U.S. patent application number 15/040004 was filed with the patent office on 2016-07-07 for conjugate based systems for controlled drug delivery.
This patent application is currently assigned to SmartCells, Inc.. The applicant listed for this patent is SmartCells, Inc.. Invention is credited to Thomas M. Lancaster, Todd C. Zion.
Application Number | 20160193351 15/040004 |
Document ID | / |
Family ID | 42395986 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160193351 |
Kind Code |
A1 |
Zion; Todd C. ; et
al. |
July 7, 2016 |
CONJUGATE BASED SYSTEMS FOR CONTROLLED DRUG DELIVERY
Abstract
Conjugates which comprise a drug and a ligand which includes a
first saccharide; wherein the conjugate is characterized in that,
when the conjugate is administered to a mammal, at least one
pharmacokinetic or pharmacodynamic property of the conjugate is
sensitive to serum concentration of a second saccharide. Exemplary
conjugates and sustained release formulations are provided in
addition to methods of use and preparation.
Inventors: |
Zion; Todd C.; (Marblehead,
MA) ; Lancaster; Thomas M.; (Stoneham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SmartCells, Inc. |
Kenilworth |
NJ |
US |
|
|
Assignee: |
SmartCells, Inc.
Kenilworth
NJ
|
Family ID: |
42395986 |
Appl. No.: |
15/040004 |
Filed: |
February 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14291021 |
May 30, 2014 |
9265838 |
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15040004 |
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13145532 |
Jul 20, 2011 |
9050370 |
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PCT/US10/22268 |
Jan 27, 2010 |
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14291021 |
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61252857 |
Oct 19, 2009 |
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Current U.S.
Class: |
530/303 |
Current CPC
Class: |
A61P 3/08 20180101; A61P
7/12 20180101; A61P 5/50 20180101; A61P 3/10 20180101; A61K 47/61
20170801; A61K 47/549 20170801; A61K 38/28 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48 |
Claims
1-387. (canceled)
388: A conjugate comprising an insulin molecule conjugated to one
or more ligands that are independently selected from the group
consisting of aminoethylglucose (AEG), aminoethylmannose (AEM),
aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),
.beta.-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose
(AEF) wherein the one or more ligands are indirectly conjugated to
the insulin molecule via a conjugate framework.
389: The conjugate of claim 388, wherein the insulin molecule is
conjugated via the A1 amino acid residue, via the B1 amino acid
residue, via the epsilon-amino group of Lys.sup.B29 or-via the
epsilon-amino group of Lys.sup.B3.
390-392. (canceled)
399: conjugate of claim 388, wherein the insulin molecule is
conjugated to two or more separate ligands.
400-402. (canceled)
403: The conjugate of claim 399, wherein the two or more separate
ligands are conjugated to a single conjugate framework that is also
conjugated to the insulin molecule.
404: The conjugate of claim 399, wherein the two or more separate
ligands and insulin molecule are each located on a separate branch
of a single branched conjugate framework.
405: The conjugate of claim 404, wherein the two or more separate
ligands and insulin molecule are each located on termini of
separate branches of a single branched conjugate framework.
406: The conjugate of claim 399, wherein the two or more separate
ligands are conjugated to the insulin molecule via two or more
different conjugation points.
407: The conjugate of claim 406, wherein the insulin molecule is
conjugated via the A1 amino acid residue and the epsilon-amino
group of Lys.sup.B29.
408: The conjugate of claim 407, wherein the insulin molecule is
conjugated to two separate conjugate frameworks that are each
conjugated to one or more separate ligands.
409. (canceled)
410: The conjugate of claim 407, wherein the insulin molecule is
conjugated to two separate branched conjugate frameworks that are
each conjugated to two ligands.
411: The conjugate of claim 410, wherein the ligands are located on
separate branches of the branched conjugate frameworks.
412: The conjugate of claim 411, wherein the ligands are located on
termini of separate branches of the branched conjugate
frameworks.
413: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are aminoethylglucose (AEG).
414: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are aminoethylmannose (AEM).
415: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are aminoethylbimannose (AEBM).
416: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are aminoethyltrimannose (AETM).
417: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are .beta.-aminoethyl-N-acetylglucosamine
(AEGA).
418: The conjugate of any one of claims 399-412, wherein the two or
more separate ligands are aminoethylfucose (AEF).
430: The conjugate of claim 388, wherein the insulin molecule is
selected from the group consisting of human insulin, porcine
insulin, and bovine insulin.
431: The conjugate of claim 388, wherein the insulin molecule is
selected from the group consisting of insulin lispro, insulin
aspart, and insulin glulisine.
432: The conjugate of claim 388, wherein the insulin molecule is
insulin glargine or insulin detemir.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/147,878 filed Jan. 28, 2009, U.S. Provisional
Application No. 61/159,643 filed Mar. 12, 2009, U.S. Provisional
Application No. 61/162,107 filed Mar. 20, 2009, U.S. Provisional
Application No. 61/163,084 filed Mar. 25, 2009, U.S. Provisional
Application No. 61/219,897 filed Jun. 24, 2009, U.S. Provisional
Application No. 61/223,572 filed Jul. 7, 2009, and U.S. Provisional
Application No. 61/252,857 filed Oct. 19, 2009, the content of each
of which is hereby incorporated by reference in its entirety.
BACKGROUND
[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 providing drugs to a patient 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 drugs in these prior art systems are thus not literally
"controlled," but simply provided in a slow release format 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 (e.g.,
see U.S. Pat. No. 4,348,387 to Brownlee et al.; U.S. Pat. Nos.
5,830,506, 5,902,603, and 6,410,053 to Taylor et al. and U.S.
Patent Application Publication No. 2004-0202719 to Zion et
al.).
[0004] Each of these systems relies on the combination of a
multivalent glucose binding molecule (e.g., the lectin Con A) and a
sugar based component that is reversibly bound by the multivalent
glucose binding molecule. Unfortunately, Con A and many of the
other readily available lectins have the potential to stimulate
lymphocyte proliferation. By binding to carbohydrate receptors on
the surfaces of certain types of lymphocytes, these so-called
"mitogenic" lectins can potentially induce the mitosis of
lymphocytes and thereby cause them to proliferate. Most mitogenic
lectins including Con A are selective T-cell mitogens. A few
lectins are less selective and stimulate both T-cells and B-cells.
Local or systemic in vivo exposure to mitogenic lectins can result
in inflammation, cytotoxicity, macrophage digestion, and allergic
reactions including anaphylaxis. In addition, plant lectins are
known to be particularly immunogenic, giving rise to the production
of high titers of anti-lectin specific antibodies. It will be
appreciated that mitogenic lectins cannot therefore be used in
their native form for in vivo methods and devices unless great care
is taken to prevent their release. For example, in U.S. Pat. No.
5,830,506, Taylor highlights the toxic risks that are involved in
using Con A and emphasizes the importance and difficulty of
containing Con A within a drug delivery device that also requires
glucose and insulin molecules to diffuse freely in and out of the
device.
