate
esters to be used in the present invention.
[0084] Preparation A: Tetrabutylammonium(TBA) Alginate
[0085] A sulfonic acid resin (Bio-Rad, AG MP-50) is converted to
the tetrabutylammonium(TBA) form by treatment with
tetrabutylammonium hydroxide (Aldrich) using a batch method at room
temperature. To a solution of 10 g of sodium salt of alginic acid
in 800 ml of distilled water is added 60 ml of sulfonic resin
(Bio-Rad, AG MP-50) in the tetrabutylammonium salt form. The
mixture is stirred at room temperature for 0.5 hours. The resin is
removed by filtration and washed with distilled water. The TBA
alginate in the filtrate is isolated by freeze-drying (yield, 16.8
g)and confirmed by .sup.1H NMR.
[0086] Preparation B: Partial Ethyl Ester of Alginic Acid, Degree
of Esterification (DE)=30 mol %.
[0087] TBA alginate( 6 g, 14.4 mmol TBA units) is dissolved in 500
ml dimethyl sulfoxide (DMSO) at room temperature. Iodoethane
(Aldrich, 673 mg, 4.3 mmol) is then added. The mixture is stirred
at 30.degree. C. for 15 hours, then cooled to room temperature. To
this solution is slowly added a solution of 2 g NaCl in 20 mL water
to completely convert the TBA to the sodium salt. After stirring
15-30 min, the solution is slowly poured into 1500 mL ethyl
acetate. The precipitate is collected by filtration and washed
three times with acetone/water (8:1 v/v) and three times with
acetone, then vacuum dried. The compound is redissolved in
distilled water (.sup..about.100 mL) and the pH adjusted to
.sup..about.6.5 with 0.2% NaHCO.sub.3 at 0.degree. C. The solution
is then dialyzed (MW cut-off 8000) overnight against distilled
water at 4.degree. C. and then freeze-dried. The yield of the
partial ester is 2.8 g and the degree of esterification is 30 +/-
1% (.sup.1H NMR, maleimide as internal standard).
[0088] Preparation C: Total and Partial Ethyl Ester of Alginic
Acid, DE=100%, 50%, 20%, 10% and 5%.
[0089] The preparation of these compounds is similar to that
described in Preparation B except that the amount of iodoethane
added is adjusted to arrive at the desired degree of
esterification.
[0090] Preparation D: Partial Propyl, Hexyl, Octyl and Dodecyl
Esters of Alginic Acid.
[0091] The preparations are similar to that described in
Preparations B and C above, but substituting 1-iodopropane,
1-iodohexane, 1-iodooctane or 1-iodododecane for iodoethane
respectively.
[0092] Preparation E: Partial Benzyl Ester of Alginic Acid,
DE=30%.
[0093] TBA alginate (2.5 g, 5.99 mmol TBA units) is dissolved in
.sup..about.200 mL DMSO at room temperature. Benzyl bromide
(Aldrich, 307 mg, 1.8 mmol) and TBA iodide (Aldrich, 30 mg) are
added. The mixture is stirred at 30.degree. C. for 15 hours, and
then cooled to room temperature. To this solution is slowly added a
solution of 0.6 g NaCl in 10 mL water to completely convert the TBA
to the sodium salt. After stirring 15-30 minutes, the solution is
slowly poured into 500 mL ethyl acetate. The precipitate is
collected by filtration and washed three times with acetone/water
(8:1 v/v) and three times with acetone, then vacuum dried. The
compound is redissolved in distilled water (.sup..about.60 mL) and
adjusted to pH .sup..about.6.5 with 0.2% NaHCO.sub.3 at 0.degree.
C., then dialyzed (MW cut-off 8000) overnight against distilled
water at 4.degree. C. The yield of the partial ester is 1.3 g and
the degree of esterification is 30 +/- 1 (.sup.1H NMR, maleimide as
internal standard).
[0094] Preparation F: Total and Partial Benzyl Ester of Alginic
Acid with Different DE.
[0095] The preparation of these compounds is similar to that
described in Preparation E except that the amount of benzyl bromide
and TBA iodide added are adjusted to arrive at the desired degree
of esterfication.
EXAMPLE 2
[0096] The following example shows the preparation of a protein
drug(Leptin)--containing alginate ethyl ester (DE=15 mol % and 10
mol %) gel and the in vitro sustained release from this gel.
