U.S. patent application number 11/583362 was filed with the patent office on 2007-05-03 for polymer-drug conjugates.
This patent application is currently assigned to SmartCells, Inc.. Invention is credited to Thomas M. Lancaster, Matthew Nalewanski, Todd C. Zion.
Application Number | 20070099820 11/583362 |
Document ID | / |
Family ID | 37963314 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070099820 |
Kind Code |
A1 |
Lancaster; Thomas M. ; et
al. |
May 3, 2007 |
Polymer-drug conjugates
Abstract
A conjugate that includes a drug covalently linked to a polymer.
Upon administration, the conjugate is digested by an enzyme that is
present at the site of administration thereby releasing a
therapeutic agent. The conjugate may demonstrate substantially the
same pharmacokinetic and pharmacodynamic behavior as the drug
itself. A material for controllably releasing a conjugate in
response to the local concentration of a molecular indicator. The
material includes a plurality of conjugates and a plurality of
multivalent cross-linking agents. The polymers of the conjugates
include an analog of the indicator within their covalent structure.
The multivalent cross-linking agents include cross-link receptors
that interact with the indicator analog and thereby cross-link the
conjugates. These non-covalent interactions are competitively
disrupted when an amount of the molecular indicator is present
thereby causing the material to release the conjugate in a manner
that is dependent on the local concentration of indicator.
Inventors: |
Lancaster; Thomas M.;
(Stoneham, MA) ; Nalewanski; Matthew; (Seabrook,
NH) ; Zion; Todd C.; (Marblehead, MA) |
Correspondence
Address: |
CHOATE, HALL & STEWART LLP
TWO INTERNATIONAL PLACE
BOSTON
MA
02110
US
|
Assignee: |
SmartCells, Inc.
|
Family ID: |
37963314 |
Appl. No.: |
11/583362 |
Filed: |
October 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60728652 |
Oct 19, 2005 |
|
|
|
Current U.S.
Class: |
514/1.3 ;
514/11.3; 514/11.7; 514/5.8; 514/5.9; 514/6.7; 514/6.9; 530/303;
530/397 |
Current CPC
Class: |
A61P 3/10 20180101; C08B
37/00 20130101; A61K 47/61 20170801; A61K 38/28 20130101; A61K
38/25 20130101; C08B 37/0084 20130101; C08B 37/0069 20130101; A61K
47/60 20170801; C08B 30/18 20130101; A61K 47/6903 20170801; C08B
37/003 20130101; C08B 37/0072 20130101; Y10S 436/827 20130101; A61K
38/26 20130101 |
Class at
Publication: |
514/003 ;
530/303; 514/012; 530/397 |
International
Class: |
A61K 38/28 20060101
A61K038/28; C07K 14/605 20060101 C07K014/605; C07K 14/61 20060101
C07K014/61; C07K 14/62 20060101 C07K014/62 |
Claims
1. A conjugate that releases a therapeutic agent within the
extracellular space of subcutaneous tissue comprising: a drug; and
a polymer covalently linked to the drug, whereupon subcutaneous
administration, the conjugate is digested by an enzyme present in
the extracellular space of subcutaneous tissue thereby releasing a
therapeutic agent.
2. The conjugate of claim 1, wherein the drug is directly linked to
the polymer and the polymer is susceptible to digestion by the
enzyme.
3. The conjugate of claim 1, wherein the drug is indirectly linked
to the polymer via a spacer that is susceptible to digestion by the
enzyme.
4. The conjugate of claim 1, wherein the therapeutic agent and the
drug are the same.
5. The conjugate of claim 1, wherein the therapeutic agent includes
the drug covalently linked to a portion of the polymer.
6. The conjugate of claim 1, wherein the therapeutic agent includes
the drug covalently linked to a portion of the polymer spacer.
7. The conjugate of claim 1, whereupon subcutaneous administration,
said conjugate releases the therapeutic agent with a serum
concentration curve that is substantially the same as when an
equivalent amount of unconjugated drug is administered.
8. The conjugate of claim 1, whereupon subcutaneous administration,
said conjugate releases the therapeutic agent with a serum
T.sub.max that is substantially the same as when an equivalent
amount of unconjugated drug is administered
9. The conjugate of claim 1, whereupon subcutaneous administration,
said conjugate releases the therapeutic agent with a serum
C.sub.max that is substantially the same as when an equivalent
amount of unconjugated drug is administered
10. The conjugate of claim 1, whereupon subcutaneous
administration, said conjugate releases the therapeutic agent with
a serum half-life that is substantially the same as when an
equivalent amount of unconjugated drug is administered.
11. The conjugate of claim 1, wherein the polymer is selected from
the group consisting of polysaccharides, polypeptides and
polynucleotides and the enzyme is selected from the group
consisting of saccharidases, peptidases and nucleases.
12. The conjugate of claim 1, wherein the polymer is selected from
the group consisting of carboxylated polysaccharides, --NH.sub.2
pendant polysaccharides, hydroxylated polysaccharides, alginate,
collagen-glycosaminoglycan, collagen, mannan, amylose, amylopectin,
glycogen, cellulose, hyaluronate, chondroitin, dextrin and
chitosan.
13. The conjugate of claim 1, wherein the polymer is selected from
the group consisting of co-polymers of aminated and non-aminated
amino acids, co-polymers of hydroxylated and non-hydroxylated amino
acids, co-polymers of carboxylated and non-carboxylated amino
acids, co-polymers of the above and adducts of the above.
14. The conjugate of claim 1, wherein the polymer is a
polysaccharide that includes 1,4-linked alpha and beta glucose
residues.
15. The conjugate of claim 14, wherein the polymer is glycogen.
16. The conjugate of claim 15, wherein the saccharidase is an
amylase.
17. The conjugate of claim 13, wherein the drug is an antidiabetic
agent.
18. The conjugate of claim 17, wherein the antidiabetic agent is
selected from the group consisting of insulin, insulin analogues,
insulin secretagogues and insulin sensitizers.
19. The conjugate of claim 17, wherein the antidiabetic agent is
insulin.
20. The conjugate of claim 17, wherein the antidiabetic agent is a
sulfonylurea.
21. The conjugate of claim 1, wherein the drug is selected from the
group consisting of growth hormones, glucagon, leptin,
glucagon-like peptide 1 (GLP-1) and GLP-1 analogues.
22. The conjugate of claim 1, wherein the conjugate is
non-antigenic when administered subcutaneously.
23. The conjugate of claim 1, wherein the conjugate is more soluble
in water than the unconjugated drug.
24. The conjugate of claim 1, wherein the conjugate is less prone
to aggregation in water than the unconjugated drug.
25. The conjugate of claim 1, wherein the polymer is glycogen and
the drug is insulin.
26. The conjugate of claim 1, wherein the polymer includes an
indicator analog within its covalent structure and whereupon mixing
of a plurality of conjugates with a plurality of multivalent
cross-linking agents that each contain two or more cross-link
receptors, said conjugates becomes cross-linked by the multivalent
cross-linking agents through interactions between the indicator
analog and the cross-link receptors and said interactions are
competitively disrupted in a manner that is dependent on the
concentration of indicator added to the mixture.
27. The conjugate of claim 26, wherein the indicator analog has
essentially the same composition as the indicator.
28. The conjugate of claim 26, wherein the indicator analog has a
higher affinity for the multivalent cross-linking agent than the
indicator.
29. The conjugate of claim 26, wherein the indicator analog is a
portion of a side group of the polymer.
30. The conjugate of claim 26, wherein the indicator analog is
incorporated into the backbone of the polymer.
31. The conjugate of claim 26, wherein the indicator is glucose and
the drug is an antidiabetic agent.
32. The conjugate of claim 26, wherein the multivalent
cross-linking agent is a multivalent binding protein.
33. The conjugate of claim 26, wherein the multivalent
cross-linking agent is a multivalent glucose-binding molecule.
34. The conjugate of claim 33, wherein the multivalent
glucose-binding molecule is selected from the group consisting of
phytohemoagglutinins, human mannan binding protein (MBP), human
pulmonary surfactant protein A (SP-A), human pulmonary surfactant
protein D, bovine serum lectin CL-43 and conglutinin.
35. The conjugate of claim 34, wherein the phytohemoagglutinin is
selected from the group consisting of concanavalin A (Con A),
succinylated Con A, and phytohemoagglutinins derived from pisum
sativum (pea), lathyrus odoratus (sweet pea), lens culinaris
(lentil), narcissus pseudonarcissus (daffodil), vicia faba (fava
bean) and vicia sativa (garden vetch).
36. The conjugate of claim 26, wherein the multivalent
cross-linking agent is comprised of a plurality of monofunctional
binding proteins or binding fragments thereof linked to one
another.
37. The conjugate of claim 36, wherein the monofunctional binding
protein is a bacterial glucose/galactose binding protein.
38. The conjugate of claim 26, wherein the polymer is a
polysaccharide and the multivalent cross-linking agent is a plant
lectin or a mammalian analog of a plant lectin.
39. The conjugate of claim 38, wherein the lectin is selected from
the group consisting of concanavalin A (Con A), succinylated Con A,
Con A covalently modified with a polyethylene glycol, and
succinylated Con A covalently modified with a polyethylene
glycol.
40. The conjugate of claim 26, wherein the polymer is glycogen, the
drug is insulin, the multivalent cross-linking agent is a
multivalent glucose-binding molecule and the indicator is
glucose.
41. The conjugate of claim 40, wherein the multivalent
glucose-binding molecule is selected from the group consisting of
concanavalin A (Con A), succinylated Con A, Con A covalently
modified with a polyethylene glycol, and succinylated Con A
covalently modified with a polyethylene glycol.
42. The conjugate of claim 41, wherein the multivalent
glucose-binding molecule is Con A covalently modified with a
polyethylene glycol.
43. A material for controllably releasing a conjugate within the
extracellular space of subcutaneous tissue in response to the local
concentration of an indicator, comprising: a plurality of
conjugates that each include a drug covalently linked to a polymer
that includes an analog of the indicator within its covalent
structure, wherein said conjugates are susceptible to digestion by
an enzyme present in the extracellular space of subcutaneous
tissue; and a plurality of multivalent cross-linking agents each
containing two or more cross-link receptors, wherein said
conjugates are cross-linked by said multivalent cross-linking
agents through interactions between the indicator analogs and the
cross-link receptors, wherein said interactions are competitively
disrupted if an amount of the indicator is present, and wherein
competitive disruption of the interactions between the indicator
analogs and the cross-link receptors causes the material to release
the conjugate in a manner that is dependent on the local
concentration of indicator.
44. The material of claim 43, wherein the material is a plurality
of particles.
45. The material of claim 44, wherein the material is an insoluble
hydrogel.
46. A method for controllably releasing a conjugate within
subcutaneous tissue in response to the local concentration of an
indicator comprising subcutaneously administering the material of
claim 43 to a patient in need thereof.
47. The method of claim 46, wherein the material is dissolved in a
biocompatible aqueous solution and the solution is injected
subcutaneously.
48. A kit for producing a material that controllably releases a
conjugate within the extracellular space of subcutaneous tissue in
response to the local concentration of an indicator comprising: a
plurality of conjugates that each include a drug covalently linked
to a polymer that includes an analog of the indicator within its
covalent structure, wherein said conjugates are susceptible to
digestion by an enzyme present in the extracellular space of
subcutaneous tissue; and a plurality of multivalent cross-linking
agents that each contain two or more cross-link receptors,
whereupon mixing, said conjugates become cross-linked by said
multivalent cross-linking agents through interactions between the
indicator analogs and the cross-link receptors, wherein said
interactions are competitively disrupted if an amount of the
indicator is added to the mixture, and wherein said competitive
disruption causes the material to release the conjugate in a manner
that is dependent on the amount of indicator added to the
mixture.
49-57. (canceled)
Description
PRIORITY INFORMATION
[0001] This application claims priority to U.S. Ser. No. 60/728,652
filed Oct. 19, 2005. The entire contents of this application are
hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The majority of "controlled-release" drug delivery systems
known in the prior art (e.g., U.S. Pat. No. 4,145,410 to Sears
which describes drug release from capsules which are enzymatically
labile) are incapable of releasing drugs at intervals and
concentrations which are in direct proportion to the amount of a
molecular indicator (e.g., a metabolite) present in the human body.
The delivery or release of drug in these prior art systems is thus
not literally "controlled," but simply a slow release which is
independent of external or internal factors.
[0003] The treatment of diabetes mellitus with injectable insulin
is a well-known and studied example where uncontrolled, slow
release of insulin is undesirable. In fact, it is apparent that the
simple replacement of the hormone is not sufficient to prevent the
pathological sequelae associated with this disease. The development
of these sequelae is believed to reflect an inability to provide
exogenous insulin proportional to varying blood glucose
concentrations experienced by the patient. To solve this problem
several biological and bioengineering approaches to develop a more
physiological insulin delivery system have been suggested.
[0004] U.S. Pat. No. 4,348,387 to Brownlee et al. discloses a
feedback controlled insulin delivery system wherein glucose-insulin
conjugates are displaced by free glucose from binding sites on a
glucose-binding molecule. The conjugated insulin retains its
biological activity once released. In practical applications,
however, the system is soluble and must be enclosed within a
membrane that is permeable to glucose and glucose-insulin but not
to the glucose-binding molecule. Without the use of a membrane or
other external device to maintain a high local concentration of the
glucose-binding molecule, the system dissociates at infinite
dilution and releases the conjugate in a non-glucose dependent
manner. Such a system that is not self-contained and requires the
use of membranes is limited to use in extracorporeal or implantable
devices and is not directly applicable to repeated administration,
e.g., by injection. Furthermore, the system works well when
confronted with short pulses of glucose such that the total amount
of glucose introduced into the system is much less than the total
amount of glucose-insulin bound to the glucose binding sites on the
binding molecule. However, in the physiological milieu, molar
glucose concentrations are approximately one million times higher
than the concentration of insulin required to achieve a
physiological effect. The net result is that when confronted with a
critical glucose concentration in vivo, there is always enough
glucose around to effectively displace and release all of the
glucose-insulin from the system. Such a system is, therefore,
incapable of responding to repeated glucose challenges in vivo,
which is ultimately required for a closed-loop delivery system.
[0005] U.S. Pat. Nos. 5,830,506, 5,902,603, and 6,410,053 to Taylor
et al. have attempted to address the lack of response to repeated
glucose challenges. Instead of enclosing a soluble competitive
binding system within a membrane, they have developed insoluble
membranes based on competitive binding that control the rate of
insulin release from a reservoir. As with the Brownlee system,
Taylor's system is designed to be used in extracorporeal or
implantable devices. The insoluble membrane is in the form of a gel
that is formed by physically crosslinking water-soluble,
glycosylated polymers with the tetravalent glucose-binding molecule
concanavalin A (Con A). Free glucose enters the gel where it
competes with the glycosylated polymer for Con A and disrupts the
crosslinks, causing a gel-to-sol transition. Insulin that is
physically trapped within the insoluble gel is thereby released.
The gel is sandwiched between two porous support membranes to
minimize leakage of the glycosylated polymer and Con A.
[0006] Taylor's system has two advantages over Brownlee's: (1) the
device is reversible and therefore capable of responding to
repeated glucose challenges and (2) the insulin does not require
chemical modification. However, the use of support membranes
ultimately leads to a complex system with slow diffusion rates.
Consequently, excessively high glucose concentrations (>400
mg/dl) are required to significantly increase insulin diffusion.
Furthermore, once glucose is removed from the system, the decrease
in insulin release rate lags behind by several hours. The Taylor
system is also severely limited because insulin release is not
directly coupled to glucose concentration. Rather, insulin release
is governed by diffusion through the glucose-responsive gel.