[0005] The risks and difficulties that are involved with these and
other in vivo uses of lectins could be significantly diminished if
an alternative controlled drug delivery system could be provided
that did not require lectins.
SUMMARY
[0006] In one aspect, the disclosure provides methods for
controlling the pharmacokinetic (PK) and/or pharmacodynamic (PD)
profiles of a drug such as insulin in a manner that is responsive
to the systemic concentrations of a saccharide such as glucose. As
discussed in the Examples, the methods are based in part on the
discovery that when certain insulin-conjugates were modified to
include high affinity saccharide ligands they could be made to
exhibit PK/PD profiles that responded to saccharide concentration
changes even in the absence of an exogenous multivalent
saccharide-binding molecule such as Con A. This finding was
unexpected and provides an unprecedented opportunity to generate
simple lectin-free saccharide-responsive drug systems. In another
aspect, the disclosure provides exemplary conjugates and methods
for making these. In general, these conjugates include a drug and
one or more separate ligands that each includes a saccharide. In
certain embodiments, the ligands are capable of competing with a
saccharide (e.g., glucose or mannose) for binding to an endogenous
saccharide-binding molecule. In certain embodiments, the ligands
are capable of competing with glucose or mannose for binding to Con
A. As discussed in more detail below, in certain embodiments, the
ligands and drug may be covalently or non-covalently attached to a
conjugate framework. In certain embodiments, the framework is
non-polymeric. In certain embodiments, a conjugate may have a
polydispersity index of one and a MW of less than about 20,000 Da.
In certain embodiments, the conjugate is long acting (i.e.,
exhibits a PK profile that is more sustained than soluble
recombinant human insulin or RHI).
[0007] As discussed in more detail below, it is to be understood
that the methods, conjugates and formulations that are described
herein are in no way limited to the delivery of insulin and that
they can be used to deliver any drug. It is also to be understood
that the methods may be used to deliver drugs in response to
saccharides other than glucose. In particular, as discussed in the
Examples, exemplary conjugates have been shown to respond to
exogenous saccharides such as alpha-methyl mannose and L-fucose. In
certain embodiments, this can be used to prepare conjugates that
can be controlled by administration of one of these exogenous
saccharides (i.e., instead of or in addition to being controlled by
fluctuations in endogenous glucose).
DEFINITIONS
[0008] Definitions of specific functional groups, chemical terms,
and general terms used throughout the specification are described
in more detail below. For purposes of this invention, the chemical
elements are identified in accordance with the Periodic Table of
the Elements, CAS version, Handbook of Chemistry and Physics,
75.sup.th Ed., inside cover, and specific functional groups are
generally defined as described therein. Additionally, general
principles of organic chemistry, as well as specific functional
moieties and reactivity, are described in Organic Chemistry, Thomas
Sorrell, University Science Books, Sausalito, 1999; Smith and March
March's Advanced Organic Chemistry, 5.sup.th Edition, John Wiley
& Sons, Inc., New York, 2001; Larock, Comprehensive Organic
Transformations, VCH Publishers, Inc., New York, 1989; Carruthers,
Some Modern Methods of Organic Synthesis, 3.sup.rd Edition,
Cambridge University Press, Cambridge, 1987.
[0009] Acyl--As used herein, the term "acyl," refers to a group
having the general formula --C(.dbd.O)R.sup.X1,
--C(.dbd.O)OR.sup.X1, --C(.dbd.O)--O--C(.dbd.O)R.sup.X1,
--C(.dbd.O)SR.sup.X1, --C(.dbd.O)N(R.sup.X1).sub.2,
--C(.dbd.S)R.sup.X1, --C(.dbd.S)N(R.sup.X1).sub.2, and
--C(.dbd.S)S(R.sup.X1), --C(.dbd.NR.sup.X1)R.sup.X1,
--C(.dbd.NR.sup.X1)OR.sup.X1, --C(.dbd.NR.sup.X1)SR.sup.X1, and
--C(.dbd.NR.sup.X1)N(R.sup.X1).sub.2, wherein R.sup.X1 is hydrogen;
halogen; substituted or unsubstituted hydroxyl; substituted or
unsubstituted thiol; substituted or unsubstituted amino;
substituted or unsubstituted acyl; cyclic or acyclic, substituted
or unsubstituted, branched or unbranched aliphatic; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
heteroaliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched alkyl; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched alkenyl; substituted or
unsubstituted alkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, aliphaticoxy,
heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,
heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,
heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or
di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or
di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino,
or mono- or di-heteroarylamino; or two R.sup.X1 groups taken
together form a 5- to 6-membered heterocyclic ring. Exemplary acyl
groups include aldehydes (--CHO), carboxylic acids (--CO.sub.2H),
ketones, acyl halides, esters, amides, imines, carbonates,
carbamates, and ureas. Acyl substituents include, but are not
limited to, any of the substituents described herein, that result
in the formation of a stable moiety (e.g., aliphatic, alkyl,
alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl,
acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro,
hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,
alkylamino, heteroalkylamino, arylamino, heteroarylamino,
alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,
heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,
heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,
heteroarylthioxy, acyloxy, and the like, each of which may or may
not be further substituted).
[0010] Aliphatic--As used herein, the term "aliphatic" or
"aliphatic group" denotes an optionally substituted hydrocarbon
moiety that may be straight-chain (i.e., unbranched), branched, or
cyclic ("carbocyclic") and may be completely saturated or may
contain one or more units of unsaturation, but which is not
aromatic. Unless otherwise specified, aliphatic groups contain 1-12
carbon atoms. In some embodiments, aliphatic groups contain 1-6
carbon atoms. In some embodiments, aliphatic groups contain 1-4
carbon atoms, and in yet other embodiments aliphatic groups contain
1-3 carbon atoms. Suitable aliphatic groups include, but are not
limited to, linear or branched, alkyl, alkenyl, and alkynyl groups,
and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl
or (cycloalkyl)alkenyl.
[0011] Alkenyl--As used herein, the term "alkenyl" denotes an
optionally substituted monovalent group derived from a straight- or
branched-chain aliphatic moiety having at least one carbon-carbon
double bond by the removal of a single hydrogen atom. In certain
embodiments, the alkenyl group employed in the invention contains
2-6 carbon atoms. In certain embodiments, the alkenyl group
employed in the invention contains 2-5 carbon atoms. In some
embodiments, the alkenyl group employed in the invention contains
2-4 carbon atoms. In another embodiment, the alkenyl group employed
contains 2-3 carbon atoms. Alkenyl groups include, for example,
ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the
like.