[0097] Leptin (100 mg/mL; 10 mM Tris HCl, pH 8.8; pH adjusted from
8.0 to 8.8 with 1M NaOH) and 6% ethyl ester alginate (15 mol %, 10
mM Tris HCl, pH 8.6) are cooled on an ice bath. Leptin (0.5 mL) is
added to the 6% ethyl ester alginate (0.18 mL) and the mixture
stirred on an ice bath for 10-15 min; the final pH is 8.6-8.8. To
this mixture is added a suspension of 1M CaCO.sub.3 (16 .mu.L) and
the resulting suspension is mixed well. To this suspension is
dropwise added, with stirring, a solution of 0.1M ZnCl.sub.2 (100
.mu.L); water is then added to bring the volume to 1 mL. The
mixture is mixed completely and kept on an ice bath for 10-20 min.
Then a solution of 1.68M .delta.-gluconolactone (Aldrich, 56 .mu.L)
is thoroughly stirred into this mixture. The final mixture (50
mg/mL leptin, 1% ethyl ester alginate; 0.1 mL) is cast on the
inside of an eppendorf tube and left overnight at 4.degree. C. to
gel. After overnight storage, in vitro release is conducted in 10
mM histidine buffer, pH 7.4. The cast gel with 15 mol % degree of
esterification exhibits minimal burst and fairly constant leptin
release showing 60% released in 6 days. The cast gel with 10 molt
degree of esterification exhibits minimal burst and fairly constant
leptin release showing 55% released in 6 days.
EXAMPLE 3
[0098] The following example shows the preparation of a protein
drug(Leptin)--containing alginate hexyl ester (DE=15 mole and 10
mol %) gel and the in vitro sustained release from this gel.
[0099] This example is performed in a similar manner as that
described in Example 2 except that the ethyl ester alginate
[0100] The hexyl ester alginate gels with 15 mole and 10 mol % of
degree of esterification exhibit minimal burst and exhibits
sustained release showing 50% a released in 6 days.
EXAMPLE 4
[0101] The following example shows the preparation of a protein
drug (Zn-Leptin)--containing alginate ethyl ester (DE=15 mol %) gel
and the in vitro sustained release from this gel.
[0102] To a solution of 4% (w/v) ethyl ester alginate (15 mol %,
0.75 mL) is added 1M Tris HCl pH 8.0 (7.5 .mu.L), 0.5 M PIPES pH
6.8 (33 .mu.L) and 0.1 M ZnCl.sub.2 (8.5 .mu.L). The mixture is
stirred well. To this solution is added Zn-leptin suspension (100
mg/mL, 675 .mu.L) and the mixture thoroughly stirred. A suspension
of 1M CaCO.sub.3 (24 .mu.L) and a solution of 1.68M
d-gluconolactone (70 .mu.L) are then thoroughly stirred into this
mixture. The final mixture (0.1 mL) is cast on the inside of an
eppendorf tube and left overnight at 4.degree. C. to gel. After
overnight storage in vitro release is conducted in 10mM histidine
buffer, pH 7.4. This cast ethyl ester alginate gel with 15 mol %
degree of esterification exhibits little burst and sustained leptin
release showing 65% released in 4 days.
EXAMPLE 5
[0103] The following example shows the preparation of a protein
drug(GCSF)--containing alginate ethyl ester (DE=30 mol %) gel and
the in vitro sustained release from this gel.
[0104] To a solution of 2.39% ethyl ester alginate (30 mol %, 0.50
mL) is added 0.1M acetate buffer (pH 4.5, 100 .mu.L), GCSF (104
.mu.L, 48.2 mg/mL, HCl pH3) and distilled water (246 mL). The
mixture is stirred well. A suspension of 1M CaHPO.sub.4 (10 .mu.L)
and a solution of 1.68M .delta.-gluconolactone (40 .mu.L) are
thoroughly stirred into this mixture. The final mixture (0.2 mL) is
cast on the inside of an eppendorf tube and left overnight at
4.degree. C. to gel. After overnight storage of the gel in vitro
release is conducted in 10mM Tris buffer, pH 7.5. This cast ethyl
ester alginate gel with 30 mol % degree of esterification exhibits
less than 5% burst and sustained release showing 20% released in 1
day and 40% released in 2 days.
EXAMPLE 6
[0105] The following example shows the preparation of a protein
drug (GCSF)--containing alginate benzyl ester (DE=30 mol %) gel and
the in vitro sustained release from this gel.
[0106] This example is performed in a similar manner that described
in Example 5 except the ethyl ester is replaced by the benzyl ester
alginate. The ester alginate gels within the overnight storage
period. The benzyl ester alginate gel with 30% of degree of
esterification exhibits less 5% burst and sustained release showing
40% released in 1 day and 80% released in 2 days.