[0007] Zion et al. (U.S. Patent Application Publication No.
2004-0202719 and "Glucose-responsive materials for self-regulated
insulin delivery", Thesis, Massachusetts Institute of Technology,
Dept. of Chemical Engineering, 2004) address the lack of response
to repeated glucose challenges in a different manner than Taylor.
In certain embodiments of their system, they combine a multivalent
glucose-binding molecule with a glycosylated polymer-insulin
conjugate. The glycosylated polymer contains multiple saccharide
binding groups and forms insoluble hydrogels or particles in the
presence of the glucose-binding molecule. In the Brownlee system,
the glucose-insulin conjugate was not polymeric and only contained
one saccharide binding group per insulin molecule, which was not
sufficient to form a cross-linked, insoluble system. In the Taylor
system, the insulin is physically immobilized within the gel
instead of being associated with a glycosylated polymer. Zion et
al. also describe uses of their system for the controlled delivery
of drugs other than insulin. These systems are responsive to the
same or a different molecular indicator that is present within the
body.
[0008] Because the Zion system is insoluble, it is self-contained
and does not require the use of membranes to function, making it
suitable for repeated dosing, e.g., through injection. Zion et al.
have also demonstrated that the rate of dissolution in their
system, and therefore the rate of polymer-drug release, is
proportional to the local concentration of the indicator molecule.
Finally, because the material dissolves from the outside inward
rather than volumetrically, the material is capable of responding
to repeated challenges, unlike the Brownlee system.
[0009] The Zion system is also superior to Taylor's because the
drug is covalently linked to the polymer rather than physically
immobilized in the system. As a result, precise control can be
obtained over the dose and rate of delivery even for very small
changes in concentration of the indicator molecule within the
physiological range. For example, where Taylor et al. demonstrate a
two- to four-fold increase in insulin release rate from 0 to 1,000
mg/dl glucose, Zion et al. have demonstrated a 50 fold increase in
insulin release rate from just 50 to 400 mg/dl glucose.
[0010] Whether used to deliver insulin or other drugs, the Zion
system suffers from one disadvantage. Indeed, the polymer-drug
conjugate that is released has a higher molecular weight (MW) than
the unmodified insulin of Taylor or the glucose-insulin conjugate
of Brownlee. The conjugate is therefore absorbed into the systemic
circulation much more slowly. In addition, once in the circulation,
the intrinsic bioactivity of the polymer-drug is diminished and the
rate of elimination is slower. Therefore, there is a need in the
art for a stimuli-responsive drug delivery system constructed from
a crosslinking molecule and a polymer-drug in which the
polymer-drug acts as rapidly and to the same extent as the
unmodified drug. Advantageously, this new polymer-drug could itself
also be used as a delivery device, i.e., without being included
within a stimuli-responsive drug delivery system.
SUMMARY OF THE INVENTION
[0011] In one aspect, the invention provides a conjugate that
includes a drug covalently linked to a polymer. The polymer and
drug can be directly linked or indirectly linked through a spacer.
Upon administration, the conjugate is digested by an enzyme that is
present at the site of administration thereby releasing a
therapeutic agent, e.g., the drug itself or the drug and a portion
of the polymer or spacer. In one embodiment the conjugate is
designed for subcutaneous administration. In one embodiment, the
polymer is susceptible to digestion by the enzyme. In other
embodiments, the spacer is susceptible to digestion by the enzyme.
In one embodiment, the rate of digestion at the site of
administration is such that the conjugate demonstrates
substantially the same pharmacokinetic and pharmacodynamic behavior
as the drug itself.
[0012] In another aspect, the invention provides a material for
controllably releasing a conjugate in response to the local
concentration of a molecular indicator. The material includes a
plurality of conjugates and a plurality of multivalent
cross-linking agents. The polymers of the conjugates include an
analog of the indicator within their covalent structure. The
multivalent cross-linking agents include cross-link receptors that
interact with the indicator analog and thereby cross-link the
conjugates. These non-covalent interactions are competitively
disrupted when an amount of the molecular indicator is present
thereby causing the material to release the conjugate in a manner
that is dependent on the local concentration of indicator.
[0013] The invention also provides methods of making and using the
conjugate, methods of making and using a material that controllably
releases a conjugate, and kits that include the conjugate and other
reagents for preparing a material that controllably releases a
conjugate.
DEFINITIONS
[0014] "Antigenic": As used herein, the term "antigenic" refers to
the ability of a substance to produce antibodies when introduced
into the body. Antigenicity is a measure of the capacity for a
particular compound to produce antibodies. Antigenicity is
generally measured as a minimum concentration of a substance to
produce a statistically significant increase in antibody levels
and/or the level of antibodies produced at a given concentration
for a particular immunization protocol.
[0015] "Biomolecule": As used herein the term "biomolecule" refers
to molecules (e.g., proteins, amino acids, peptides,
polynucleotides, nucleotides, carbohydrates, sugars, lipids,
nucleoproteins, glycoproteins, lipoproteins, steroids, etc.)
whether naturally-occurring or artificially created (e.g., by
synthetic or recombinant methods) that are commonly found in cells
and tissues. Specific classes of biomolecules include, but are not
limited to, enzymes, receptors, neurotransmitters, hormones,
cytokines, cell response modifiers such as growth factors and
chemotactic factors, antibodies, vaccines, haptens, toxins,
interferons, ribozymes, anti-sense agents, plasmids, DNA, and
RNA.
[0016] "Biocompatible": As used herein, "biocompatible" materials
and solutions are materials and solutions that do not elicit an
undesirable detrimental response in vivo.
[0017] "Drug": As used herein, the term "drug" refers to small
molecules or biomolecules that alter, inhibit, activate, or
otherwise affect biological or chemical events. For example, drugs
may include, but are not limited to, anti-AIDS substances,
anti-cancer substances, antibiotics, anti-diabetic substances,
immunosuppressants, anti-viral substances, enzyme inhibitors,
neurotoxins, opioids, hypnotics, anti-histamines, lubricants,
tranquilizers, anti-convulsants, muscle relaxants and
anti-Parkinson substances, anti-spasmodics and muscle contractants
including channel blockers, miotics and anti-cholinergics,
anti-glaucoma compounds, anti-parasite and/or anti-protozoal
compounds, modulators of cell-extracellular matrix interactions
including cell growth inhibitors and anti-adhesion molecules,
vasodilating agents, inhibitors of DNA, RNA or protein synthesis,
anti-hypertensives, analgesics, anti-pyretics, pyretics, steroidal
and non-steroidal anti-inflammatory agents, anti-angiogenic
factors, anti-secretory factors, anticoagulants and/or
anti-thrombotic agents, local anesthetics, ophthalmics,
prostaglandins, anti-depressants, anti-psychotic substances,
anti-emetics, and imaging agents. A more complete listing of
exemplary drugs suitable for use in the present invention may be
found in "Pharmaceutical Substances: Syntheses, Patents,
Applications" by Axel Kleemann and Jurgen Engel, Thieme Medical
Publishing, 1999; the "Merck Index: An Encyclopedia of Chemicals,
Drugs, and Biologicals", edited by Susan Budavari et al., CRC
Press, 1996, and the United States Pharmacopeia-25/National
Formulary-20, published by the United States Pharmcopeial
Convention, Inc., Rockville Md., 2001.
[0018] "Enzyme": As used herein, the term "enzyme" is used to refer
to any of numerous proteins or conjugated proteins produced by
living organisms that function as biochemical catalysts. Enzymes
capable of catalyzing the hydrolytic cleavage of particular
polymers or specific chemical bonds are of particular interest for
this specific application. Examples of enzyme families include
saccharidases that are capable of cleaving polysaccharide linkages,
peptidases that are capable of cleaving polypeptide linkages, and
nucleases that are capable of cleaving polynucleotide linkages.
[0019] "Growth Factors": As used herein, "growth factors" are
chemicals that regulate cellular metabolic processes, including but
not limited to differentiation, proliferation, synthesis of various
cellular products, and other metabolic activities. Growth factors
may include several families of chemicals, including but not
limited to cytokines, eicosanoids, and differentiation factors.
[0020] "Polymer": As used herein, a "polymer" is a compound that
includes a string of covalently linked monomers. A polymer can be
made from one type of monomer or more than one type of monomer. The
term "polymer" therefore encompasses copolymers, including
block-copolymers in which different types of monomer are grouped
separately within the overall polymer. A polymer can be linear or
branched.
[0021] "Polynucleotide": As used herein, a "polynucleotide" is a
polymer of nucleotides. The terms "polynucleotide", "nucleic acid",
and "oligonucleotide" may be used interchangeably. The polymer may
include natural nucleosides (i.e., adenosine, thymidine, guanosine,
cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine,
and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,
2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,
5-methylcytidine, C5-bromouridine, C5-fluorouridine,
C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,
C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine,
4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, dihydrouridine,
methylpseudouridine, 1-methyl adenosine, 1-methyl guanosine,
N6-methyl adenosine, and 2-thiocytidine), chemically modified
bases, biologically modified bases (e.g., methylated bases),
intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose,
2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose), or
modified phosphate groups (e.g., phosphorothioates and
5'-N-phosphoramidite linkages).
[0022] "Polypeptide": As used herein, a "polypeptide" is a polymer
of amino acids. The terms "polypeptide", "protein", "oligopeptide",
and "peptide" may be used interchangeably. Polypeptides may contain
natural amino acids, non-natural amino acids (i.e., compounds that
do not occur in nature but that can be incorporated into a
polypeptide chain) and/or amino acid analogs as are known in the
art. Also, one or more of the amino acid residues in a polypeptide
may be modified, for example, by the addition of a chemical entity
such as a carbohydrate group, a phosphate group, a farnesyl group,
an isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. These modifications
may include cyclization of the peptide, the incorporation of
D-amino acids, etc.
[0023] "Polysaccharide": As used herein, a "polysaccharide" is a
polymer of sugars. The terms "polysaccharide", "carbohydrate", and
"oligosaccharide", may be used interchangeably. The polymer may
include natural sugars (e.g., glucose, fructose, galactose,
mannose, arabinose, ribose, and xylose) and/or modified sugars
(e.g., 2'-fluororibose, 2'-deoxyribose, and hexose).
[0024] "Saccharide": As used herein, the term "saccharide" refers
to monomers of sugars. A saccharide can be a natural sugar (e.g.,
glucose, fructose, galactose, mannose, arabinose, ribose, and
xylose) or a modified sugar (e.g., 2'-fluororibose, 2'-deoxyribose,
hexose, etc.).
[0025] "Small molecule": As used herein, the term "small molecule"
refers to molecules, whether naturally-occurring or artificially
created (e.g., via chemical synthesis), that have a relatively low
molecular weight. Typically, small molecules are monomeric and have
a molecular weight of less than about 1500 g/mol. Preferred small
molecules are biologically active in that they produce a local or
systemic effect in animals, preferably mammals, more preferably
humans. In certain preferred embodiments, the small molecule is a
drug. Preferably, though not necessarily, the drug is one that has
already been deemed safe and effective for use by the appropriate
governmental agency or body. For example, drugs for human use
listed by the FDA under 21 C.F.R. .sctn..sctn. 330.5, 331 through
361, and 440 through 460; drugs for veterinary use listed by the
FDA under 21 C.F.R. .sctn..sctn. 500 through 589, incorporated
herein by reference, are all considered acceptable for use in
accordance with the present invention.
BRIEF DESCRIPTION OF THE DRAWING
[0026] The invention is described with reference to the several
figures of the drawing, in which:
[0027] FIG. 1 is a schematic of an exemplary controlled release
material comprising an insulin-glycosylated polymer conjugate and a
multivalent glucose-binding molecule.
[0028] FIG. 2 shows serum total Ig-class anti-insulin antibody
titers obtained for male SD rats after week 8 of an immunization
protocol using saline, insulin, insulin-glycogen conjugate, and
insulin plus adjuvant solutions. Titers are expressed as .mu.U of
insulin bound per ml of serum assayed. Solutions were injected s.c.
into male Sprague-Dawley (SD) rats weighing 200-250 g (n=5 or 6)
each week for four weeks. Blood was collected each week for up to
eight weeks and pooled to quantify the titer of all Ig-class
anti-insulin antibodies using a radioimmunoassay (RIA) technique
(Esoterix, Inc., Calabasas Hills, Calif.).
[0029] FIG. 3 shows a scatter plot of insulin/conjugate loading w/w
% as measured by amino acid analysis for various synthesis schemes
arbitrarily delineated 1-10. The circled point corresponds to
conjugate used in repeated dosage HbA.sub.1c study: 7.0.+-.0.1 wt %
insulin/conjugate.
[0030] FIG. 4 shows (a) Blood glucose depression profiles in male
SD rats (n=3) for subcutaneously injected (.tangle-solidup.)
insulin-dextran (70 K), (.diamond-solid.) insulin-glycogen (Type II
oyster), and (.box-solid.) unmodified human recombinant insulin.
Insulin-polymers were injected at 2.5 U of conjugated insulin/kg;
unmodified insulin was injected at 2.0 U/kg. (b) (.diamond.) Blood
glucose depression and (.quadrature.) serum insulin profiles for
subcutaneously injected insulin-glycogen at 2.5 U of conjugated
insulin/kg. T.sub.max .about.15-45 min.; T.sub.nadir .about.30-45
min.; and T.sub.70% BGL .about.150 min. Insulin levels were
measured using a Porcine Insulin ELISA kit (ALPCO Diagnostics,
Windham, N.H.) and blood glucose levels were measured using a
Medisense.RTM. Precision Xtra.TM. glucometer.
[0031] FIG. 5 shows (a) denaturing SDS-PAGE demonstration of the
amylase-induced breakdown of intact conjugate into low molecular
weight products and insulin. Lanes: (1) MW standards, (2)
recombinant insulin at 10 mg/ml, (3) recombinant insulin at 1
mg/ml, (4) intact conjugate at 7% wt. insulin loading, (5) saliva
control, (6 through 12) intact conjugate mixed with dilutions of
human salivary amylase; dilutions of human saliva are at 1, 4, 16,
64, 128, 256 and 512.times., respectively; (b) serum fluorescence
and insulin levels for a bolus FITC-conjugate injection at t=0 in
normal SD rats (n=2). (.quadrature.) Serum Fluorescence
corresponding to FITC-conjugate absorption and elimination measured
at .quadrature..sub.ex=485 nm and .quadrature..sub.em=525 nm.
(.diamond-solid.) Serum insulin levels (mU/L) as measured by
RIA.
[0032] FIG. 6 shows (a) euglycemic glucose clamp results on n=5
normal felines dosed with 0.5 U/kg conjugate, (.quadrature.)
glucose infusion rate (GIR) and (.diamond-solid.) serum insulin
concentration as measured by porcine insulin ELISA (ALPCO
Diagnostics, Inc., Windham, N.H.) (b) serum insulin concentration
versus time for n=5 normal cats dosed with 0.5 U/kg of
(.diamond-solid.) human recombinant insulin and (.box-solid.)
IPC.
[0033] FIG. 7 shows the results of serum fluorescence measurements
of FITC labeled glycogen (MW.about.1,000K) and dextran (MW=70K or
500K).
[0034] FIG. 8 shows glucose set point (GSP) curves for Con
A-insulin-glycogen conjugate gels containing (.diamond-solid.) 0.0,
(.tangle-solidup.) 0.1, (.box-solid.) 0.5, (.circle-solid.) 0.9,
and (*) 1.0 weight fraction of dimeric, succinylated Con A.