[0012] Alkyl--As used herein, the term "alkyl" refers to optionally
substituted saturated, straight- or branched-chain hydrocarbon
radicals derived from an aliphatic moiety containing between 1-6
carbon atoms by removal of a single hydrogen atom. In some
embodiments, the alkyl group employed in the invention contains 1-5
carbon atoms. In another embodiment, the alkyl group employed
contains 1-4 carbon atoms. In still other embodiments, the alkyl
group contains 1-3 carbon atoms. In yet another embodiment, the
alkyl group contains 1-2 carbons. Examples of alkyl radicals
include, but are not limited to, methyl, ethyl, n-propyl,
isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl,
tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl,
n-octyl, n-decyl, n-undecyl, dodecyl, and the like.
[0013] Alkynyl--As used herein, the term "alkynyl" refers to an
optionally substituted monovalent group derived from a straight- or
branched-chain aliphatic moiety having at least one carbon-carbon
triple bond by the removal of a single hydrogen atom. In certain
embodiments, the alkynyl group employed in the invention contains
2-6 carbon atoms. In certain embodiments, the alkynyl group
employed in the invention contains 2-5 carbon atoms. In some
embodiments, the alkynyl group employed in the invention contains
2-4 carbon atoms. In another embodiment, the alkynyl group employed
contains 2-3 carbon atoms. Representative alkynyl groups include,
but are not limited to, ethynyl, 2-propynyl (propargyl),
1-propynyl, and the like.
[0014] Aryl--As used herein, the term "aryl" used alone or as part
of a larger moiety as in "aralkyl", "aralkoxy", or "aryloxyalkyl",
refers to an optionally substituted monocyclic and bicyclic ring
systems having a total of five to 10 ring members, wherein at least
one ring in the system is aromatic and wherein each ring in the
system contains three to seven ring members. The term "aryl" may be
used interchangeably with the term "aryl ring". In certain
embodiments of the present invention, "aryl" refers to an aromatic
ring system which includes, but not limited to, phenyl, biphenyl,
naphthyl, anthracyl and the like, which may bear one or more
substituents.
[0015] Arylalkyl--As used herein, the term "arylalkyl" refers to an
alkyl group substituted with an aryl group (e.g., an aromatic or
heteroaromatic group).
[0016] Bivalent hydrocarbon chain--As used herein, the term
"bivalent hydrocarbon chain" (also referred to as a "bivalent
alkylene group") is a polymethylene group, i.e.,
--(CH.sub.2).sub.z--, wherein z is a positive integer from 1 to 30,
from 1 to 20, from 1 to 12, from 1 to 8, from 1 to 6, from 1 to 4,
from 1 to 3, from 1 to 2, from 2 to 30, from 2 to 20, from 2 to 10,
from 2 to 8, from 2 to 6, from 2 to 4, or from 2 to 3. A
substituted bivalent hydrocarbon chain is a polymethylene group in
which one or more methylene hydrogen atoms are replaced with a
substituent. Suitable substituents include those described below
for a substituted aliphatic group.
[0017] Carbonyl--As used herein, the term "carbonyl" refers to a
monovalent or bivalent moiety containing a carbon-oxygen double
bond. Non-limiting examples of carbonyl groups include aldehydes,
ketones, carboxylic acids, ester, amide, enones, acyl halides,
anhydrides, ureas, carbamates, carbonates, thioesters, lactones,
lactams, hydroxamates, isocyanates, and chloroformates.
[0018] Cycloaliphatic--As used herein, the terms "cycloaliphatic",
"carbocycle", or "carbocyclic", used alone or as part of a larger
moiety, refer to an optionally substituted saturated or partially
unsaturated cyclic aliphatic monocyclic or bicyclic ring systems,
as described herein, having from 3 to 10 members. Cycloaliphatic
groups include, without limitation, cyclopropyl, cyclobutyl,
cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,
cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In
some embodiments, the cycloalkyl has 3-6 carbons.
[0019] Halogen--As used herein, the terms "halo" and "halogen"
refer to an atom selected from fluorine (fluoro, --F), chlorine
(chloro, --Cl), bromine (bromo, --Br), and iodine (iodo, --I).
[0020] Heteroaliphatic--As used herein, the terms "heteroaliphatic"
or "heteroaliphatic group", denote an optionally substituted
hydrocarbon moiety having, in addition to carbon atoms, from one to
five heteroatoms, that may be straight-chain (i.e., unbranched),
branched, or cyclic ("heterocyclic") and may be completely
saturated or may contain one or more units of unsaturation, but
which is not aromatic. Unless otherwise specified, heteroaliphatic
groups contain 1-6 carbon atoms wherein 1-3 carbon atoms are
optionally and independently replaced with heteroatoms selected
from oxygen, nitrogen and sulfur. In some embodiments,
heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon
atoms are optionally and independently replaced with heteroatoms
selected from oxygen, nitrogen and sulfur. In yet other
embodiments, heteroaliphatic groups contain 1-3 carbon atoms,
wherein 1 carbon atom is optionally and independently replaced with
a heteroatom selected from oxygen, nitrogen and sulfur. Suitable
heteroaliphatic groups include, but are not limited to, linear or
branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.
[0021] Heteroaralkyl--As used herein, the term "heteroaralkyl"
refers to an alkyl group substituted by a heteroaryl, wherein the
alkyl and heteroaryl portions independently are optionally
substituted.
[0022] Heteroaryl--As used herein, the term "heteroaryl" used alone
or as part of a larger moiety, e.g., "heteroaralkyl", or
"heteroaralkoxy", refers to an optionally substituted group having
5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10,
or 14 it electrons shared in a cyclic array; and having, in
addition to carbon atoms, from one to five heteroatoms. Heteroaryl
groups include, without limitation, thienyl, furanyl, pyrrolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,
oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,
pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,
naphthyridinyl, and pteridinyl. The terms "heteroaryl" and
"heteroar-", as used herein, also include groups in which a
heteroaromatic ring is fused to one or more aryl, carbocyclic, or
heterocyclic rings, where the radical or point of attachment is on
the heteroaromatic ring. Non limiting examples include indolyl,
isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl,
benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl,
phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl,
carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl,
tetrahydroquinolinyl, and tetrahydroisoquinolinyl. A heteroaryl
group may be mono- or bicyclic. The term "heteroaryl" may be used
interchangeably with the terms "heteroaryl ring", "heteroaryl
group", or "heteroaromatic", any of which terms include rings that
are optionally substituted.
[0023] Heteroatom--As used herein, the term "heteroatom" refers to
nitrogen, oxygen, or sulfur, and includes any oxidized form of
nitrogen or sulfur, and any quaternized form of a basic nitrogen.
The term "nitrogen" also includes a substituted nitrogen.