EXAMPLE 7
[0107] This example shows the preparation of alginate ethyl ester
beads.
[0108] The gel beads are prepared by adding dropwise a 2% ester
alginate solutions into 100 mM calcium chloride solutions
(distilled water or 1M Tris HCl pH 7.0 buffer). The formed beads
are washed with distilled water or buffer. Beads are prepared using
either 30% or 50% degree of esterification.
EXAMPLE 8
[0109] This example shows the preparation of Leptin-containing
alginate ester beads.
[0110] The beads are prepared by adding dropwise a solution of 25
mg/mL Leptin in 2% ethyl ester alginate (Tris HC1, pH 8.7) into a
solution of 100 mM calcium chloride and 25 mM zinc chloride. The
beads are prepared using 30% degree of esterification. The beads
demonstrate sustained leptin release in vitro.
EXAMPLE 9
[0111] This example shows the molecular weight breakdown (or
degradation) of ester alginates in buffers at neutral physiological
pH.
[0112] Alginate esters (1% solution) are dissolved in either
phosphate buffer (0.1M sodium phosphate, pH 6.8) or 0.1M Tris
buffer (pH 7.0) and incubated at 37.degree. C. The molecular weight
breakdown is determined by measuring the decrease in the solution
viscosity (Brookfield, 25.degree. C) at selected time intervals.
Unmodified sodium alginate is found to be relatively stable in that
its viscosity decreased only 5% in 8 days (phosphate buffer);
however with ethyl and benzyl esters of alginic acid (DE=30%) the
viscosity drops 35 in 8 days in same buffer. The amount of
degradation of esters of alginic acid is also dependent on the
degree of esterification, e.g., with ethyl ester of lower degree of
esterification (DE=15%) the viscosity decreases 25% in 8 days. Thus
the molecular weight breakdown is directly related to the degree of
esterification.
EXAMPLE 10
[0113] This example shows the in vivo degradation (or gradual
disappearance) of alginate ester hydrogels without protein and
hydrogels containing protein.
[0114] The ester alginate gels are prepared in a similar manner to
that described in Example 3 but the final mixture is taken up in a
syringe and allowed to gel in the syringe at 4.degree. C.
overnight. Then 100 .mu.L of gel is injected subcutaneously into
the back of the neck of mice (Charles River, 12 week old female,
BDF1, 20 g, 5 mice per group) and the site surgically examined
periodically on different members of the group.
[0115] Using ethyl and benzyl ester alginate material with DE=30%,
the results of the single injection site study show that the ester
alginate hydrogels disappear within 2 weeks. Using ethyl ester
alginate gel with DE=15%, the gels are still present at 30 and 61
days, but reduced in size. Using ethyl ester alginate material with
DE=5%, the gels are still present at 30 and 61 days with little
reduction in size. Using the unsubstituted sodium alginate
material, the gel persists relatively unchanged through day 61.
[0116] The rate of disappearance of the ester alginate gels is
similar with or without protein.
EXAMPLE 11
[0117] This example provides weight loss and pharmacokinetics data
for leptin-containing alginate ester hydrogels in rats.
[0118] The ethyl ester alginate gels are prepared in a similar
manner to that described in Example 4 but the final mixture is
taken up in a syringe and allowed to gel in the syringe at
4.degree. C. overnight. Rats are given a bolus dose of 0 mg/kg
(control) and 100 mg/kg, then blood levels and weight loss are
monitored for seven days.
[0119] The results show: the ethyl ester alginate with DE=5 mol %
exhibits a steady blood level of .sup..about.2000 ng/mL for 3 days,
then declines to 2.sup..about.3 ng/mL over the next 3-4 days; the
ethyl ester alginate with DE=15 mol % exhibits a steady blood level
of .sup..about.2000 ng/mL for 2 days, then declines to 2-3 ng/mL at
5 days; the ethyl ester alginate with DE=30 mole exhibits a blood
level of .sup..about.2000 ng/mL for 1 day, which decreases to 2-3
ng/mL at 4 days; the blood level of Zn-leptin suspention peaks at
12 hr, then decreases to 1-2 ng/mL at 6 days. All animals exhibit
weight loss showing that the Zn-leptin is active. The results also
show that incorporating Zn-leptin in the ethyl ester alginate gels
(DE=5 mol % and 15 mol %) nearly doubles (factor of 1.8-1.9) the
area under the curve (AUC) of the Zn-leptin, suggesting a doubling
of the bioavailability; and use of ethyl ester alginate gel (DE=30
mol %) shows a similar bioavailability to Zn-leptin, based on
AUC.
* * * * *