[0035] FIG. 9 shows (a) glucose set point (GSP) curve for dimeric
Con A-insulin-glycogen gels (insulin loading=0.6% w/w) and (b)
corresponding in vitro glucose-responsive release kinetics for
glucose concentrations of (.diamond-solid.) 50, (.box-solid.) 100,
(.tangle-solidup.) 200, and (.circle-solid.) 400 mg/dl. Inlay: Rate
Sensitivity data corresponding to the ratio of pseudo-first order
release for a particular glucose concentration (R.sub.x, where
x=100, 200, and 400 mg/dl) to that obtained at 50 mg/dl
(R.sub.50).
[0036] FIG. 10 shows (a) GSP curves for (.box-solid.) lower set
point and (.diamond-solid.) higher set point formulations and (b)
corresponding CGS traces in STZ-diabetic rats.
[0037] FIG. 11 shows CGS traces for STZ-diabetic rats (n=2 for each
group) treated with (left) (a) 10, (b) 20, and (c) 30 U/kg NPH and
(right) (a) 50 ul, (b) 150 ul, and (c) 200 ul of a inventive gel
formulation.
[0038] FIG. 12 shows average CGS values for STZ-diabetic rats (n=6)
treated with an average daily dose of 150.+-.30 .mu.l of an
inventive material. The arrow represents the time of the last gel
injection (Day 6 of the study). The average glucose value (solid
line) for all six rats from Day 0 to Day 7=98 mg/dl.+-.29 mg/dl
(dotted lines). Inlay: Average body weights on day 0 and day 7
demonstrating a statistically significant 6% increase in weight
over the study (p<0.05).
[0039] FIG. 13 shows images of tissue samples extracted on Day 3
from the injection site. (a) Con A-gel: Injection site contains (1)
an acellular area that is presumably part of the gel; (2) A large
region of necrotic neutrophils; and (3) a thick capsule of
proliferating fibroblasts, blood capillaries and some macrophages.
(b) PEG-Con A-gel: Injection site appears normal with the exception
that the muscle layer contained a mild to moderate population of
lymphocytes and fewer plasma cells. (c) Saline: Injection site
appears normal.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0040] This application refers to a number of published documents
including patents, patent applications and articles. Each of these
published documents is hereby incorporated by reference.
A. Polymer-Drug Conjugates
[0041] In one aspect, the invention provides a conjugate that
includes a drug covalently linked to a polymer. The polymer and
drug can be directly linked or indirectly linked through a spacer.
The spacer may itself be a polymer but can also be a coupling
agent, etc. Upon administration, the conjugate is digested by an
enzyme that is present at the site of administration thereby
releasing a therapeutic agent, e.g., the drug itself or the drug
and a portion of the polymer and/or spacer. It is to be understood
that the conjugate may be digested by more than one enzyme present
at the site of administration. In addition, it will be appreciated
that digestion of the conjugate may produce a population of
different species, each digested at different points within the
spacer and/or polymer and each digested to different extents. These
species may be further digested over time after the initial
cleavage. This further digestion may occur at the site of
administration and/or after they have been absorbed into the
systemic circulation. In general, because of the enzymatic
degradation, the therapeutic agent is absorbed into circulation
more rapidly than the conjugate would be without enzymatic
action.
[0042] In one set of embodiments, the degradation rate of the
conjugate is such that the pharmacokinetic and/or pharmacodynamic
behavior of the therapeutic agent are substantially the same as the
unconjugated drug. Generally, absorption of the therapeutic agent
will lag behind absorption of the unconjugated drug because of the
need for enzymatic digestion. In certain embodiments however the
lag is minimal. In one embodiment, the similarity in
pharmacokinetic and/or pharmacodynamic behavior is observed when
the conjugated and unconjugated drugs are administered
subcutaneously. For example, from a pharmacokinetic (PK)
perspective, the serum concentration curve of the therapeutic agent
may be substantially the same as when an equivalent amount of
unconjugated drug is administered. Additionally or alternatively,
the conjugate may release the therapeutic agent to yield a serum
T.sub.max, a serum C.sub.max, a mean serum residence time (MRT), a
mean serum absorption time (MAT) and/or a serum half-life that is
substantially the same as when the unconjugated drug is
administered. From a pharmacodynamic (PD) perspective, the
conjugate may act on substances within the body in substantially
the same way as the unconjugated drug. For example, in the case of
an insulin conjugate, the conjugate may affect blood glucose levels
in substantially the same way as unconjugated insulin. In this
case, substantially similar pharmacodynamic behavior can be
observed by comparing the time to reach minimum blood glucose
concentration (T.sub.nadir), the duration over which the blood
glucose level remains below a certain percentage of the initial
value (e.g., 70% of initial value or T.sub.70% BGL), etc. It will
be appreciated that these PK and PD characteristics can be
determined according to any of a variety of published
pharmacokinetic and pharmacodynamic methods (e.g., see Baudys et
al., Bioconjugate Chem. 9:176-183 (1998) for methods suitable for
subcutaneous delivery).
[0043] In one embodiment, an inventive conjugate produces
pharmacokinetic (PK) parameters such as time to reach maximum serum
drug concentration (T.sub.max), mean drug residence time (MRT),
serum half-life, and mean drug absorption time (MAT) that are
within 20% of those values determined for the unconjugated drug.
More preferably a conjugate produces PK parameters that are within
15% or even 10% of those produce by the unconjugated drug.
[0044] For example, in embodiments involving an insulin conjugate
for subcutaneous delivery the conjugate may produce an insulin
T.sub.max between 15-30 minutes, a mean insulin residence time
(MRT) of less than 50 minutes, and a mean insulin absorption time
(MAT) of less than 40 minutes, all of which are within 20% of those
values determined from the human recombinant insulin treatment
group. In certain embodiments, the conjugate may produce an insulin
T.sub.max between 20-25 minutes, a mean insulin residence time
(MRT) of less than 45 minutes, and a mean insulin absorption time
(MAT) of less than 35 minutes. In certain embodiment, the conjugate
may produce a serum half-life of less than 120 minutes, e.g., less
than 100 minutes.
[0045] In one embodiment, an inventive conjugate produces
pharmacodynamic (PD) parameters such as time to reach
minimum/maximum blood concentration of a substance
(T.sub.nadir/T.sub.max) or duration over which the blood level of
the substance remains below/above 70% /130% of the initial value
(T.sub.70 % BL/T.sub.130% AL).
[0046] For example, in embodiments involving an insulin conjugate
for subcutaneous delivery the conjugate may produce a glucose
T.sub.nadir between 45-60 minutes and a glucose T.sub.70% BGL of
less than 180 minutes, both of which are within 20% of those
determined from the human recombinant insulin treatment group. In
certain embodiments the conjugate may produce a glucose T.sub.nadir
between 50-55 minutes and a glucose T.sub.70% BGL of less than 160
minutes.
[0047] In one embodiment, the polymer is susceptible to digestion
by the enzyme present at the site of administration. In other
embodiments, the spacer is susceptible to digestion by the enzyme.
In yet other embodiments, both the polymer and spacer are
susceptible to digestion by the enzyme. One skilled in the art will
recognize that a number of enzymes are present in the body that
could cleave the polymer and/or spacer. Without limitation, these
include saccharidases, peptidases, and nucleases. Exemplary
saccharidases include, but are not limited to, maltase, sucrase,
amylase, glucosidase, glucoamylase, and dextranase. Exemplary
peptidases include, but are not limited to, dipeptidyl
peptidase-IV, prolyl endopeptidase, prolidase, leucine
aminopeptidase, and glicyl glycine dipeptidase. Exemplary nucleases
include, but are not limited to, deoxyribonuclease I, ribonuclease
A, ribonuclease T1, and nuclease S1.
[0048] One skilled in the art will also recognize that, depending
on the choice of enzyme, there are a number of polymers and spacers
that are susceptible to enzymatic cleavage. For example, in cases
where saccharidase degradation is desired, polysaccharide polymers
and spacers can be used. For example, Sauer et al. describe the
structure/function relationships between the sacchridase
glucoamylase with regard to its binding and catalytic behavior
(Biochim Biophys Acta. 2000 Dec. 29; 1543(2): 275-293). Thus,
without limitation, a conjugate that includes a polysaccharide
comprising repeating chains of 1,4-linked alpha-D-glucose residues
will be degraded by alpha-amylases. Suitable polysaccharides
include glycogen and partially digested glycogen derived from any
number of sources, including but not limited to, sweet corn,
oyster, liver (human, bovine, rabbit, rat, horse), muscle (rabbit
leg, rabbit abdominal, fish, rat), rabbit hair, slipper limpet,
baker's yeast, and fungus. Other polysaccharide polymers and
spacers that one could use include carboxylated polysaccharides,
--NH.sub.2 pendant polysaccharides, hydroxylated polysaccharides,
alginate, collagen-glycosaminoglycan, collagen, mannan, amylose,
amylopectin, cellulose, hyaluronate, chondroitin, dextrin,
chitosan, etc. In cases where peptidase cleavage is desired,
polypeptide polymers or polypeptide spacers that contain amino acid
sequences recognized by the cleaving enzyme can be used. For
example, Thoma et al. describe the structural basis for
proline-specific exopeptidases such as human dipeptidyl peptidase
IV (Structure 11: 947-59, 2003). Thus, without limitation, a
conjugate that includes a [-Glycine-Proline-] sequence will be
degraded by prolidase. In certain embodiments one could use
co-polymers of aminated and non-aminated amino acids, co-polymers
of hydroxylated and non-hydroxylated amino acids, co-polymers of
carboxylated and non-carboxylated amino acids, co-polymers of the
above or adducts of the above. In cases where nuclease degradation
is desired, polynucleotide polymers and spacers can be used. For
example, Beers describes the role of pancreatic ribonuclease in
hydrolyzing polyadenylic acid sequences (J Biol Chem. 235:2393-8,
1960). Thus, without limitation, a conjugate that includes a
polynucleotide containing an oligomer of sequential adenosine
residues will be degraded by ribonuclease A.
[0049] The drug used will depend on the disease or disorder to be
treated. The conjugates are not limited to any particular drug and
may include small molecule drugs or biomolecular drugs. As
described below and in the Examples, in certain embodiments, the
conjugates may be used to treat diabetes mellitus in which case
antidiabetic drugs would be used. Biomolecules that are suitable
for this purpose include, but are not limited to, therapeutic
proteins and peptides, e.g., insulin, growth hormones, glucagon,
leptin, glucagon-like peptide 1 (GLP-1) and GLP-1 analogues. For
example, in one embodiment, the conjugate may include insulin
covalently linked to glycogen. Instead of insulin one could also
use insulin analogues, insulin secretagogues (e.g., sulfonylureas
or meglinitides) or insulin sensitizers. Sulfonylureas currently
known in the art include chlorpropamide, glibenclamide, gliclazide,
glimepiride, glipizide, gliquidone, tolazamide and tolbutamide.
Meglinitides currently known in the art include nateglinide and
repaglinide. Biguanides (e.g., metformin) are antidiabetic agents
that decrease glucose levels by reducing hepatic glucose and
increasing preipheral uptake of glucose. These and any other drug
can be used in a conjugate of the invention.
[0050] The present invention encompasses conjugates that are loaded
with one or more drugs and to differing levels. In one embodiment,
loading levels in the range of 0.5-15% w/w of drug to conjugate are
suitable. As described in the Examples, this range has been found
to produce insulin-glycogen conjugates with beneficial
characteristics. In certain embodiments loading levels within the
narrower range of 5-10% are suitable, and in yet other embodiments
a range of 6-8% can be used.
[0051] It is to be understood that the techniques of the invention
may also be exploited to produce conjugates that are more stable
and/or soluble in solution than unconjugated drugs. This can be
advantageous in a number of applications besides long term storage.
For example, the solubility of native insulin in water is limited
which is problematic for pumps and devices that require high
concentrations of insulin in order to function (e.g., see Wolpert
et al. in BMJ 324:1253, 2002). In addition, insulin tends to
aggregate at higher temperatures. Jens Brange has reviewed the
issues of insulin instability under high temperature, agitation,
and non-physiological pH in Galenics of insulin: The
Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin
Preparations. Springer-Verlag, Berlin, 1987. In this context, the
inventors have found that conjugation of insulin to a polymer such
as glycogen can produce a conjugate that is more soluble and/or
less prone to aggregation than native insulin.
B. Methods of Making Conjugates
[0052] In another aspect, the invention provides methods for making
inventive conjugates. In general, these methods involve producing a
covalent link between a drug and a polymer (optionally via a
degradable spacer which may or may not be polymeric). It is to be
understood that the drug may be linked to the desired polymer
through any number of chemical linkages, including but not limited
to amide, ester, ether, isourea, and imine bonds. Exemplary methods
are discussed in more detail below and in the Examples.
[0053] In certain embodiments, the drug can be linked to the
polymer via a natural or chemically added pendant group. For
example, in one embodiment, polymers with --COOH pendant groups
(carboxyl bearing polymers, or "CBP's") and carboxyl-derivatives
with increased reactivity (e.g., acid halides, esters, etc.) may be
used. Such polymers may naturally include carboxyl groups or may be
modified to include them. Exemplary CBPs include but are not
limited to carboxylated polysaccharides (CPS) such as
carboxymethylated glycogen. Naturally occurring carboxylated
polymers include but are not limited to carboxylated poly(amino
acids) such as poly-L-glutamate and poly-L-aspartate that contain
peptide linkages that are recognized and cleaved by serum
peptidases. The carboxylate content may be varied between 1 and
100% mol COOH/mol amino acid residue by copolymerizing carboxylated
amino acids (e.g., amino acids with a carboxyl group in addition to
the carboxyl group which becomes part of the polymer backbone) with
non-carboxylated amino acids (e.g., amino acids whose only carboxyl
group becomes part of the polymer backbone).
[0054] In another embodiment, polymers having --NH.sub.2 pendant
groups (--NH.sub.2 bearing polymers, or "NBP's") may be used. Such
polymers may be naturally occurring or may be chemically modified
to include a primary amine. Examples of the latter type of NBP
include, but are not limited to, --NH.sub.2 pendant polysaccharides
(NPS) such as amino-derivatized glycogen. The degree of --NH.sub.2
substitution with respect to monomer may vary between 1 and 100%
mol. Other suitable NBP's include, but are not limited to,
polynucleotides where one or more of the purine bases has been
derivatized with an amine group at the 2' location. Naturally
occuring aminated polymers include poly(amino acids) such as
copolymers poly-L-lysine (PLL) containing peptide linkages that are
recognized and cleaved by serum peptidases. The amine content may
be varied between 1 and 100% mol NH.sub.2/mol amino acid residue by
copolymerizing an aminated amino acid (e.g., an amino acid with an
amine in addition to the amine group that eventually becomes part
of the polymer backbone) with non-aminated amino acids (e.g., an
amino acid whose only amine is that which eventually becomes part
of the polymer backbone). Proteins that include epsilon-NH.sub.2
lysine groups (and which naturally have alpha-NH.sub.2 terminal
groups) and peptide linkages that are recognized and cleaved by
serum peptidases may also be used.
[0055] In another embodiment, polymers having --OH pendant groups
(--OH bearing polymers, or "OBP's") may be used. Such polymers may
be naturally hydroxylated or may be chemically modified using
standard organic chemistry techniques to include a hydroxyl group.
Naturally occurring OBP's include, but are not limited to,
polysaccharides such as glycogen and maltodextrin and all
polynucleotides. In addition, poly(amino acids) such as
poly(serine), poly(threonine), poly(tyrosine), and
poly(4-hydroxyproline) may also be employed as hydroxylated
polymers, provided that they contain peptide linkages that are
recognized and cleaved by serum peptidases. The hydroxyl content of
the poly(amino acids) may be varied between 1 and 100% mol --OH/mol
AA residue by co-polymerizing hydroxylated amino acids with
non-hydroxylated amino acids. Of course, carboxyl, amino, and
hydroxyl pendant groups may be mixed in a single polymer by
co-polymerizing the appropriate amino acids in desired ratios.