[0024] Heterocyclic--As used herein, the terms "heterocycle",
"heterocyclyl", "heterocyclic radical", and "heterocyclic ring" are
used interchangeably and refer to a stable optionally substituted
5- to 7-membered monocyclic or 7- to 10-membered bicyclic
heterocyclic moiety that is either saturated or partially
unsaturated, and having, in addition to carbon atoms, one or more
heteroatoms, as defined above. A heterocyclic ring can be attached
to its pendant group at any heteroatom or carbon atom that results
in a stable structure and any of the ring atoms can be optionally
substituted. Examples of such saturated or partially unsaturated
heterocyclic radicals include, without limitation,
tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl,
piperidinyl, pyrrolinyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl,
piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl,
thiazepinyl, morpholinyl, and quinuclidinyl. The terms
"heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclic
group", "heterocyclic moiety", and "heterocyclic radical", are used
interchangeably herein, and also include groups in which a
heterocyclyl ring is fused to one or more aryl, heteroaryl, or
carbocyclic rings, such as indolinyl, 3H-indolyl, chromanyl,
phenanthridinyl, or tetrahydroquinolinyl, where the radical or
point of attachment is on the heterocyclyl ring. A heterocyclyl
group may be mono- or bicyclic. The term "heterocyclylalkyl" refers
to an alkyl group substituted by a heterocyclyl, wherein the alkyl
and heterocyclyl portions independently are optionally
substituted.
[0025] Unsaturated--As used herein, the term "unsaturated", means
that a moiety has one or more double or triple bonds.
[0026] Partially unsaturated--As used herein, the term "partially
unsaturated" refers to a ring moiety that includes at least one
double or triple bond. The term "partially unsaturated" is intended
to encompass rings having multiple sites of unsaturation, but is
not intended to include aryl or heteroaryl moieties, as herein
defined.
[0027] Optionally substituted--As described herein, compounds of
the invention may contain "optionally substituted" moieties. In
general, the term "substituted", whether preceded by the term
"optionally" or not, means that one or more hydrogens of the
designated moiety are replaced with a suitable substituent. Unless
otherwise indicated, an "optionally substituted" group may have a
suitable substituent at each substitutable position of the group,
and when more than one position in any given structure may be
substituted with more than one substituent selected from a
specified group, the substituent may be either the same or
different at every position. Combinations of substituents
envisioned by this invention are preferably those that result in
the formation of stable or chemically feasible compounds. The term
"stable", as used herein, refers to compounds that are not
substantially altered when subjected to conditions to allow for
their production, detection, and, in certain embodiments, their
recovery, purification, and use for one or more of the purposes
disclosed herein.
[0028] Suitable monovalent substituents on a substitutable carbon
atom of an "optionally substituted" group are independently
halogen; --(CH.sub.2).sub.0-4R.sup..largecircle.;
--(CH.sub.2).sub.0-4OR.sup..largecircle.;
--O--(CH.sub.2).sub.0-4C(O)OR.sup..largecircle.;
--(CH.sub.2).sub.0-4CH(OR.sup..largecircle.).sub.2;
--(CH.sub.2).sub.0-4SR.sup..largecircle.; --(CH.sub.2).sub.0-4Ph,
which may be substituted with R.sup..largecircle.;
--(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1Ph which may be substituted
with R.sup..largecircle.; --CH.dbd.CHPh, which may be substituted
with R.sup..largecircle.; --NO.sub.2; --CN; --N.sub.3;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.).sub.2;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)R.sup..largecircle.;
--N(R.sup..largecircle.)C(S)R.sup..largecircle.;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)NR.sup..largecircle..sub.2;
--N(R.sup..largecircle.)C(S)NR.sup..largecircle..sub.2;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)OR.sup..largecircle.;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)R.sup..largecircle.;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)NR.sup..largecircle..su-
b.2;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)OR.sup..largecircle-
.; --(CH.sub.2).sub.0-4C(O)R.sup..largecircle.;
--C(S)R.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)OR.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)SR.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)OSiR.sup..largecircle..sub.3;
--(CH.sub.2).sub.0-4OC(O)R.sup..largecircle.;
--OC(O)(CH.sub.2).sub.0-4SR--, SC(S)SR.sup..largecircle.;
--(CH.sub.2).sub.0-4SC(O)R.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)NR.sup..largecircle..sub.2;
--C(S)NR.sup..largecircle..sub.2; --C(S)SR.sup..largecircle.;
--SC(S)SR.sup..largecircle.,
--(CH.sub.2).sub.0-4OC(O)NR.sup..largecircle..sub.2;
--C(O)N(OR.sup..largecircle.)R.sup..largecircle.;
--C(O)C(O)R.sup..largecircle.;
--C(O)CH.sub.2C(O)R.sup..largecircle.;
--C(NOR.sup..largecircle.)R.sup..largecircle.;
--(CH.sub.2).sub.0-4SSR.sup..largecircle.;
--(CH.sub.2).sub.0-4S(O).sub.2R.sup..largecircle.;
--(CH.sub.2).sub.0-4S(O).sub.2OR.sup..largecircle.;
--(CH.sub.2).sub.0-4OS(O).sub.2R.sup..largecircle.;
--S(O).sub.2NR.sup..largecircle..sub.2;
--(CH.sub.2).sub.0-4S(O)R.sup..largecircle.;
--N(R.sup..largecircle.)S(O).sub.2NR.sup..largecircle..sub.2;
--N(R.sup..largecircle.)S(O).sub.2R.sup..largecircle.;
--N(OR.sup..largecircle.)R.sup..largecircle.;
--C(NH)NR.sup..largecircle..sub.2; --P(O).sub.2R.sup..largecircle.;
--P(O)R.sup..largecircle..sub.2; --OP(O)R.sup..largecircle..sub.2;
--OP(O)(OR.sup..largecircle.).sub.2; SiR.sup..largecircle..sub.3;
--(C.sub.1-4 straight or branched
alkylene)O--N(R.sup..largecircle.).sub.2; or --(C.sub.1-4 straight
or branched alkylene)C(O)O--N(R.sup..largecircle.).sub.2, wherein
each R.sup..largecircle. may be substituted as defined below and is
independently hydrogen, C.sub.1-6 aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, or, notwithstanding the
definition above, two independent occurrences of
R.sup..largecircle., taken together with their intervening atom(s),
form a 3-12-membered saturated, partially unsaturated, or aryl
mono- or bicyclic ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur, which may be substituted
as defined below.