[0056] In another embodiment, polymers having --SH pendant groups
(--SH bearing polymers, or "SBP's") may be used. Such polymers may
be naturally sulfhydrylated or may be chemically modified using
standard organic chemistry techniques to include a sulfhydryl
group.
[0057] Co-polymers, mixtures, and adducts of the above polymers may
also be used in the practice of the invention. Indeed, such
combinations may be particularly useful for optimizing the
mechanical and chemical properties of the matrix. Both the choice
of polymer and the ratio of polymers in a co-polymer may be
adjusted to optimize the stiffness of the matrix and the
degradation rate of the component polymer.
[0058] Exemplary drugs suitable for use in this invention include
but are not limited to those containing --COOH, --NH.sub.2, --OH,
and --SH reactive moieties. Specific examples include therapeutic
peptides and proteins bearing alpha-terminal NH.sub.2 and/or
epsilon-NH.sub.2 lysine groups.
[0059] --COOH bearing drugs can be conjugated to OBP's using the
procedure outlined by Kim et al. Biomaterials 24:4843 (2003).
Briefly, the OBP is dissolved in DMSO along with the --COOH
functionalized drug and reacted by means of
N',N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) as catalysts under a dry atmosphere. The resulting product
is precipitated in ethanol, then dissolved in water, and filtered
to remove insoluble particles, followed by lyophilization to obtain
the pure conjugate. --COOH functionalized drugs can be conjugated
to NBP's using a carbodiimide (EDAC) coupling procedure. Using this
procedure, the --COOH bearing drug is functionalized by reaction
with EDAC in a pH 5 buffer followed by the addition of NBP. The
resulting product is ultrafiltered and lyophilized to obtain the
pure conjugate
[0060] --NH.sub.2 bearing drugs can be conjugated to CBP's to
produce a stable amide bond as described by Baudys et al., Bioconj.
Chem. 9:176-183 (1998). This reaction can be achieved by adding
tributylamine (TBA) and isobutylchloroformate to a solution of the
CBP and an --NH.sub.2 bearing drug in dimethylsulfoxide (DMSO)
under anhydrous conditions followed by separation of conjugated and
unconjugated drug using HPLC size exclusion chromatography
(HPLC-SEC). --NH.sub.2 bearing drugs can alternatively be coupled
to OBP's through cyanalation using reagents including, but not
limited to, cyanogen bromide (CNBr), N-cyanotriethylammonium
tetrafluoroborate (CTEA), 1-Cyano-4-(Dimethylamino)-pyridinium
tetrafluorborate (CDAP), and p-nitrophenylcyanate (pNPC). CNBr
reactions can be carried out at mildly basic pH in aqueous solution
followed by ultrafiltration, separation by HPLC-SEC, and
lyophilization. CDAP reactions are carried out in a mixture of DMSO
and water at mildly basic pH using triethylamine (TEA) as a
catalyst followed by ultrafiltration, separation by HPLC-SEC, and
lyophilization. --NH.sub.2 bearing drugs can be conjugated to NBP's
through glutaraldehyde coupling in aqueous buffered solutions
containing pyridine followed by quenching with glycine,
ultrafiltration, and lyophilization.
[0061] --OH functionalized therapeutic agents can be conjugated to
OBP's according to the divinylsulfone (DVS) procedure. Using this
procedure, OBP is added to a pH 11.4 bicarbonate buffer and
activated with DVS followed by addition of an --OH functionalized
drug after which glycine is added to neutralize and quench the
reaction. The resulting polymer is dialyzed exhaustively against
deionized water and finally lyophilized to obtain the pure
conjugate.
[0062] --SH functionalized drugs can be conjugated to NBP's
according to a method described by Thoma et al., J. Am. Chem. Soc.
121:5919-5929 (1999). This reaction involves suspending the NBP in
anhydrous dimethylformamide (DMF) followed by the addition of
2,6-lutidine and acid anhydride and subsequent precipitation and
purification of the reactive intermediate. A --SH functionalized
drug is then added to a solution of the intermediate in DMF with
triethylamine followed by extensive ultrafiltration and
lyophylization to obtain the pure polymer-therapeutic agent
conjugate.
[0063] In general, the amount of drug that is loaded onto the
polymer can be controlled by adjusting the molecular weight of the
polymer and/or the level of chemical activation (i.e., when pendant
groups are added to the polymer). As discussed in the Examples,
this can in turn be used to control the pharmacokinetic and
pharmacodynamic profile of the therapeutic agent that is released
from the conjugate upon administration. In certain embodiments it
may prove advantageous to include a step of purifying the polymer
prior to activation and reaction with the drug. This will be
particularly advantageous when using naturally occuring polymers
(e.g., partially digested glycogen from natural sources) since it
will allow for the removal of contaminants, e.g., antigenic
proteins.
[0064] As discussed in the Examples, the present invention has been
exemplified using insulin as a conjugated drug. Thus, in one
particular embodiment, insulin is coupled to glycogen (an OBP)
using a CNBr coupling procedure. Briefly, glycogen is dissolved in
deionized water after which solid CNBr is added to the resulting
solution and the pH is maintained constant at 10.7 using 3N sodium
hydroxide (NaOH) solution. After stirring for 15 minutes, solid
CNBr is added again and the pH maintained constant at 10.7 while
stirring for 45 minutes. Insulin is then added to the solution and
the pH adjusted to 9.15 using solid sodium bicarbonate. The
solution is stirred overnight, ultrafiltered exhaustively against
deionized, and lyophilized. The resulting powder is then purified
from unconjugated insulin by HPLC-SEC using a 1 M acetic acid
mobile phase over a Superdex.TM. 30 HiLoad 16/60 (Amersham
Biosciences, Piscataway, N.J.) packed column. The insulin-glycogen
fraction is then lyophilized to obtain the conjugate as a pure
white powder.
[0065] In another embodiment, the weight percentage of insulin
loading can be increased by using CDAP conjugation. Briefly,
glycogen is dissolved in a 50:50 mixture of deionized water and
dimethylsulfoxide (DMSO). 1-cyano-4-dimethylaminopyridinium
tetrafluoroborate (CDAP) is then added dropwise to the glycogen
solution at 0.degree. C. After stirring for 5 minutes,
triethylamine is added and the solution stirred for another 5
minutes. At this time, dilute HCl is added to remove carbamate
groups, neutralize triethylamine, and control the resulting ability
of the polymer to react with insulin. The mixture is allowed to
stir for 15 minutes. The pH is then adjusted to 10.4 followed by
the addition of insulin to the solution and final pH adjustment to
9.15. The solution is stirred overnight, ultrafiltered exhaustively
against deionized water, and lyophilized. The resulting powder is
then purified from unconjugated insulin by HPLC-SEC using a 1 M
acetic acid mobile phase over a Superdex.TM. 30 HiLoad 16/60
(Amersham Biosciences, Piscataway, N.J.) packed column. The
insulin-glycogen fraction is then lyophilized to obtain the
conjugate as a pure white powder. Removal of the dilute HCl acid
treatment step prior to increasing the pH to 10.4 allows for even
higher loading of insulin. As discussed in the Examples, the
inventors have also found that adding an amount of glycine during
the reaction can increase the solubility of the conjugate. For
example, when drugs are conjugated to highly branched
functionalized polymers, they are presented in some as high density
multivalent chemical entities. If in one case the drug has limited
solubility in aqueous solution or in another case, has the tendency
to self aggregate, such as is the case with insulin, solution
instability and insolubility will be enhanced by the conjugate
multivalency. The inventors have discovered that covalent addition
of ionically charged side chains to the polymer, such as glycine
and ethylenediamine, greatly improves the drug-conjugate solubility
and stability in solution.
C. Materials for Controllably Releasing a Conjugate
[0066] In another aspect, the invention provides a material for
controllably releasing an inventive conjugate in response to the
local concentration of a molecular indicator. The material includes
a plurality of conjugates and a plurality of multivalent
cross-linking agents. According to this aspect, the polymeric
component of the conjugate includes an analog of the indicator
within its covalent structure. The multivalent cross-linking agents
include cross-link receptors that interact with the indicator
analog and thereby cross-link the conjugates. These non-covalent
interactions are competitively disrupted when an amount of the
molecular indicator is present thereby causing the material to
release the conjugate in a manner that is dependent on the local
concentration of indicator.
[0067] In one embodiment, the accessibility and thus the enzymatic
degradability of the conjugates within the cross-linked material is
minimal. However, as the local concentration of indicator
increases, the multivalent cross-linking agents bind to free
indicator molecules thereby weakening surface cross-links and
allowing conjugates to erode away from the surface of the material.
Once released from the cross-linked material, the conjugates are
degraded by enzymes present at the site of administration thereby
releasing a therapeutic agent which can be absorbed into the
circulation. This controlled release scheme is illustrated in FIG.
1.
Indicator and Indicator Analog
[0068] As noted above, in this aspect of the invention, the
polymeric component of the conjugate includes an analog of the
indicator within its covalent structure. As used herein, the term
"indicator analog" refers to a chemical group that interacts with a
cross-link receptor of the multivalent cross-linking agent in a
similar manner to the corresponding indicator molecule. One skilled
in the art will recognize that the indicator analog may have
essentially the same composition as the indicator itself or may be
a chemically related species. It will be appreciated that where the
affinity of the cross-link receptor for the indicator and the
indicator analog are different, the density of cross-links may need
to modified so that the desired amount of conjugate is released for
a given local concentration of indicator. The indicator analog may
be naturally present within the covalent structure of the polymer
(e.g., as part of the backbone or as a side group). Alternatively
(or additionally) it may be artificially incorporated into the
covalent structure post-polymerization (i.e., as a chemical group
that is covalently linked to the polymer through its backbone or
side groups). For example, when the indicator is glucose the
indicator analog can be a derivative of glucose, lactose, maltose,
mannose, mannobiose, mannotriose, etc. In one embodiment the
indicator is glucose, the polymer is glycogen (which includes
glucose moieties as indicator analogs), and the conjugate is
released from the material in response to the local concentration
of glucose.
[0069] Sugars can be conjugated to polymeric --NH.sub.2 groups as
described in Thoma et al., J. Am. Chem. Soc., 121:5919 (1999).
Briefly, NBP (1 mmol based on --NH.sub.2 groups) is suspended in a
mixture of dimethylformamide (DMF) and 1 ml of 2,6-lutidine under a
dry argon atmosphere. At 0.degree. C., a solution of acid anhydride
(3.0 mmol) in 1 ml of DMF is added within 15 minutes, and the
resulting clear solution stirred for 16 hr at 0.degree. C. The
product is precipitated by dropwise addition to 40 ml of a stirred
1:1 mixture of ethanol and ether. The solid is filtered, washed,
and dried under vacuum. 10.0 mg (0.050 mmol) of dried solid is
dissolved in 2 ml of DMF containing 2 equivalents of thioglucose
(varying alpha/beta anomer ratio), obtained from Sigma Aldrich.
Triethylamine is then added at 2 equivalents and stirred at room
temperature for 16 h. The mixture is then added dropwise to 30 ml
of a 1:1 mixture of ethanol and ether. The precipitate is washed
with ethanol and dried under vacuum. The crude product may then be
dissolved in deionized water and ultrafiltered exhaustively against
fresh deionized water, followed by lyophilization to produce dry
NBP-glucose polymer. The degree of glycosylation is easily adjusted
by varying the equivalents of thioglucose used in the reaction
mixture. In this case, the unreacted --NH.sub.2 groups are capped
with glycerol by adding an excess (3.0 to 5.0 equivalents) of
thioglycerol, obtained from Sigma Aldrich.
[0070] Sugars such as glucose or mannose can be conjugated to OBPs
using a divinylsulfone (DVS) procedure. Briefly, the OBP is added
to a pH 11.4 bicarbonate buffer and activated with DVS. D-mannose
or D-glucose is then added and allowed to react for .about.1 hr at
room temperature, after which glycine is added to neutralize and
quench the reaction. The resulting polymer is dialyzed exhaustively
against deionized water and finally lyophilized to obtain
glycosylated-OBP. OBPs may also be modified with sugars using
periodate coupling as described in Mislovicova et al., Bioconjugate
Chem., 13:136-142 (2002). 100 mg of OBP is dissolved in 1-3.5 ml of
a 0.05 M aqueous solution of sodium periodate (NaIO.sub.4) and
stirred in the dark at 4.degree. C. for one hour. The volume of
periodate solution is varied depending on the degree of
hydroxylation of the OBP and the desired extent of reaction. The
reaction is stopped by adding 1 ml of ethylene glycol and stirring
for one hour. The resulting mixture is dialyzed against water and
lyophilized. The resulting dry dialdehyde form of OBP is dissolved
in 4 ml of a 0.05 M phosphate buffer, pH 7 at 10 mg/ml. To this
solution, 4 ml of a solution of GA or MA in 0.05 M phosphate
buffer, pH 7 at 10-50 mg/ml is added along with 2.5 ml of a 10
mg/ml sodium cyanoborohydride (NaCNBH.sub.3) solution and the
resulting mixture stirred at room temperature for 24 hours. The
reaction is then stopped by adding a sodium borohydride
(NaBH.sub.4) solution in 0.05 M pH 9.5 borate buffer at a
concentration of 5 mg/ml to reduce the remaining aldehyde groups.
The resulting mixture is stirred for 6 hours at room temperature,
after which the pH is adjusted to 7 using 4 M hydrochloric acid
(HCl). The resulting solution is ultrafiltered exhaustively against
deionized water and lyophilized to obtain pure glycosylated-OBP. In
this embodiment, equal volumes of the two reactant (polymer and
sugar) solutions are employed to prevent the components from
reacting too quickly. The degree of conjugation is controlled by
adjusting the concentrations of the solutions.
Multivalent Cross-Linking Agent
[0071] Conjugates are cross-linked within the material through
interactions between the indicator analogs of the polymers and the
cross-link receptors of multivalent cross-linking agents. These
interactions are non-covalent and thus reversible. As the local
concentration of the indicator increases, it competes with the
indicator analog for interactions with the cross-link receptor
eventually causing the conjugates to become detached from the
multivalent cross-linking agent.
[0072] In general, the multivalent cross-linking agent will be
selected based on its binding properties for the indicator and its
analog. For example, if the indicator is a peptidic hormone then
the multivalent cross-linking agent might be prepared by chemically
linking two or more copies of a receptor protein or antibody for
the hormone. Similarly, if the indicator is a saccharide then the
multivalent cross-linking agent might be a multivalent saccharide
binding protein. Exemplary multivalent saccharide binding proteins
include plant lectins, or phytohemoagglutinins (PHA's), such as
concanavalin A (Con A) and those derived from pisum sativum (pea),
lathyrus odoratus (sweet pea), lens culinaris (lentil), narcissus
pseudonarcissus (daffodil), vicia faba (fava bean), and vicia
sativa (garden vetch) as well as human analogues of plant lectins
such as human mannan binding protein (MBP, also called mannan
binding lectin, Sheriff et al., Structural Biology, 1:789-794
(1994); Dumestre-Perard et al., Molecular Immunology, 39:465-473
(2002)), human pulmonary surfactant protein A (SP-A, Allen, et al.,
Infection and Immunity, 67:4563-4569 (1999)), human pulmonary
surfactant protein D (SP-D, Persson et al., The Journal of
Biological Chemistry, 265:5755-5760 (1990)), CL-43 (a human serum
protein), and conglutinin. One skilled in the art will recognize
that any multivalent binding protein may be exploited for use with
the invention.