[0029] Suitable monovalent substituents on R.sup..largecircle. (or
the ring formed by taking two independent occurrences of
R.sup..largecircle. together with their intervening atoms), are
independently halogen, --(CH.sub.2).sub.0-2R.sup. , -(haloR.sup. ),
--(CH.sub.2).sub.0-2OH, --(CH.sub.2).sub.0-2OR.sup. ,
--(CH.sub.2).sub.0-2CH(OR.sup. ).sub.2; --O(haloR.sup. ), --CN,
--N.sub.3, --(CH.sub.2).sub.0-2C(O)R.sup. ,
--(CH.sub.2).sub.0-2C(O)OH, --(CH.sub.2).sub.0-2C(O)OR.sup. ,
--(CH.sub.2).sub.0-2SR.sup. , --(CH.sub.2).sub.0-2SH,
--(CH.sub.2).sub.0-2NH.sub.2, --(CH.sub.2).sub.0-2NHR.sup. ,
--(CH.sub.2).sub.0-2NR.sup. .sub.2, --NO.sub.2, --SiR.sup. .sub.3,
--OSiR.sup. .sub.3, --C(O)SR.sup. , --(C.sub.1-4 straight or
branched alkylene)C(O)OR.sup. , or --SSR.sup. wherein each R.sup.
is unsubstituted or where preceded by "halo" is substituted only
with one or more halogens, and is independently selected from
C.sub.1-4 aliphatic, --CH.sub.2Ph, --O(CH.sub.2).sub.0-1Ph, or a
5-6-membered saturated, partially unsaturated, or aryl ring having
0-4 heteroatoms independently selected from nitrogen, oxygen, or
sulfur. Suitable divalent substituents on a saturated carbon atom
of R.sup..largecircle. include .dbd.O and .dbd.S.
[0030] Suitable divalent substituents on a saturated carbon atom of
an "optionally substituted" group include the following: .dbd.O,
.dbd.S, .dbd.NNR*.sub.2, .dbd.NNHC(O)R*, .dbd.NNHC(O)OR*,
.dbd.NNHS(O).sub.2R*, .dbd.NR*, .dbd.NOR*,
--O(C(R*.sub.2)).sub.2-3O--, or --S(C(R*.sub.2)).sub.2-3S--,
wherein each independent occurrence of R* is selected from
hydrogen, C.sub.1-6 aliphatic which may be substituted as defined
below, or an unsubstituted 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur. Suitable divalent
substituents that are bound to vicinal substitutable carbons of an
"optionally substituted" group include: --O(CR*.sub.2).sub.2-3O--,
wherein each independent occurrence of R* is selected from
hydrogen, C.sub.1-6 aliphatic which may be substituted as defined
below, or an unsubstituted 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0031] Suitable substituents on the aliphatic group of R* include
halogen, --R.sup. , -(haloR.sup. ), --OH, --OR.sup. ,
--O(haloR.sup. ), --CN, --C(O)OH, --C(O)OR.sup. , --NH.sub.2,
--NHR.sup. , --NR.sup. .sub.2, or --NO.sub.2, wherein each R.sup.
is unsubstituted or where preceded by "halo" is substituted only
with one or more halogens, and is independently C.sub.1-4
aliphatic, --CH.sub.2Ph, --O(CH.sub.2).sub.0-1Ph, or a 5-6-membered
saturated, partially unsaturated, or aryl ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
[0032] Suitable substituents on a substitutable nitrogen of an
"optionally substituted" group include --R.sup..dagger.,
--NR.sup..dagger..sub.2, --C(O)R.sup..dagger.,
--C(O)OR.sup..dagger., --C(O)C(O)R.sup..dagger.,
--C(O)CH.sub.2C(O)R.sup..dagger., --S(O).sub.2R.sup..dagger.,
--S(O).sub.2NR.sup..dagger..sub.2, --C(S)NR.sup..dagger..sub.2,
--C(NH)NR.sup..dagger..sub.2, or
--N(R.sup..dagger.)S(O).sub.2R.sup..dagger.; wherein each
R.sup..dagger. is independently hydrogen, C.sub.1-6 aliphatic which
may be substituted as defined below, unsubstituted --OPh, or an
unsubstituted 5-6-membered saturated, partially unsaturated, or
aryl ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, or, notwithstanding the definition
above, two independent occurrences of R.sup..dagger., taken
together with their intervening atom(s) form an unsubstituted
3-12-membered saturated, partially unsaturated, or aryl mono- or
bicyclic ring having 0-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
[0033] Suitable substituents on the aliphatic group of
R.sup..dagger. are independently halogen, --R.sup. , -(haloR.sup.
), --OH, --OR.sup. , --O(haloR.sup. ), --CN, --C(O)OH,
--C(O)OR.sup. , --NH.sub.2, --NHR.sup. , --NR.sup. .sub.2, or
--NO.sub.2, wherein each R.sup. is unsubstituted or where preceded
by "halo" is substituted only with one or more halogens, and is
independently C.sub.1-4 aliphatic, --CH.sub.2Ph,
--O(CH.sub.2).sub.0-1Ph, or a 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
[0034] Suitable protecting group--As used herein, the term
"suitable protecting group," refers to amino protecting groups or
hydroxyl protecting groups depending on its location within the
compound and includes those described in detail in Protecting
Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,
3.sup.rd edition, John Wiley & Sons, 1999.
[0035] Suitable amino-protecting groups include methyl carbamate,
ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),
9-(2-sulfo)fluorenylmethyl carbamate,
9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),
1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl
carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl
carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl
carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate,
benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl
derivative, N'-p-toluenesulfonylaminocarbonyl derivative,
N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide,
chloroacetamide, trichloroacetamide, trifluoroacetamide,
phenylacetamide, 3-phenylpropanamide, picolinamide,
3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,
p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,
acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,
4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide
(Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,
N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),
5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one,
5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one,
1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine,
N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine,
amine N-oxide, diphenylphosphinamide (Dpp),
dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt),
dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl
phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide
(Nps), 2,4-dinitrobenzenesulfenamide,
pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,
triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), .beta.-trimethyl silylethanesulfonamide
(SES), 9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and
phenacylsulfonamide.
[0036] Suitable hydroxyl protecting groups include methyl,
methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,
(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),
p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),
guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),
siloxymethyl, 2-methoxyethoxymethyl (MEM),
2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,
2-(trimethyl silyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),
3-bromotetrahydropyranyl, tetrahydrothiopyranyl,
1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP),
4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl
S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl
(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethyl silylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),
2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl
carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl
p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl
p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate,
alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl
S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl
dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,
4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,
2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,
4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxycarbonyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts). For protecting 1,2- or 1,3-diols, the protecting groups
include methylene acetal, ethylidene acetal, 1-t-butylethylidene
ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene
acetal, 2,2,2-trichloroethylidene acetal, acetonide,
cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene
ketal, benzylidene acetal, p-methoxybenzylidene acetal,
2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,
2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene
acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho
ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene
ortho ester, .alpha.-methoxybenzylidene ortho ester,
1-(N,N-dimethylamino)ethylidene derivative,
.alpha.-(N,N'-dimethylamino)benzylidene derivative,
2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),
1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),
tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic
carbonates, cyclic boronates, ethyl boronate, and phenyl
boronate.
[0037] In any case where a chemical variable (e.g., an R group) is
shown attached to a bond that crosses a bond of ring, this means
that one or more such variables are optionally attached to the ring
having the crossed bond. Each R group on such a ring can be
attached at any suitable position, this is generally understood to
mean that the group is attached in place of a hydrogen atom on the
parent ring. This includes the possibility that two R groups can be
attached to the same ring atom. Furthermore, when more than one R
group is present on a ring, each may be the same or different than
other R groups attached thereto, and each group is defined
independently of other groups that may be attached elsewhere on the
same molecule, even though they may be represented by the same
identifier.