[0073] As suggested above, other multivalent cross-linking agents
may be constructed by chemically linking multiple monovalent
binding proteins, for example, antibodies, cell membrane receptors,
lectins, collecting, etc. Still other multivalent molecules may be
constructed by chemically linking specific binding fragments of
proteins, for example, antibodies, cell membrane receptors,
lectins, collecting, etc. Exemplary protein fragments include
truncated MBP (Eda et al., Biosci. Biotechnol. Biochem.,
62:1326-1331 (1998)), truncated conglutinin (Eda et al., Biochem.
J. 316:43 (1996)), truncated SP-D (Eda et al., Biochem. J. 323:393
(1997)), and the glucose/galactose binding protein of E. Coli
(Salins et al., Analytical Biochemistry 294:19-26 (2001)). In
addition, a variety of monovalent ligand-binding proteins are
available commercially from Sigma-Aldrich, including folate-binding
protein, thyroxine-binding globulin, and lactoferrin.
[0074] Monovalent molecules and fragments may be linked directly to
one another or to polymer scaffolds. Suitable scaffold materials
include but are not limited to the CBPs, NBPs, and OBPs described
above. In certain embodiments, proteins may be attached to these
polymers to form multivalent cross-linking agents using the
insulin-conjugation procedures that are described in the Examples
or other standard organic chemistry reactions (see March, "Advanced
Organic Chemistry", 5th ed. John Wiley and Sons, New York, N.Y.,
2001).
[0075] In certain embodiments, mono- or multivalent binding
proteins can be synthesized by rational computational design
followed by site directed mutagenesis of ligand-binding proteins as
described in Looger et al., Nature 423:185-190 (2003). For example,
in one embodiment a soluble L-Lactate binding protein can be
synthesized by introducing random modification to wild-type glucose
binding protein (GBP), ribose binding protein (RBP),
arabinose-binding protein (ABP), glutamine binding protein (QBP),
and/or histidine binding protein (HBP). The monovalent L-lactate
binding protein is made multivalent by attaching several protein
molecules to a polymer. A conjugate with a polymer comprising
lactate moieties can be produced based on the procedure of de Jong
(de Jong et al., Journal of Controlled Release 71:261-275 (2001)).
The lactate functionalized polymer can then be cross-linked with
the multivalent lactate-binding protein to produce a
lactate-responsive delivery system. Elevated L-lactate
concentrations are indicative of several medical conditions
including extreme muscle fatigue.
[0076] It is to be understood that any of these multivalent
cross-linking agents may be chemically modified with short-chain
polymers, e.g., polyethyleneglycol (PEG), to reduce in vivo
immunogenic responses (e.g., reduced mitogenicity and/or
antigenicity). For example, the terminal-NH.sub.2 and epsilon-amino
lysine groups of protein-based cross-linking agents can be reacted
with activated PEG molecules (e.g., without limitation
N-hydroxysuccinimide activated PEG, succinimidyl ester of PEG
propionic acid, succinimidyl ester of PEG butanoic acid,
succinimidyl ester of PEG alpha-methylbutanoate), in aqueous
solution at room temperature followed by ultrafiltration to remove
unreacted PEG and lyophilization to obtain the pure pegylated
crosslinking agent. Other exemplary monovalent chemical compounds
that can be used to modify a multivalent cross-linking agent
include natural and synthetic amino acids, other water soluble but
non-PEG-containing polymers such as poly(vinyl alcohol), reagents
that can be easily coupled to lysines, e.g., through the use of
carbodiimide reagents, and perfluorinated compounds.
[0077] In one embodiment, the cross-linked material becomes an
insoluble hydrogel. The hydrogel degrades as free indicator
molecules compete for the interactions between the cross-linking
agent and the indicator analog. The hydrogel degrades in a
layer-by-layer fashion from the outside in, allowing the conjugate
to be released at an approximately constant rate as the hydrogel
degrades. The hydrogel may be injected into a patient. In one
embodiment, injection is done subcutaneously. Even where a patient
requires long term treatment, the degradation mechanism of the
material can reduce the frequency of injections by preventing the
therapeutic agent from being wasted. The agent is only released as
needed, not constantly. As described in U.S. Patent Application
Publication No. 2004-0202719, the cross-linked material may also be
in the form of particles. These can be prepared in aqueous solution
through self assembly by mixing dilute solutions of the conjugate
and multivalent cross-linking agent.
[0078] In one embodiment, the conjugate includes insulin (drug)
covalently linked to glycogen (polymer) and the multivalent
cross-linking agent is a multivalent glucose-binding molecule
(multi-GBM). The glycogen naturally contains glucose moieties
within the backbone and on side groups that serve as the indicator
analogs for the multi-GBM. In certain embodiments, Con A or
pegylated Con A may be used as the multi-GBM, and when combined
with the insulin-glycogen conjugate will form an insoluble
hydrogel. While incorporated within the cross-linked hydrogel, the
insulin-glycogen amylase degradation occurs at a fraction of the
rate of uncrosslinked insulin-glycogen. However, as the glucose
concentration in the environment of the hydrogel increases, the
insulin-glycogen is de-crosslinked and released from the material.
The insulin-glycogen released by glucose from the matrix is then
degraded by amylases present at the site of administration thereby
releasing insulin molecules (optionally with a portion of the
glycogen polymer attached) with essentially the same
pharmacokinetic and pharmacodynamic behavior as unconjugated
insulin.
D. Methods of Using Conjugates and Materials for Controllably
Releasing a Conjugate
[0079] In another aspect, the present invention provides methods of
using inventive conjugates and materials for controllably releasing
a conjugate. The inventive conjugates and materials can be used in
any application. Generally, the conjugates and materials are
suitable for delivering drugs. Thus, inventive conjugates can be
used alone to deliver a drug or can be used in the context of an
inventive material that controllably releases the conjugate in
response to an indicator at the site of administration.
[0080] When used alone, an inventive conjugate can be administered
as a pharmaceutical composition with one or more inactive agents
such as a sterile, biocompatible carrier including, but not limited
to, sterile water, saline or buffered saline. The invention
encompasses treating a disease by administering the pharmaceutical
compositions of the invention. Although the pharmaceutical
compositions of the present invention can be used for treatment of
any subject (e.g., any animal) in need thereof, they are most
preferably used in the treatment of humans. The conjugates of this
invention can be administered to humans and other animals by a
variety of routes including oral, intravenous, intramuscular,
intra-arterial, subcutaneous, intraventricular, transdermal,
rectal, intravaginal, intraperitoneal, topical (as by powders,
ointments, or drops), buccal, or as an oral or nasal spray or
aerosol. In general the most appropriate route of administration
will depend upon a variety of factors including the nature of the
drug (e.g., its stability in the environment of the
gastrointestinal tract), the condition of the patient (e.g.,
whether the patient is able to tolerate oral administration), etc.
Subcutaneous injection is a preferred route of administration.
General considerations in the formulation and manufacture of
pharmaceutical compositions may be found, for example, in
Remington's Pharmaceutical Sciences, 19.sup.th ed., Mack Publishing
Co., Easton, Pa., 1995.
[0081] When used in the context of an inventive material, the
conjugates can be used to deliver a therapeutic agent to a patient
in a controlled manner. Thus in one embodiment the present
invention provides a method for controllably releasing a conjugate.
This method simply involves administering an inventive cross-linked
material to a patient in need thereof. In one embodiment, the
accessibility and thus the enzymatic degradability of the
conjugates within the administered material is minimal. However, as
the local concentration of indicator increases, the multivalent
cross-linking agents of the material bind to free indicator
molecules thereby weakening surface cross-links and allowing
conjugates to erode away from the surface of the material. Once
released from the cross-linked material, the conjugates are
degraded by enzymes present at the site of administration thereby
releasing a therapeutic agent which can be absorbed into the
circulation.
[0082] In certain embodiments, the material is administered
subcutaneously, e.g., by injection. The material can be dissolved
in a biocompatible carrier for ease of delivery. For example, the
biocompatible carrier can be an aqueous solution including, but not
limited to, sterile water, saline or buffered saline.
[0083] According to the methods of treatment of the present
invention, the disease of interest is treated in a patient such as
a human or other mammal by administering to the patient a
therapeutically effective amount of a drug in the form of a
conjugate, in such amounts and for such time as is necessary to
achieve the desired result. In one embodiment, the material
includes an insulin-glycogen conjugate and the material is used to
treat diabetes mellitus. By a "therapeutically effective amount" of
a drug is meant a sufficient amount of the drug to treat (e.g., to
ameliorate the symptoms of, delay progression of, prevent
recurrence of, delay onset of, etc.) the disease at a reasonable
benefit/risk ratio, which involves a balancing of the efficacy and
toxicity of the therapeutic agent. In general, therapeutic efficacy
and toxicity may be determined by standard pharmacological
procedures in cell cultures or with experimental animals, e.g., by
calculating the ED.sub.50 (the dose that is therapeutically
effective in 50% of the treated subjects) and the LD.sub.50 (the
dose that is lethal to 50% of treated subjects). The
ED.sub.50/LD.sub.50 represents the therapeutic index of the agent.
Although in general therapeutic agents having a large therapeutic
index are preferred, as is well known in the art, a smaller
therapeutic index may be acceptable in the case of a serious
disease, particularly in the absence of alternative therapeutic
options. Ultimate selection of an appropriate range of doses for
administration to humans is determined in the course of clinical
trials.
[0084] It will be understood that the total daily usage of an
inventive conjugate for any given patient will be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
patient will depend upon a variety of factors including the
disorder being treated and the severity of the disorder; the
activity of the specific drug employed; the specific composition
employed; the age, body weight, general health, sex and diet of the
patient; the time of administration, route of administration and
rate of excretion of the specific drug employed; the duration of
the treatment; drugs used in combination or coincidental with the
specific drug employed; and like factors well known in the medical
arts.
E. Kits
[0085] In another aspect the present invention provides kits that
include an inventive conjugate and other reagents for preparing a
material that controllably releases a conjugate in response to the
local concentration of an indicator. The kit includes separate
containers that include a plurality of conjugates and a plurality
of multivalent cross-linking agents. The polymeric component of the
conjugate includes an analog of the indicator within its covalent
structure. The multivalent cross-linking agents include cross-link
receptors that can interact with the indicator analog and thereby
cross-link the conjugates. When the conjugates and multivalent
cross-linking agents of the kit are mixed a cross-linked material
is formed. The non-covalent interactions between the indicator
analogs of the conjugate and the cross-link receptors of the
multivalent cross-linking agents are competitively disrupted when
an amount of the molecular indicator is present thereby causing the
material to release the conjugate in a manner that is dependent on
the local concentration of indicator. In one embodiment, the
material is designed for subcutaneous delivery and the kit includes
a syringe. The kit may also include instructions for mixing the
conjugates and multivalent cross-linking agents to produce the
cross-linked material.
EXAMPLES
I. Methods of Making Exemplary Conjugates
[0086] This first set of examples describes various methods for
making exemplary conjugates. The examples also include assays for
purifying and assaying the starting ingredients and final products.
It is to be understood that these methods can be modified to
produce other conjugates that fall within the scope of the
invention.
Example 1
Insulin and Glycogen
Insulin
[0087] Human recombinant insulin (HRI) is a well established and
well characterized biologic protein made from E. coli. HRI is
readily available in large, pharmaceutical-grade quantities from a
number of manufacturers, including Eli Lilly, Novo Nordisk, and
Diosynth. Insulin activity can be determined using an in vitro
insulin receptor binding assay (e.g., see Example 13) and in vivo
pharmacokinetic and pharmacodynamic studies using SD rat models
(e.g., see later Examples).
Glycogen
[0088] Glycogen can be produced from Golden Bantam sweet corn
(Curry Seed Company, Elk Point, S.Dak.) according to a modified
procedure (Morris and Morris, J. Biol. Chem. 130:535-544, 1939).
Briefly, 10 g of moist sweet corn kernels are ground and extracted
with 3.times.100 ml of deionized water. The combined extracts are
strained to remove coarse particles, and filtered through a fritted
funnel to remove insoluble starches. The extract is then
concentrated to one third the volume by boiling, and the hot
solution is then filtered by vacuum filtration to remove
insolubles. The .about.100 ml of extract is allowed to cool to room
temperature, and 300 ml of glacial acetic acid is added, which
causes glycogen to precipitate out of solution. The glycogen
precipitate is recovered by centrifugation at 5,000.times.g
(Allegra 21R, Beckman Coulter, Fullerton, Calif.). The recovered
glycogen is dissolved in 10M potassium hydroxide, and the resulting
solution is boiled for 1 hour to destroy soluble protein. The
solution is cooled, and the glycogen is precipitated with an excess
of ethanol and recovered by centrifugation at 5,000.times.g. The
glycogen precipitate is redissolved in potassium hydroxide and the
process repeated once more to eliminate any remaining protein in
the sample. The final glycogen precipitate is then lyophilized
(Freezemobile, Virtis, Gardiner, N.Y.) to remove water and ethanol,
and the synthesis provides 1.2 g (12% of initial weight of corn) of
purified, protein-free corn glycogen as a dry powder. The final
product can be characterized by nuclear magnetic resonance (NMR)
for purity, carbon, hydrogen, and nitrogen (CHN) analysis for
residual protein content and purity, and size exclusion
chromatography for molecular weight distribution analysis. If
further purification is desired, the lyophilized corn glycogen
product can be made free of impurities according to the method of
Example 4. The highly purified glycogen fractions are lyophilized
and then used subsequently in the synthesis of inventive
conjugates.
Example 2
Generalized CNBr Conjugation Method
[0089] This example describes a generalized method for making
insulin-glycogen conjugates using cyanogen bromide (CNBr) as a
coupling agent. Briefly, a known mass of glycogen is dissolved in
deionized water at a concentration of 10 mg/ml. Solid CNBr is added
to the resulting solution at a CNBr to glycogen mass ratio between
0.05 and 1.5 and the pH maintained constant at 10.7+/-0.2 using 3N
sodium hydroxide (NaOH) solution. After stirring for 15 minutes,
another equal mass of solid CNBr equal is added and the pH
maintained constant at 10.7+/-0.2 while stirring for 45 minutes.
Insulin is then added to the solution at an insulin to glycogen
mass ratio between 0.05 and 0.60 and the pH adjusted to 9.15 using
solid sodium bicarbonate. The solution is stirred overnight,
ultrafiltered exhaustively against deionized water using a 50 kDa
MWCO polyethersulfone disc membrane filter (Millipore, Bedford,
Mass.), and lyophilized. The resulting powder is then purified from
unconjugated insulin by gel filtration HPLC (Waters, Milford,
Mass.) using a 1 M acetic acid mobile phase over a Superdex.TM. 30
HiLoad 16/60 (Amersham Biosciences, Piscataway, N.J.) packed
column. The insulin-glycogen fraction is then lyophilized to obtain
the conjugate as a pure white powder.