[0038] Biodegradable--As used herein, the term "biodegradable"
refers to molecules that degrade (i.e., lose at least some of their
covalent structure) under physiological or endosomal conditions.
Biodegradable molecules are not necessarily hydrolytically
degradable and may require enzymatic action to degrade.
[0039] Biomolecule--As used herein, the term "biomolecule" refers
to molecules (e.g., polypeptides, amino acids, polynucleotides,
nucleotides, polysaccharides, sugars, lipids, nucleoproteins,
glycoproteins, lipoproteins, steroids, metabolites, 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.
[0040] Drug--As used herein, the term "drug" refers to small
molecules or biomolecules that alter, inhibit, activate, or
otherwise affect a biological event. 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, 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.
[0041] Exogenous--As used herein, an "exogenous" molecule is one
which is not present at significant levels in a patient unless
administered to the patient. In certain embodiments the patient is
a mammal, e.g., a human, a dog, a cat, a rat, a minipig, etc. As
used herein, a molecule is not present at significant levels in a
patient if normal serum for that type of patient includes less than
0.1 mM of the molecule. In certain embodiments normal serum for the
patient may include less than 0.08 mM, less than 0.06 mM, or less
than 0.04 mM of the molecule.
[0042] Hyperbranched--As used herein, a "hyperbranched" structure
is a covalent structure that includes at least one branched branch
(e.g., a dendrimeric structure). A hyperbranched structure may
include polymeric and/or non-polymeric substructures.
[0043] Normal serum--As used herein, "normal serum" is serum
obtained by pooling approximately equal amounts of the liquid
portion of coagulated whole blood from five or more non-diabetic
patients. A non-diabetic human patient is a randomly selected 18-30
year old who presents with no diabetic symptoms at the time blood
is drawn.
[0044] Polymer--As used herein, a "polymer" or "polymeric
structure" is a structure that includes a string of covalently
bound 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.
[0045] 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-deazadenosine, 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).
[0046] 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.
[0047] Polysaccharide--As used herein, a "polysaccharide" is a
polymer of saccharides. The terms "polysaccharide", "carbohydrate",
and "oligosaccharide", may be used interchangeably. The polymer may
include natural saccharides (e.g., arabinose, lyxose, ribose,
xylose, ribulose, xylulose, allose, altrose, galactose, glucose,
gulose, idose, mannose, talose, fructose, psicose, sorbose,
tagatose, mannoheptulose, sedoheptulose, octolose, and sialose)
and/or modified saccharides (e.g., 2'-fluororibose, 2'-deoxyribose,
and hexose). Exemplary disaccharides include sucrose, lactose,
maltose, trehalose, gentiobiose, isomaltose, kojibiose,
laminaribiose, mannobiose, melibiose, nigerose, rutinose, and
xylobiose.
[0048] 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 Da. 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, are all considered
acceptable for use in accordance with the present invention.
[0049] Treat--As used herein, the term "treat" (or "treating",
"treated", "treatment", etc.) refers to the administration of a
conjugate of the present disclosure to a subject in need thereof
with the purpose to alleviate, relieve, alter, ameliorate, improve
or affect a condition (e.g., diabetes), a symptom or symptoms of a
condition (e.g., hyperglycemia), or the predisposition toward a
condition.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1: Comparison between RP-HPLC chromatograms obtained
for (a) exemplary conjugate synthesized using TSAT-C6 as the
scaffold, AEM as the ligand, and NH.sub.2--B1-BOC2(A1,B29)-insulin
as the drug (conjugate I-1) and (b) an insulin-glycogen conjugate
synthesized according to Example 32.
[0051] FIG. 2: Accelerated stability testing (AST) aggregation
assay for conjugate I-1 (.quadrature.), conjugate I-16 (.DELTA.),
and RHI (.diamond-solid.) in PBS buffer. The conjugates demonstrate
greatly enhanced stability over pharmaceutical grade RHI.
[0052] FIG. 3: Accelerated stability testing (AST) chemical
stability results: (a) RP-HPLC AST conjugate stability and (b)
LC/MS data on AST conjugates.
[0053] FIG. 4: In vivo bioactivity in (n=4) non-diabetic, male
Sprague-Dawley (SD) rats for fresh conjugate (.tangle-solidup.) and
72 hr AST conjugate (.largecircle.). The 72 hr AST conjugate
bioactivity was indistinguishable from that of the fresh conjugate
(p>0.21 for all timepoints).
[0054] FIG. 5: Blood glucose depression profile in non-diabetic,
male SD rats (n=3) for subcutaneously injected (.tangle-solidup.)
insulin-dextran (70 K) at a dose of .about.20 U of insulin
equivalents/kg.
[0055] FIG. 6: Blood glucose depression profile in non-diabetic,
male SD rats (n=3) for subcutaneously injected (.box-solid.)
insulin-glycogen (Type II oyster) at a dose of .about.2.5 U of
insulin equivalents/kg.
[0056] FIG. 7: Blood glucose levels resulting from a 3.5 U
equivalent insulin/kg subcutaneous dose of (.diamond-solid.)
TSAT-C6-AEM-2 insulin conjugate I-1 and (.quadrature.) soluble
recombinant human insulin (RHI) in male non-diabetic SD rats. Each
set of data represents the average and standard deviation for n=6
rats.
[0057] FIG. 8: Serum insulin concentrations resulting from a 3.5 U
equivalent insulin/kg subcutaneous dose of (.diamond-solid.)
TSAT-C6-AEM-2 insulin conjugate I-1 and (.quadrature.) soluble
recombinant human insulin (RHI) in male non-diabetic SD rats. Each
set of data represents the average and standard deviation for n=6
rats.
[0058] FIG. 9: Plot of (.diamond-solid.) serum insulin and
(.largecircle.) blood glucose levels following subcutaneous
injection in non-diabetic, male SD rats at time 0 with
TSAT-C6-AEM-2 (B29-substituted) insulin conjugate I-7 (5 U/kg).
Data represents the average and standard deviation for n=3
rats.
[0059] FIG. 10: Chemical structures of AEG, AEM, AEBM and AETM. The
affinity of these saccharide based ligands for Con A increases as
shown.
[0060] FIG. 11: Chemical structures of some exemplary
non-dendrimeric conjugate intermediates. Exemplary conjugate
ligands that include a saccharide are also shown for illustrative
purposes (AEG, AEM, AEBM, AETM, AEGA and AEF).
[0061] FIG. 12: Plot of serum insulin (left) and blood glucose
(right) levels following subcutaneous injection in non-diabetic,
male SD rats at time 0 with TSAT-C6-AEM-2 insulin conjugate I-1
(.diamond-solid.), soluble recombinant human insulin,
(.largecircle.) and insulin lispro (.DELTA.) (all 3.5 U/kg). Data
represents the average and standard deviation for n=6 rats.
[0062] FIG. 13: Plot of blood glucose levels following subcutaneous
injection in non-diabetic, male SD rats (n=3 for each formulation)
at time 0 with TSAT-C6 based insulin conjugates with the different
ligands as shown. The glucose lowering response decreases as the
affinity of the ligand increases.
[0063] FIG. 14: Plot of serum insulin (left) and blood glucose
(right) levels following subcutaneous injection in non-diabetic,
male SD rats (n=3) at time 0 with TSAT-C6-AEM-2 conjugates I-1 (3.5
U/kg).
[0064] FIG. 15: Plot of serum insulin (left) and blood glucose
(right) levels following subcutaneous injection in non-diabetic,
male SD rats (n=3) at time 0 with TSAT-C6-AEBM-2 I-3 conjugate (5
U/kg).
[0065] FIG. 16: Plot of serum insulin (left) and blood glucose
(right) levels following subcutaneous injection in non-diabetic,
male SD rats (n=3) at time 0 with TSAT-C6-AEBM-1 AETM-1 conjugate
I-4 (5 U/kg).
[0066] FIG. 17: Plot of serum insulin (left) and blood glucose
(right) levels following subcutaneous injection in non-diabetic,
male SD rats (n=3) at time 0 with TSAT-C6-AETM-2 conjugate I-2 (5
U/kg).
[0067] FIG. 18: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSAT-C6-AETM-2 conjugate I-2 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AETM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is very significant
(p<0.05).
[0068] FIG. 19: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with soluble recombinant human insulin (RHI)
followed by IP injection of alpha-methyl mannose (left) or saline
(right) after 15 minutes. As shown, no change in PK/PD profile
results from injection of alpha-methyl mannose (p>>0.05).
[0069] FIG. 20: Plot of serum insulin levels following subcutaneous
injection in non-diabetic, male SD rats (n=3 for each expt.) at
time 0 with TSAT-C6-AETM-2 conjugate I-2 followed by IP injection
of alpha-methyl mannose (.diamond-solid.), L-fucose (.box-solid.)
or saline (.tangle-solidup.) after 15 minutes. As shown,
alpha-methyl mannose and L-fucose appear to exhibit the same kind
of effect.
[0070] FIG. 21: Plot of serum insulin levels following subcutaneous
injection in non-diabetic, male SD rats at time 0 with
TSAT-C6-AETM-2 conjugate I-2 followed by IP injection of glucose
(.diamond-solid.), galactose (.box-solid.) or saline
(.tangle-solidup.) after 15 minutes. As shown, galactose exhibits
no effect as compared to saline. Glucose appears to exhibit a small
effect; however, this is complicated by the fact that the exogenous
insulin from the conjugate quickly lowers the glucose, so the
sustained effect observed with alpha-methyl mannose and L-fucose
does not occur.
[0071] FIG. 22: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSAT-C6-AETM-2 conjugate I-2 solution at 5
U/kg dissolved in either (a) buffered saline containing 1M
alpha-methyl mannose or (b) buffered saline. In (a) the rats were
subsequently injected at 15 min. with the same volume of saline
solution used in (b) at a different subcutaneous site than the one
used for the conjugate solution. In (b) the rats were subsequently
injected at 15 min. with the same volume of 1M alpha-methyl mannose
solution used in (a) at a different subcutaneous site than the one
used for the conjugate solution. As shown, the serum insulin levels
do not increase and the blood glucose levels do not decrease in
experiment (a) relative to experiment (b).
[0072] FIG. 23: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSAT-C6-AETM-2 conjugate I-2 solution at 5
U/kg. At 15 min, 60 min, 120 min, or 240 min after the conjugate
injection, the rats were given a 4 g/kg IP a-MM injection.
[0073] FIG. 24: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSAT-C6-AEM-2 conjugate I-7 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is very significant
(p<0.05).
[0074] FIG. 25: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSAT-C6-GA-2 conjugate I-5 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is not significant.
[0075] FIG. 26: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with DSS-C6-AEM-1 conjugate I-8 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is as not significant.
[0076] FIG. 27: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSPE-AEM-3 conjugate I-9 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is significant (p<0.05).
[0077] FIG. 28: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with DSS-AETM-1 conjugate I-10 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is significant (p<0.05).
[0078] FIG. 29: Plot of serum insulin and blood glucose levels
following subcutaneous injection in non-diabetic, male SD rats
(n=3) at time 0 with TSPE-AETM-3 conjugate I-11 followed by IP
injection of alpha-methyl mannose (left) or saline (right) after 15
minutes. Alpha-methyl mannose is a very high affinity saccharide
which is capable of competing with AEM for binding to lectins such
as Con A. As shown, the change in PK/PD profile that results from
injection of alpha-methyl mannose is significant (p<0.05).
[0079] FIG. 30: Plot of serum insulin concentration as a function
of time for 0.4 mg/kg i.v. injections of (.diamond-solid.) RHI and
(.tangle-solidup.) TSAT-C6-AETM-2 conjugate I-6 into non-diabetic,
male SD rats (n=3 per group). Data (average of n=3) is fit using a
two-compartment bi-exponential model. The TSAT-C6-AETM-2 conjugate
is eliminated from serum much faster than RHI.
[0080] FIG. 31: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared as described in Example 51: (a) 1.times.P-1.times.Z, (b)
4.times.P-4.times.Z, and (c) 10.times.P-4.times.Z.
[0081] FIG. 32: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared as described in Example 52: (a) 4.times.P-1.times.Z and
(b) 4.times.P-2.times.Z.
[0082] FIG. 33: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared as described in Example 52: (a) 10.times.P-1.times.Z and
(b) 10.times.P-2.times.Z.
[0083] FIG. 34: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared as described in Example 53: (a) no cresol and (b) 4.times.
cresol.
[0084] FIG. 35: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared as described in Example 54: (a) no salt, (b) 3.3.times.
salt, and (c) glycerol.
[0085] FIG. 36: Plots of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting TSAT-C6-AETM-2 (I-6) conjugates followed by IP
injection of glucose (4 g/kg) at 240 minutes. Formulations were
prepared containing increasing amounts of unmodified insulin as
described in Example 55: (a) 1/24, (b) 1/12, and (c) 1/6.
[0086] FIG. 37: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with a
long-acting TSAT-C6-AETM-2 conjugate I-6 prepared according to
Example 56 followed by IP injection of glucose (4 g/kg) at 240
minutes.
[0087] FIG. 38: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with a
long-acting TSAT-C6-AETM-2 conjugate I-6 prepared according to
Example 57 followed by IP injection of glucose (4 g/kg) at 240
minutes. The material was injected after storage at 2-8 C for (a)
one week or (b) two weeks.
[0088] FIG. 39: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with a
long-acting TSAT-C6-AETM-2 conjugate I-6 prepared according to
Example 57 followed by IP injection of glucose (4 g/kg) at 240
minutes. The material was injected after storage at room
temperature for (a) one week or (b) two weeks.