Example 3
Generalized CDAP Conjugation Method
[0090] This example describes a generalized method for making
insulin-glycogen conjugates using cyanodimethylamino-pyridinium
tetrafluoroborate (CDAP) as a coupling agent. This synthesis was
used to prepare various insulin-glycogen conjugates that are
described in later Examples. The inventors have found that this
method produces increased insulin loading as compared to the CNBr
method of Example 2. Briefly, 8.0 g of glycogen is dissolved in 160
ml of 25 mM HEPES, 0.15 M NaCl, pH 9.0. 2.4-12.0 mL of a 1.0M CDAP
solution in DMSO is added dropwise to the glycogen solution at
0.degree. C. After stirring for 1 minute, a volume equal to that of
the CDAP solution consisting of 0.2M triethylamine (TEA) is added
dropwise over one minute. At this time, the pH of the reaction
solution is adjusted to 9.0 using 1.2N HCl. Then, 80-2000 ml of a
10 mg/ml insulin solution in a 20 mM HEPES (pH 9.0) solution is
added over the next three minutes and the pH adjusted again to 9.0
using 0.3N NaOH. Finally, in some cases 1.6 ml of a 100 mg/ml
glycine solution in 100 mM HEPES buffer containing 0.15M NaCl (pH
9.0) can be added. In other cases, amine-functionalized sugars such
as mannosamine, glucosamine, and other glucose or
mannose-containing sugars. The inventors have found that the
addition of the glycine step increases the solubility of the
conjugate. The entire reaction mixture is slowly stirred at room
temperature overnight.
[0091] The resulting mixture is then diluted by a factor of two
with 1 M acetic acid and purified from unconjugated insulin, low MW
reaction byproducts, and reagents by gel filtration HPLC (Waters,
Milford, Mass.) using a 1 M acetic acid mobile phase over a
Superdex.sup.198 30 HiLoad 16/60 (Amersham Biosciences, Piscataway,
N.J.) packed column. In some cases, it is necessary to repeat this
procedure one or to more times to ensure complete removal of
unconjugated insulin. The final mixture is then purified from
acetic acid by gel filtration HPLC (Waters, Milford, Mass.) using a
pH 8.0, 20 mM HEPES buffer containing 0.150 M NaCl mobile phase
over a Superdex.TM. 30 HiLoad 16/60 (Amersham Biosciences,
Piscataway, N.J.) packed column. Finally, the collected fraction is
precipitated by adding two volumes of a 50:50 (v/v) mixture of
ethanol and ether, washed, and dried under vacuum at room
temperature overnight.
Example 4
Insulin Conjugated to Glycogens from Different Sources
[0092] Insulin-glycogen conjugates can be prepared using glycogen
or partially digested glycogen derived from any number of sources,
including but not limited to, sweet corn, oyster, liver (human,
bovine, rabbit, rat, horse), muscle (rabbit leg, rabbit abdominal,
fish, rat), rabbit hair, slipper limpet, baker's yeast, and fungus.
For example, these glycogens can be covalently linked to insulin
using the CNBr or CDAP methods of Examples 2 and 3.
[0093] Commercial glycogen preparations generally contain protein
contaminants. In certain embodiments it is advantageous to remove
these contaminants before the drug is loaded onto the glycogen. The
resulting conjugate preparations are purer and have a higher drug
load per gram of glycogen. This example describes a generalized
method for producing protein-free glycogen. The purification
procedure involves heating the glycogen in concentrated alkali.
This takes advantage of the fact that macromolecules other than
polysaccharides (particularly proteins) are destroyed by alkali. In
addition, oxygen has minimal solubility in hot alkali, so random
oxidative damage to the glycogen is minimized. First, 30 g of
glycogen is dissolved in 200 ml of 30% potassium hydroxide and
heated in a covered Erlenmeyer flask in a boiling water bath for 2
hours. Any sediment that is produced after cooling is removed by
centrifugation. Thereafter, the glycogen solution is transferred to
a 1 L beaker and three volumes of ethanol are slowly stirred in.
The resulting precipitate is centrifuged and washed twice with a
75% ethanol solution in water. It is then redissolved in 200 ml of
water and the precipitation process repeated until the alkali is
removed. The final traces of alkali are removed by neutralizing the
glycogen solution with acetic acid before the final precipitation.
Thereafter, the glycogen is washed twice with pure ethanol and then
twice with ether and allowed to dry to produce protein-free
glycogen. The inventors have found that insulin loading is
increased when purified glycogen is used instead of commercial
unpurified glycogen.
Example 5
Conjugation of Glycogen with Non-Human Insulin, Insulin Analogues,
etc.
[0094] Instead of human insulin-glycogen conjugates one can prepare
glycogen conjugates that include non-human insulin or insulin
analogues (i.e., peptides with insulin like bioactivity that differ
from insulin by one or more substitutions, additions or deletions).
For example, these conjugates can be prepared using the CNBr or
CDAP methods of Examples 2 and 3.
Example 6
Symlin-Glycogen Conjugate
[0095] The peptidic anti-diabetic drug symlin (pramlintide acetate)
is derived from the natural peptide amylin. It can also be
conjugated with glycogen (and other OBPs), e.g., using the CNBr or
CDAP methods of Examples 2 and 3.
Example 7
Secretagogue-Glycogen Conjugates
[0096] Peptidic insulin secretagogues (e.g., GLP-1 or the GLP-1
analogue exanitide) can be coupled to glycogen (and other OBPs) by
the CNBr method used to conjugate insulin and glycogen (see Example
2). These peptidic secretagogues can also be conjugated with
glycogen using the CDAP coupling method of Example 3.
[0097] Sulfonylureas (SU), such as glibenclamide, can be conjugated
to glycogen (and other OBPs) according to the procedure outlined in
Kim et al. Biomaterials 24:4843 (2003). Briefly, the OBP is
dissolved in dimethylsulfoxide (DMSO) with the sulfonylurea and
reacted by means of N',N'-dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) as catalysts. The reaction is
carried out for 48 hours under argon and the dicyclohexylurea
removed by filtration. The product is precipitated in ethanol, then
dissolved in water, and filtered to remove insoluble particles.
Following lyophilization, the product is further purified by
dialysis, and lyophilized to obtain a pure OBP-SU product. An
SU-conjugated pullulan obtained by this procedure was shown to
possess dose-dependent insulinotropic action (see Kim et al.
Biomaterials 24:4843 (2003)).
Example 8
rHGH-Glycogen Conjugates
[0098] The peptidic drug symlin recombinant human growth hormone
(rHGH) can also be conjugated with glycogen (and other OBPs), e.g.,
using the CNBr or CDAP methods of Examples 2 and 3.
Example 9
Glucagon-Glycogen Conjugates
[0099] The peptidic drug glucagon can also be conjugated with
glycogen (and other OBPs), e.g., using the CNBr or CDAP methods of
Examples 2 and 3.
Example 10
Degree of Conjugation by Amino Acid Analysis
[0100] This example describes a generalized method for determining
the degree of conjugation of a peptidic drug (e.g., insulin) by
amino acid analysis (UCLA Biopolymers Laboratory, Los Angeles,
Calif.). This method was used to characterize some of the
conjugates that are described in later Examples. Purified conjugate
is dissolved at 2.5 mg per mL in 1.times. PBS buffer free from
calcium and zinc ions. The samples are dried, hydrolyzed with 6N
hydrochloric acid, and the contents analyzed by an amino acid
analyzer (e.g., Beckman 6300 model, Beckman-Coulter, Fullerton,
Calif.). The peptidic content is calculated based on the relative
concentrations of detected amino acids, and the samples are run in
duplicate to ensure an accurate reading.
Example 11
Degree of Purity by SDS-PAGE
[0101] This example describes a generalized method for determining
the amount of unconjugated peptidic drug (e.g., insulin) in a
sample of conjugate using SDS-PAGE. This method was used to
characterize some of the conjugates that are described in later
Examples. First, Laemmli sample buffer is made by mixing the sample
with 950 .mu.l of 1.times. Laemmli Sample Buffer (Bio-Rad,
Hercules, Calif.). No mercaptoethanol is used in this particular
assay. The desired sample is diluted in 1.times. PBS buffer to a
concentration between 0.5 and 5 mg/ml. 25 .mu.l of this solution is
pipetted into a microcentrifuge tube, followed by 50 .mu.l of
1.times. Laemmli Sample Buffer solution. The centrifuge tube is
closed, vortexed briefly, and then placed into boiling water for 5
min. After boiling, the sample is cooled in ice-cold water. The
desired samples are pipetted into a BioRad 15-well (15 .mu.l
capacity) precast 15% polyacrylamide gel and run at 150V for 70
minutes. The desired samples are run against a mixture of proteins
that act as molecular weight standards. After running the gel, the
protein bands are fixed in the gel using a 40:10:50 methanol:acetic
acid:water mixture by volume for 15 minutes, followed by washing
twice with water. The bands are revealed by staining with colloidal
Coomassie blue stain for 1.5 hours. The background staining is
removed from the gel by 3.times. washing in DI water. The
drug-polymer conjugate, due to its high molecular weight remains
near the top of the gel, while "free" drug and other protein
impurity bands, if present, migrate to the bottom of the gel.
Pictures of the bands are taken by placing the developed gels on a
light table and capturing images of the gel with a digital camera
attached to a personal computer. If necessary, the relative
densities of electrophoretic bands are quantitatively measured by
AlphaImage software.
Example 12
Degree of Purity by Analytical SEC-HPLC
[0102] This example describes the use of gel filtration
chromatography (GFC) to determine the amounts of unconjugated drug
remaining in a sample of conjugate after purification. This method
was used to characterize some of the conjugates that are described
in later Examples. Briefly, a 10-50 mg/ml solution of the conjugate
dissolved in aqueous buffer is injected into an HPLC (Waters
Corporation, Milford, Mass.) equipped with a Sephacryl HiPrep 16/60
column (Amersham Biosciences, Piscataway, N.J.) equilibrated at
room temperature with a 5-20% acetic acid mobile phase and a UV
absorbance detector operating at 280 nm. The column is eluted with
the same buffer at a flow rate of 1.0 ml/min. When used to assay an
insulin-glycogen conjugate, the conjugated insulin generally elutes
between 40 and 90 min under these conditions, whereas unconjugated
insulin elutes between 90 and 180 min. The relative amount of
conjugated versus unconjugated drug is determined by the ratio of
the unconjugated peak area to the total peak area
(unconjugated+conjugated). The absolute amount of unconjugated drug
in the sample is determined by comparing the peak area to a peak
area calibration curve obtained for a set of known drug standards.
The mass of unconjugated drug divided by the total mass of injected
conjugate yields the mass % of unconjugated drug per conjugate.
II. In Vitro Assays of Exemplary Conjugates
[0103] This second set of examples describes various experiments
investigating the in vitro properties of some exemplary
conjugates.
Example 13
Generalized Assay for Insulin-Polymer Conjugate Bioactivity in
Vitro
[0104] This example describes a generalized method for assaying the
bioactivity of insulin-polymer conjugates using Hep G-2 cells that
express a human insulin receptor. This method was used to
characterize some of the insulin containing conjugates that are
described in later Examples. The in vitro assay can be used to
measure the insulin activity and pharmacologic action of a
particular insulin-polymer conjugate. Hep G-2 (hepatocytes) are
seeded in 24-well plates at a density of 4.times.105 cells/well,
fed three days after subculture, and used within 3 days for
experimentation.
[0105] Cell culture media is removed from the wells, and the cells
are rinsed with calcium and magnesium free phosphate-buffered
saline. 450 .mu.L of binding buffer (118 mM sodium chloride, 5 mM
potassium chloride, 1.2 mM magnesium sulfate, 8.8 mM dextrose), 1
mg/mL bovine serum albumin, and 100 mM Hepes buffer at pH 8, and 50
uL of unlabeled conjugate at various concentrations 0.1-1000 ug/mL
in binding buffer (1.9% wt. insulin) are added per well. Next, 50
uL of a 2 ug/mL .sup.125I-labeled recombinant human insulin
(1-2.times.10.sup.4 cpm, Amersham Biosciences, Piscataway, N.J.) is
added to each well. The plates are then incubated at 15.degree. C.
for 6 hours. The buffer is aspirated, and the cells are washed
3.times. with ice-cold sodium chloride solution, and the contents
of each well are solubilized with 0.1% sodium dodecyl sulfate
solution. The plates are shaken gently to detach the cells and the
contents of each well are transferred into counting tubes.
Radioactivity is determined using a gamma counter (20/20 series,
Isodata, Inc., Rolling Meadows, Ill.). The radioactivity in
solution allows for a determination of the ratio of bound to free
.sup.125I-insulin versus the concentration of unlabeled
insulin-polymer conjugate, a measure of the relative
bioactivity.
[0106] The assay can be repeated with insulin-polymer conjugates
that have been subjected to enzymatic digestion. For example, when
analyzing an insulin-glycogen conjugate one group can be
preincubated at 37.degree. C. with a 10 U/mL solution of
.alpha.-amylase (porcine, Sigma Aldrich, St. Louis, Mo.), while
another group is preincubated at 37.degree. C. with rat serum
(Sigma Aldrich, St. Louis, Mo.) so that the insulin-glycogen
conjugate is digested in the presence of .alpha.-amylase and serum
amylases prior to addition to the wells containing Hep G-2
cells.
Example 14
Insulin-Glycogen Conjugate Digestion in Vitro
[0107] This example demonstrates insulin-glycogen degradation in
the presence of amylases. As previously discussed, once an
insulin-glycogen conjugate is released from an inventive
glucose-responsive material, it is digested by amylases that
breakdown the large molecular weight insulin-glycogen conjugate
into low molecular weight conjugates in vivo. As shown in other
Examples, these conjugates have been found to absorb, act, and
eliminate in substantially the same manner as human recombinant
insulin. An in vitro amylase digestion assay was performed to
qualify the proposed insulin-glycogen degradation mechanism.
Briefly, insulin-glycogen synthesized according to the CDAP
coupling method of Example 3 using Type IX glycogen from bovine
liver (Sigma Aldrich, St. Louis, Mo.) was incubated with a number
of different human saliva concentrations to demonstrate how it
degrades into free insulin in the presence of amylase. The saliva
sample was serially diluted 1:2 in 1.times. PBS buffer to 1:512. 90
.mu.L aliquots of each of the 1:4, 1:16, 1:64, 1:128, 1:256, and
1:512 dilutions were added to pre-prepared microcentrifuge tubes
containing 90 .mu.L of undiluted insulin-glycogen. These samples as
well as a sample containing 90 .mu.L of 1.times. PBS and 90 .mu.L
of undiluted conjugate were vortexed and then placed in an
incubator/shaker set at 37.degree. C. for 15 min. After incubation,
25 .mu.L of each sample was added to 50 .mu.L of Lamelli buffer and
the combination was vortexed prior to being loaded on a 4-15%
Tris-HCl SDS PAGE gel. In addition to the incubated samples, a
molecular weight ladder and high and low insulin standards were
also run (with the high insulin standard at 2.5 mg/mL and the low
insulin standard at 0.25 mg/mL). The gels were run in SDS running
buffer for 45 minutes to an hour at a constant voltage of 150 V.
Under these conditions, the amylase completely digested the
insulin-glycogen conjugate into native MW insulin at the 1:16
dilution level as quantified by the relative band density of high
MW conjugate to that of native MW insulin.
III. In Vivo Assays of Exemplary Conjugates
[0108] This third set of examples describes various experiments
investigating the in vivo properties of some exemplary
conjugates.
Example 15
Generalized Assay for Insulin-Polymer Conjugate Bioactivity in
Vivo
[0109] This example describes a generalized method for assaying the
bioactivity of insulin-polymer conjugates using an enzyme-linked
immunosorbent assay (ELISA). This method was used to characterize
some of the insulin containing conjugates that are described in
later Examples.
[0110] Subcutaneous injections of insulin-polymer conjugates or
insulin controls are administered at a dose between 1 and 10
equivalent U insulin/kg body weight behind the neck of fasted
normal diabetic rats (Male Sprague-Dawley, 200-250 g, n=2-4). Blood
samples are collected via tail vein bleeding at -15 and 0 minutes,
and at 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 minutes after
injection. Blood glucose values are measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). The remaining blood is centrifuged to obtain serum and
stored in a freezer until measured for serum insulin levels. Serum
insulin is measured using an ELISA kit (ALPCO Diagnostics, Windham,
N.H.). Pharmacodynamic (PD) parameters such as time to reach
minimum blood glucose concentration (T.sub.nadir) and duration over
which the blood glucose level remains below 70% of initial value
(T.sub.70% BGL), are determined according to published methods for
subcutaneous delivery. Pharmacokinetic (PK) parameters such as time
to reach maximum serum insulin concentration (T.sub.max), mean
insulin residence time (MRT), and mean insulin absorption time
(MAT), are determined according to published methods for
subcutaneous delivery using commercially available ELISA assays
(ALPCO Diagnostics, Windham, N.H.). The blood glucose depression
curve and corresponding serum insulin profile for each formulation
may then be compared to those obtained for an insulin control to
determine any differences in the timing and extent of the insulin
action.
Example 16
In Vivo Bioactivity of Insulin-Glycogen Conjugate vs. Insulin
[0111] This example compares the in vivo bioactivity of an
insulin-glycogen conjugate with that of unmodified recombinant
human insulin (RHI). The insulin-glycogen conjugate was synthesized
according to the general method described in Example 2 using 1 g of
commercially available, unpurified oyster glycogen (Type II,
Sigma-Aldrich, St. Louis, Mo.), a CNBr to glycogen mass ratio of
0.68, and a human recombinant insulin (Sigma-Aldrich, St. Louis,
Mo.) to glycogen mass ratio of 0.60. The resulting purified
material contained 1.0 wt % of insulin per insulin-glycogen
conjugate as measured using the method described in Example 10. The
bioactivity of the insulin-glycogen conjugate was evaluated at 2.5
equivalent U of insulin/kg according to the general method
described in Example 13. In addition, the bioactivity of unmodified
human recombinant insulin was evaluated at 1 U/kg.
Example 17
In Vivo Safety of Insulin-Glycogen Conjugate
[0112] Subcutaneous injections of (i) tris buffer, pH 7.4 with 150
mM sodium chloride (negative control), (ii) insulin-glycogen
conjugate in tris buffer, (iii) human recombinant insulin in tris
buffer, and (iv) human recombinant insulin plus complete Freund's
adjuvant (positive control), were each administered using a 0.25 ml
injection behind the neck of two separate fasted normal rat groups
(Sprague Dawley, male, 200-225 g, n=4) on days 0, 7, 14, 21, and
28. The insulin-glycogen conjugates were prepared according to the
method of Example 3. As rats gained weight over the course of the
study, the volume of injection was adjusted to keep the dose per
kilogram of body weight constant. On days 7-28, positive control
groups received mixtures of recombinant human insulin plus
incomplete Freund's adjuvant.
[0113] Serum samples were collected via tail vein bleeding on days
28 and 56, and serum concentrations of specific anti-human insulin
antibodies of all Ig classes were measured using a Total Insulin
Antibody (Total IAB) assay (Esoterix, Calabasa Hills, Calif.).
Briefly, test and control serum samples were first treated with
acidified-charcoal to remove unbound insulin and strip the insulin
from IAB complexes. The insulin-bound charcoal was removed from the
serum by centrifugation. The IAB-containing supernatant was then
incubated for 72 hours at 4.degree. C. with radiolabeled insulin.
As a control for nonspecific binding (NSB), unlabeled insulin was
added to replicate samples to compete with radiolabeled insulin for
IAB binding. Polyethylene glycol (PEG) was added to precipitate
antigen-antibody complexes, which were then pelleted by
centrifugation, washed and counted by gamma counting. Specific IAB
binding was calculated by subtracting the NSB counts from the total
counts of the corresponding tubes. Specific counts precipitated
were converted to .mu.U of insulin based on the specific activity
of the radiolabeled insulin used in the assay. Data are presented
in FIG. 2 as total IAB binding capacity in .mu.U of insulin bound
per ml of serum tested.
Example 18
Insulin-Glycogen Conjugate Behavior in Vivo
[0114] The inventors have demonstrated controllable insulin
loadings from 0.5-15% w/w of insulin-glycogen conjugates and found
that a 7% w/w loading is suitable for long-term repeated dose
studies in STZ-diabetic rats (FIG. 3). As noted previously, other
competitive binding applications attempt to release unmodified
insulin from a reservoir by trapping a glucose-responsive gel
between porous membranes. However, the use of support membranes
ultimately leads to a complex system with slow diffusion rates.
Consequently, excessively high glucose concentrations (>400
mg/dl) are required to significantly increase insulin diffusion.
Furthermore, once glucose is removed from the system, the decrease
in insulin release rate lags behind by several hours. In addition,
devices such as those based on glucose-responsive
membrane-controlled reservoirs will always require maintenance and
therefore repeated invasive surgery.
[0115] The in vivo digestion behavior of the insulin-glycogen
conjugate was assessed via subcutaneous injection (alone without a
glucose-binding molecule such as Con A) into normal male SD rats.
As shown in FIG. 4, the times to reach the serum insulin peak
(T.sub.max) and glucose nadir (T.sub.nadir) concentrations were
found to be between 30-45 minutes after injection, serum
immunoreactive insulin half-life was less than 120 minutes, and the
time to return to within 70% of fasting glucose levels (T.sub.70%
BGL) was less than 180 minutes. Therefore, despite being chemically
bound to a >100,000 kDa polymer, the insulin acts just as
rapidly as unconjugated insulin in vivo.
[0116] SDS-PAGE has confirmed that the insulin-glycogen conjugate
is essentially free from unconjugated insulin (FIG. 5a). SDS-PAGE
on insulin-glycogen incubated with amylase, however, shows an
increase in native insulin band intensity and a corresponding
decrease in high MW insulin-glycogen band intensity (FIG. 5a).
Therefore, unlike insulin-dextran, insulin-glycogen is rapidly
degraded by amylases in vitro and in vivo to release bioactive
insulin with a MW nearly identical to unmodified insulin, thereby
preserving its pharmacological activity. To further illustrate this
point, conjugate constructed from FITC-labeled glycogen was
injected s.c. at a dose of 12 U/kg into n=2 non-diabetic JV/JV rats
and the glucose clamped at 111+23 mg/dl for the following 7 hrs.
Serum was collected from the other catheter as a function of time
and analyzed for fluorescence (fmax, Molecular Devices, Sunnyvale,
Calif.) to determine glycogen absorption rate and insulin levels
(RIA, Joslin Diabetes Center). FIG. 5b demonstrates the rapid
glycogen absorption and elimination rate and nearly a one-to-one
correspondence between serum fluorescence and insulin
concentrations. Furthermore, the inventors have also confirmed the
conjugate bioactivity in non-diabetic felines using a euglycemic
clamp protocol and verified that the amylase degradation-enabled
PK/PD parameters hold between species (FIG. 6).
Comparative Example 19
Insulin-Dextran vs. Insulin-Glycogen Conjugates
[0117] U.S. Patent Application Publication No. 2004-0202719 to Zion
et al. describes insulin-dextran conjugates. These conjugates fail
to form insoluble cross-linked gels in the presence of multivalent
glucose-binding agents unless the molecular weight (MW) of the
dextran component is greater than 70K. However, subcutaneously
administered conjugates with dextran MW >70K are very slowly
absorbed into the systemic circulation. If the MW is decreased to a
sufficiently low value the insulin-dextran conjugate will be
absorbed more rapidly, but the system will be soluble (i.e., like
the Brownlee system of U.S. Pat. No. 4,348,387). Although Zion et
al. make reference to increasing the biological activity of the
insulin-dextran conjugate by releasing insulin from a cleavable
conjugate, they state that the cleavable conjugate is first
absorbed into systemic circulation where it is then rapidly
cleaved. Zion et al. do not describe how the high MW
insulin-dextran conjugates can be absorbed into systemic
circulation prior to cleaving.
[0118] This example compares the in vivo pharmacodynamic profiles
of subcutaneously administered insulin-glycogen (Oyster Type II,
Sigma-Aldrich, MW.about.1,000K) and insulin-dextran (Sigma-Aldrich,
MW.about.70K). The insulin-glycogen conjugates are more rapidly
absorbed than the insulin-dextran conjugates because the former are
digested by enzymes at the site of administration.
[0119] Insulin-dextran was synthesized using a modified cyanogen
bromide (CNBr) coupling reaction. Briefly, 500 mg of dextran
(MW=70K, Sigma-Aldrich) was dissolved in 50 ml of deionized water.
56 mg of solid CNBr was added to the resulting solution and the pH
was maintained at 10.7.+-.0.2 using 5 N NaOH solution. After
stirring for 15 min, another 56 mg of solid CNBr was added and the
pH was maintained at 10.7.+-.0.2 while stirring for 45 min. 300 mg
of human recombinant insulin was then added to the solution, and
the pH was adjusted to 9.15 using solid sodium bicarbonate. The
solution was stirred overnight, ultrafiltered exhaustively against
DI water using a 10K MWCO polyethersulfone disc membrane filter
(Millipore, Bedford, Mass.), and lyophilized. The resulting powder
was then purified from unconjugated insulin by high-performance
liquid chromatography (Waters, Milford, Mass.) using a 1 M acetic
acid mobile phase over a Superdex.TM. 75 packed column (Amersham
Biosciences, Piscataway, N.J.). The insulin-dextran fraction was
then lyophilized to obtain the conjugate as a pure powder
(InsFITC-Dex70K or InsTRITC-ManDex70K). The degree of insulin
conjugation was 10% (w/w) as determined by the method of Example
10.
[0120] Subcutaneous injections of insulin-dextran were administered
using 0.25 ml of a sterilized 1.times. PBS solution (0.88
equivalent mg insulin/ml) behind the neck of two fasted normal rat
groups (Male Sprague-Dawley, 200-250 g, n=4). Measurements of
glucose and insulin values from tail vein blood samples were
performed at -15 and 0 minutes, and at 15, 30, 45, 60, 90, 120,
180, 240, 300 and 360 minutes after injection.
[0121] As shown in FIG. 4, the times to reach the glucose nadir
(T.sub.nadir) concentration was found to be about 3 h after
injection, and the serum glucose levels remain depressed for at
least five hours post injection. Therefore, with this type of
protracted pharmacodynamic profile, it is difficult to obtain other
parameters such as time to return to within 70% of fasting glucose
levels (T.sub.70% BGL) which one can conclude was at least greater
than 2.5 hours. These results are in stark contrast with the
results shown on the same graph in FIG. 4 that were obtained under
the same conditions with an insulin-glycogen conjugate.
Comparative Example 20
FITC-Polymer Pharmacokinetic Profile
[0122] This example reinforces comparative Example 19 by using
serum fluorescence measurements to further demonstrate the
difference in pharmacokinetic profiles of glycogen (which is
rapidly degraded by enzymes) and dextran (which cannot be broken
down by enzymes). Each polymer was fluorescently labeled and
injected subcutaneously into male SD rats. The concentration of
polymer and/or breakdown products absorbed into systemic
circulation was determined by measuring the amount of fluorescence
in serum as a function of time after the injection.
[0123] Fluorescein isothiocyanate glycogen (FITC-glycogen) was
synthesized by first preparing a 2 mg/ml solution of glycogen in
0.1 M sodium carbonate buffer (pH 9) and a solution of fluoroscein
isothiocyanate (FITC) in anhydrous DMSO at 1 mg/ml. To 1 ml of the
glycogen solution, 50 .mu.L of FITC solution was added slowly in 5
.mu.L aliquots under gentle stirring at room temperature. The
reaction was then incubated in the dark for 8 hours at 4.degree. C.
followed by the addition of NH.sub.4Cl to a final concentration of
50 mM. After another 4.degree. C. incubation for 2 hours, xylene
cyanol and glycerol were added to 0.1% and 5%, respectively.
Finally, the FITC-glycogen was separated from the reaction medium
by ethanol precipitation and centrifugation followed by
redissolution in DI water. The FITC-glycogen water solution was
then purified 3.times. by repeated ethanol precipitation and
redissolution, followed by lyophilization to obtain the essentially
pure powder.
[0124] Fluorescein isothiocyanate dextrans of MW 70K, 500K
(FITC-Dex-70, FITC-Dex-500) were purchased from Sigma-Aldrich (St.
Louis, Mo.) and used without further purification.
[0125] Each FITC-polymer was dissolved in 1.times. PBS buffer at 10
mg/ml and injected sub-Q into n=3 rats at time 0. Serum was
collected every 15-30 min over the next 3.5 hours and serum
fluorescence measured using a fluorescence spectrophotometer plate
reader (Fmax, Molecular Devices, Sunnyvale, Calif.) FIG. 7
demonstrates that the rapidly degradable FITC-glycogen, despite
having a MW of .about.1000K, absorbs significantly faster into
systemic circulation than even the FITC-Dex-70 having a MW of 70K,
which is non-degradable.
IV. Materials for Controllably Releasing a Conjugate
[0126] This fourth set of examples describes the preparation of
exemplary materials for controllable releasing conjugates. The
examples also describe some of their in vitro and in vivo
properties.
Example 21
Insulin-Glycogen Based Hydrogel (I)
[0127] This example describes the preparation of an exemplary
glucose-sensitive controlled release material from insulin-glycogen
conjugates and pegylated Concanavalin A (PEG-Con A).
Insulin-Glycogen Conjugate
[0128] The insulin-glycogen conjugate was prepared according to the
CDAP coupling method of Example 3 using the insulin and glycogen of
Example 1.
Concanavalin A
[0129] Concanavalin A (Con A) is a glucose/mannose binding lectin
that exists as a tetramer at physiological pH and temperature. Con
A was produced via extraction from the jack bean (Canavalia
ensiformis) by the method of Agrawal and Goldstein (Biochim.
Biophys. Acta 147:262-271, 1967). Several commercial sources of Con
A exist, with one of the largest manufacturers being (EY Labs, San
Mateo, Calif.) which can produce between 1 g and 1 kg scales of
purified Con A. Typically, the purified Con A received from
commercial sources still contains small molecular weight protein
impurities that need to be removed before use. These impurities
were removed as described by Sophianopoulos and Sophianopoulos
(Prep. Biochem. 11:413-435, 1981). Briefly, Con A was dissolved at
a concentration of 20 mg/ml in 80 mM glycine buffer containing 1.0M
sodium chloride, 3 mM of manganese chloride, and 3 mM of calcium
chloride at a pH of 3.1. The solution was cooled to 6.degree. C.
using an ice bath, and the solution was stirred for 17 hours
overnight at 6.degree. C. The next day the solution was warmed to
room temperature, followed by heating at 45.degree. C. for 2 hours.
Upon cooling the solution was centrifuged at 1500.times.g for 15
minutes (Allegra 21R, Beckman Coulter, Fullerton, Calif.), and the
precipitate was discarded. The supernatant solution was adjusted to
a pH of 4.9 using 1 M sodium acetate solution, and the supernatant
was then ultrafiltered using an Amicon 400 ml cell (Millipore,
Bedford, Mass.) equipped with a 25 kDa molecular weight cutoff
membrane. Dialysis was carried out using pH 7 distilled water with
0.05 mM calcium chloride and 0.05 mM manganese chloride, after
which time the Con A solution was lyophilized to give a nearly
salt-free Con A powder that is free of the low molecular weight
protein impurities. The final powder purity was confirmed via
denaturing polyacrylamide gel electrophoresis (SDS-PAGE), ion
exchange chromatography, and circular dichroism (CD)
spectroscopy.
Pegylation Reagent
[0130] Pegylation reagents consist of poly(ethylene glycol) (PEG)
chains attached to a linker group that activates the PEG chains for
covalent attachment of proteins through reaction with the
.epsilon.-amino lysine residues. In this Example, PEG2-NHS-5k was
used which has a "V-shaped" structure comprising two 5 kDa
molecular weight chains of PEG attached to an NHS ester. The NHS
ester is an activated molecule that reacts easily with lysine
groups to form a covalent amide linkage. The PEG2-NHS-5k reagent is
readily available in 5 g quantities (e.g., from Nektar
Therapeutics, San Carlos, Calif.).
Pegylated Con A
[0131] Pegylated Con A (PEG-Con A) was prepared by dissolving 500
mg of Con A (EY Labs, San Mateo, Calif.) in 100 ml of a 100 mM
borate buffer (pH=10). After dissolution, the contents were heated
to 37.degree. C. using a heated water bath and temperature
controller. Next, a desired amount of PEG2-NHS-5k (Nektar
Therapeutics, San Carlos, Calif.), was dissolved in 7.67 ml of
deionized water. The PEGylation agent solution was slowly added
dropwise via a pipette to the heated Con A solution. The resulting
solution was allowed to react at 37.degree. C. for one hour, after
which time the reaction mixture was poured into 300 mL of 150 mM
phosphate buffered saline. The solution was then ultrafiltered
using a 400 ml Amicon cell (Millipore, Bedford, Mass.), a 50 kDa
MWCO membrane (Millipore, Bedford, Mass.), and a pH 7.4 aqueous
solution of 0.05 mM calcium chloride and 0.05 mM manganese chloride
to remove unreacted PEGylation agent and salts. The ultrafiltered
solution was then lyophilized (Freezemobile, Virtis, Gardiner,
N.Y.) to give the pure protein as a dry white powder.
Glucose-Sensitive Controlled Release Material
[0132] The glucose-sensitive controlled release material was
produced using a dual-syringe setup comprising two 1 ml syringes.
Briefly, 1 g of purified insulin-glycogen conjugate was dissolved
in 20 ml of 200 mM BES buffer, pH 7.4, 150 mM sodium chloride to
give a 50 mg/ml conjugate solution. In another vial, 1 g of
purified PEG-Con A was dissolved in 10 ml of 20 mM BES buffer, pH
7.4, 0.1 mM manganese chloride, and 0.1 mM calcium chloride to give
a 100 mg/ml solution.
[0133] Each solution was centrifuged at 5000.times.g (Allegra 21R,
Beckman Coulter, Fullerton, Calif.), and the supernatant was
coarse-filtered through a 0.45 .mu.m filter followed by sterile
filtration through a 0.22 .mu.m filter. 200 .mu.l of each
individual solution was mixed in a sterile centrifuge tube and
allowed to react for one hour. The resulting gel was centrifuged
and washed exhaustively under aseptic conditions followed by
aseptic loading into a 0.5 ml 27G 1/2'' insulin syringe which was
then used to administer the gel subcutaneously.
Example 22
Insulin-Glycogen Hydrogel Release Dynamics in Vitro
[0134] In certain embodiments, the glucose sensitivity of an
inventive material (e.g., a material prepared according to the
method of Example 21) can be adjusted by modifying the
characteristics of its components. Thus, modification of the
valency of the multivalent glucose-binding molecule, the degree and
MW of PEG modification, the molecular weight or insulin loading of
the insulin-glycogen conjugate, and/or substituting glucose units
with higher affinity mannose units allows precise adjustment of
glucose sensitivity. In this way, the glucose sensitivity can be
adjusted from physiologically hypoglycemic to hyperglycemic
concentrations (50-500 mg/dl). For example, chemically modified
dimeric Con A when mixed at varying ratios with tetrameric Con A
effectively shifts the glucose set point (GSP) from over 1,000
mg/dl to under 100 mg/dl (FIG. 8). Manipulation of the glucose
setpoint and the loading of the conjugate enables precise tuning of
the glucose-responsive insulin release profile.
[0135] Furthermore, the inventors have correlated the GSP curve
(FIG. 9a) to in vitro release rates in buffered saline solutions
containing varying concentrations of glucose such that the rate of
insulin-glycogen release from the gels increases by over a factor
of 50, when the glucose concentration increases from 50 to 400
mg/dl (FIG. 9b), closely resembling the relative increase in
insulin release rate for isolated islet cell cultures.
[0136] The inventors have also shown how materials with
increasingly "leaky" or low glucose set points translate into more
hypoglycemia in vivo as measured by CGS on STZ-rats. FIG. 10a shows
a GSP curve for two different formulations, which were then
administered to STZ-diabetic rats by subcutaneous injection and
tracked over time by CGS. The higher set point formulation was
capable of cycling between 50 and 120 mg/dl while the lower set
point formulation pushed the rat into hypoglycemia during the
entire duration of the experiment (FIG. 10b).
Example 23
Improved Therapeutic Window Compared to Conventional Insulin
[0137] By virtue of its glucose-responsive kinetics and ability to
shut off below a critical glucose concentration, the
glucose-sensitive material of Example 21 has a much higher
therapeutic window than commercialized long-acting insulins such as
insulin NPH (Novolin.RTM.ge NPH, Novo Nordisk A/S). Longer glucose
control with insulin NPH can only be achieved with larger doses
which ultimately lead to more hypoglycemia. FIG. 11 (left) shows
continuous glucose sensor (CGS, Guardian RT Wireless, Medtronic
Minimed) traces of STZ-rats (60 mg/kg STZ after overnight fast)
responding to increasing doses of insulin NPH ((a) 10, (b) 20 and
(c) 30 U/kg). The 10 U/kg dose is the only one that does not cause
hypoglycemia, but the rats (n=2 for each dose) returned to
hyperglycemia several hours later. The highest dose of NPH was
active for 18-20 hours but caused hypoglycemia for the entire
duration of the experiment. On the other hand, increasing the
dosage volume of the glucose-sensitive material of Example 21
extended the duration of control while maintaining normal blood
glucose levels (FIG. 11 (right), (a) 50 ul, (b) 150 ul, and (c) 200
ul gel). Most importantly, with just a 200 ul size gel, the
inventors were able to control blood glucose levels for over 24
hours without causing hypoglycemia.
Example 24
HbA.sub.1c Reduction Over One Week with Minimal Hypoglycemia
[0138] The gold-standard for determining the extent of long-term
glycemic control is a normalized HbA.sub.1c level. Achieving
normalized HbA.sub.1c values with conventional insulin preparations
is nearly impossible without risking frequent hypoglycemia.
However, the inventors have been able to reduce HbA.sub.1c to near
normal levels in STZ-induced diabetic rats (60 mg/kg STZ after
overnight fast) with a single daily subcutaneous (s.c.) injection
of the glucose-sensitive material of Example 21 while maintaining
average blood glucose values at 98+29 mg/dl and causing little to
no hypoglycemia (FIG. 12). The arrow in FIG. 12 represents the time
of the last gel injection (Day 6 of the study). The average glucose
value (solid line, 98 mg/dl) is shown with error ranges (dotted
lines, +29 mg/dl). The inlay to FIG. 12 shows the average body
weights on day 0 and day 7 demonstrating a statistically
significant 6% increase in weight over the study (p<0.05). One
week after STZ-injection, the rats were enrolled on a two-week long
daily dose regimen of 30 U/kg NPH and then switched to three weeks
of daily dosing of the inventive material. The two weeks of NPH
injections did not significantly reduce HbA.sub.1c values versus
baseline. The first two weeks of dosing with the inventive material
were used to determine the optimal volume and glucose set-point for
24-h control. In the final week, the rats were equipped with CGS
systems and tracked continuously for the final week (FIG. 12). Rats
were dosed at the same time each day with the same formulation
except in cases where the gel was found to burn out, in which case
a larger volume of the same formulation was administered earlier in
the day to return to the same dosing schedule. On average, the rats
received 150.+-.30 .mu.l of inventive gel per day. At the end of
the final week, the rats had gained weight (FIG. 12) and exhibited
HbA.sub.1c values that were almost indistinguishable from
non-diabetic rats monitored over the same period of time. After the
final day 6 injection, the rats were tracked for three more days
until they returned to hyperglycemia to demonstrate that
spontaneous islet regeneration had not occurred during the
treatment.
Example 25
Rapid On/Off Characteristics Via Glucose Clamps
[0139] The inventors have developed a method for clamping blood
glucose levels in double jugular vein (JV/JV) catheterized rats at
a desired value for extended periods of time by infusing a 50%
dextrose solution through the catheter line. In this type of
experiment two key parameters are followed: (a) the target blood
glucose level which is set by design and maintained at a particular
level by adjusting (b) the glucose infusion rate (GIR). The
diabetic rats do not produce enough endogenous insulin to achieve
blood glucose levels below 300 mg/dl even under fasting conditions.
Therefore, exogenously administered insulin (NPH or the
glucose-sensitive material of Example 21) is required to achieve
blood glucose levels below 300 mg/dl. As insulin is delivered
during the experiment, the GIR is adjusted to compensate for the
glucose-lowering activity of the insulin.
[0140] Blood glucose levels were sampled frequently via tail vein
bleeding and the glucose infusion rate (GIR) varied to compensate
for any drift. Once the GIR was dropped to zero, the NPH dosed rats
could not rapidly return to 100 mg/dl, because the insulin delivery
rate was too slow. On the other hand, a GIR increase of 4.times.
was observed when rats were dosed with an inventive gel and held at
300 vs. 100 mg/dl, indicating a glucose-responsive increase in
insulin delivery rate between the two levels. In addition, unlike
the NPH-dosed rats, each time the GIR was dropped to zero, the
gel-injected rats were capable of rapidly returning to 100 mg/dl
(<30 min), albeit with a slight overshoot. Finally, complete
dissolution of the inventive material was observed during the last
cycle when the GIR required to maintain glucose levels at 300 mg/dl
eventually decreases to zero.
[0141] In a separate experiment, three different non-diabetic rats
were clamped at 100, 200, and 300 mg/dl glucose, respectively, for
8 hrs. Blood samples were taken through the JV catheter
approximately every 60 min, centrifuged to separate cells from
serum, and analyzed for insulin content by radioimmunoassay (RIA,
Joslin Diabetes Center). The insulin levels were averaged to obtain
a steady state serum insulin level as a function of glucose
concentration. The insulin levels more than tripled as the glucose
concentration was increased from 100 to 300 mg/dl in vivo.
Example 26
Injection Site Compatibility
[0142] As described in Example 21 and previously, in certain
embodiments the multivalent binding agents of an inventive material
are chemically modified to reduce undesirable immunogenic
reactions. For example, the otherwise mitogenic, inflammogenic, and
toxic plant lectin, Con A can be modified with polyethylene glycol
(PEG) to prepare a safer multivalent binding agent (PEG-Con A). In
vivo histopathology studies have showed that specific PEG-Con A
formulations are free from fibrous and necrotic tissue at the
injection site with minimal increase in lymphocyte and macrophage
local density. Two rats from each group were euthanized on Days 3,
10, and 28. The injection site tissue was excised, fixed in
formalin, embedded in paraffin, sectioned and stained with
hematoxylin and eosin. The density of fibrous and/or necrotic
tissue as well as the presence of granulocytes, macrophages, and
lymphocytes were evaluated by a veterinary pathologist. The images
of FIG. 13 are images of the tissue samples extracted on Day 3 from
the injection site. (a) Con A-gel: Injection site contains (1) an
acellular area that is presumably part of the gel; (2) a large
region of necrotic neutrophils; and (3) a thick capsule of
proliferating fibroblasts, blood capillaries and some macrophages.
(b) PEG-Con A-gel: Injection site appears normal with the exception
that the muscle layer contained a mild to moderate population of
lymphocytes and fewer plasma cells. (c) Saline: Injection site
appears normal. Subcutaneous biocompatibility was assessed by
injecting the target material (saline, Con A-gel, or PEG-Con A-gel)
under the skin of six male SD rats.
Example 27
Formulation of an Injectable, Completely Resorbable Material
[0143] The inventive gel of Example 21, has viscosity
characteristics that allow easy loading and injection through a
standard 271/2 G insulin syringe obviating the need for invasive
surgery. Unlike a device which delivers active components from an
inactive, implantable or insertable object, all of the components
of the gel are dissolved and exposed to the body at rates that
depend on the environmental glucose concentration. This is
necessary to avoid unwanted material accumulation after repeated
daily dosing. To illustrate this point, gels were synthesized from
(i) fluorescently-labeled insulin-glycogen conjugate
(FITC-conjugate)/unlabeled Con A and (ii) unlabeled
insulin-glycogen conjugate/fluorescently-labeled Con A (FITC-Con A)
in the presence of India ink (SpeedBall.RTM. Superblack) and
injected under the skin of both normal and STZ-diabetic rats on
Days 2, 1, and 0 in order to simultaneously observe the injection
site post-mortem and monitor the rate of disappearance of each of
the components by fluorescence imaging.
[0144] The insulin-glycogen conjugate was completely eliminated
from the injection site in STZ-diabetic rats both one and two days
after injection but still remained at the injection site after one
day in normal rats. Taken together, not only is the
insulin-glycogen conjugate removed completely from the injection
site, its rate of disappearance is dependent on the glycemic state
of the animal (i.e. hyperglycemia leads to more rapid elimination
of material from the site). Similarly, the FITC-Con A was
completely eliminated two days after injection in the STZ-diabetic
rats but remained even after two days in the normal rats.
Example 28
Insulin-Glycogen Based Hydrogel (II)
[0145] This example describes the preparation of another exemplary
glucose-sensitive controlled release material from insulin-glycogen
conjugates and succinylated-Concanavalin A (s-Con A). Briefly,
0.100 ml of a 50 mg/ml insulin-glycogen conjugate solution
(prepared according to the method of Example 3, using Type II
oyster glycogen from and containing 1 wt % insulin) in 200 mM pH
7.4 BES buffer containing 1 mM CaCl.sub.2 and MnCl.sub.2 was mixed
with 0.100 ml of a 100 mg/ml succinylated-Concanavalin A solution
(EY Labs, San Mateo, Calif.) in 20 mM pH 7.4 BES buffer containing
1M NaCl, 1 mM CaCl.sub.2 and MnCl.sub.2. The two solutions were
mixed together in a centrifuge tube and allowed to gel. The
resulting gel was centrifuged and washed exhaustively to remove any
uncrosslinked insulin-glycogen and s-Con A.
[0146] 1.0 ml of 1.times. PBS containing 0 mg/dl of D-glucose was
added to one of the twenty-four 3 ml wells of a Multiwell.TM. plate
(Becton Dickinson, Franklin Lakes, N.J.). The gel was then added to
the solution and agitated for 1 hr using a microplate
incubator/shaker ("Jitterbug," Boekel Industries, Philadelphia,
Pa.) set at 37.degree. C. After 1 hr, 0.5 ml of release medium was
removed and the insulin-glycogen concentration determined by ELISA
(ALPCO Diagnostics, Windham, N.H.). The release medium was
supplemented with 0.5 ml of a 100 mg/dl glucose solution in
1.times. PBS to make a 50 mg/dl solution, and the gels were
agitated for another hour. This process was repeated for release
media with glucose concentrations of 100, 200, 400, and 800 mg/dl
for a total of 6 concentrations over 6 hr. The percent
insulin-glycogen dissolution was then calculated by normalizing the
cumulative concentration of insulin in the release medium as
measured by ELISA by that released at 100% gel dissolution. In all
cases, 100% dissolution was obtained after the 800 mg/dl glucose
incubation.
Other Embodiments
[0147] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of the specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims.
* * * * *