[0089] FIG. 40: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting conjugate formulations prepared according to Example 58
followed by IP injection of glucose (4 g/kg) at 240 minutes. The
conjugates are DSS-AEM-1 (I-8), DSS-AETM-1 (I-10), TSAT-C6-AEM-2
(I-7), C6-amide-AEM-2 (I-17), TSPE-AEM-3 (I-9), and TSPE-AETM-3
(I-11).
[0090] FIG. 41: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with
long-acting conjugate formulations prepared according to Example 59
followed by IP injection of alpha-methyl mannose (4 g/kg) at 240
minutes. The conjugates are (a) TSAT-C6-AETM-2 (I-6) and (b)
TSAT-C6-GA-2.
[0091] FIG. 42: Plot of blood glucose levels following subcutaneous
injection in non-diabetic (normals) and diabetic (DM's) male SD
rats at time 0 with TSAT-C6-AETM-2 (B29-substituted, PZI) conjugate
I-6. The conjugate was administered at 5, 10 and 20 U/kg. As shown,
the non-diabetic male SD rats did not show any hypoglycemia while
the glucose levels in diabetic male SD rats showed a clear dose
proportional response that lasted for over 8 hours at the highest
dose.
[0092] FIG. 43: Plot of blood glucose levels over 24 hours
following subcutaneous injection in non-diabetic (normals) and
diabetic (DM's) male SD rats at time 0 with TSAT-C6-AETM-2
(B29-substituted, PZI) conjugate I-6. The conjugate was
administered at 7, 14 and 28 U/kg.
[0093] FIG. 44: Plot of serum insulin (.diamond-solid.) and blood
glucose (.largecircle.) levels following subcutaneous injection in
non-diabetic, male SD rats (n=3 per dose) at time 0 with a
long-acting conjugate I-17 formulation prepared according to
Example 67 followed by IP injection of glucose (4 g/kg) at 240
minutes.
[0094] FIG. 45: Structures of exemplary insulin-conjugates. As
described in the Examples, these conjugates were each prepared with
recombinant wild-type human insulin (see FIG. 63 for the structure
of wild-type human insulin). The symbol "insulin" inside an oval as
shown in FIG. 45 is therefore primarily intended to represent a
wild-type human insulin. As discussed herein, it is to be
understood that the present disclosure also encompasses inter alia
versions of these and other conjugates that include an insulin
molecule other than wild-type human insulin.
[0095] FIG. 46: Plots of serum insulin concentration as a function
of time following injection of conjugate I-6 or RHI (left) and
conjugate I-6 with and without glucose or .alpha.-methyl mannose
(right).
[0096] FIG. 47: The first two panels show plots of serum insulin
(.diamond-solid.) and blood glucose (.largecircle.) levels
following constant intravenous (i.v.) infusion of RHI (3.5 mU/min)
or I-6 (15 mU/min) in non-diabetic, male SD rats (n=3). IP
injection of glucose (4 g/kg) was given at 240 minutes. The next
three panels compare plots of serum insulin (.diamond-solid.) and
blood glucose (.largecircle.) levels following constant intravenous
(i.v.) infusion of RHI (3.5 mU/min) or I-6 (15 mU/min) in
non-diabetic, male SD rats (n=3) when an IP injection of glucose
(4, 2, or 1 g/kg) was given at 240 minutes.
[0097] FIG. 48: Plots of serum insulin concentration as a function
of time following injection of conjugates with and without glucose
or .alpha.-methyl mannose. Rats were infused i.v. with sugar
solution at t=-60 min and throughout study. Each conjugate was
injected at 10 U/kg i.v. at time 0, and serum conjugate
concentrations were measured.
[0098] FIG. 49: Composition of insulin conjugates tested in
non-diabetic minipig sugar-dependent elimination half-life studies.
As described in the Examples, these conjugates were each prepared
with recombinant wild-type human insulin (see FIG. 63 for the
structure of wild-type human insulin). The schematic in FIG. 49 is
therefore primarily intended to represent a wild-type human
insulin. As discussed herein, it is to be understood that the
present disclosure also encompasses inter alia versions of these
and other conjugates that include an insulin molecule other than
wild-type human insulin.
[0099] FIG. 50: .beta.-phase elimination half-life results in
non-diabetic minipigs during glucose, .alpha.-methyl mannose or
saline infusion.
[0100] FIG. 51: Plots of serum concentrations of (a) recombinant
human insulin (RHI) and (b) Di-Sub-AETM-2 insulin conjugate II-2
following a 0.1 U/kg intravenous (i.v.) injection into non-diabetic
male Yucatan minipigs equipped with dual vascular access ports (n=3
per study). In each experiment, the animals were infused with
(.diamond-solid.) i.v. alpha methyl mannose (a-MM) solution (25%
w/v infused at constant rate of 80 ml/h) or () no solution. Data
are plotted as the average values fit with a curve derived from the
two-compartment, bi-exponential model.
[0101] FIG. 52: Blood glucose depression curves in non-diabetic
male Yucatan minipigs equipped with dual vascular access ports (n=3
per study) following i.v. injection of conjugates at 0.1 U/kg under
conditions of (a) no i.v. sugar infusion or (b) i.v. alpha methyl
mannose (a-MM) infusion (25% w/v infused at constant rate of 80
ml/h). () RHI, () I-7, () I-6, () I-11, and ( ) II-2.
[0102] FIG. 53: Blood glucose levels in (a, -, closed symbols)
alloxan-diabetic Yucatan minipigs (n=3 per dose) and (b, - - - -,
open symbols) non-diabetic Yucatan minipigs (n=3 per dose) under
fasting conditions after a sub-Q injection at time 0 with soluble
Di-Sub-AETM-2 insulin conjugate II-2 at doses of 0.25, 0.50, and
1.00 U/kg. Data are plotted as the average values.+-.one standard
deviation. NOTE: FIG. 53(b) scale is enlarged for clarity.
[0103] FIG. 54: Blood glucose levels in (a, -, closed symbols)
alloxan-diabetic Yucatan minipigs (n=3 per dose) and (b, - - - -,
open symbols) non-diabetic Yucatan minipigs (n=3 per dose) under
fasting conditions after a sub-Q injection at time 0 with soluble
recombinant human insulin (RHI) at doses of ( ,.DELTA.) 0.063 and
() 0.125 U/kg. Data are plotted as the average values.+-.one
standard deviation. NOTE: FIG. 54(b) scale is enlarged for
clarity.
[0104] FIG. 55: Additional insulin conjugates for use in
non-diabetic minipig sugar-dependent elimination half-life studies.
As described in the Examples, these conjugates were each prepared
with recombinant wild-type human insulin (see FIG. 63 for the
structure of wild-type human insulin). The schematic in FIG. 55 is
therefore primarily intended to represent a wild-type human
insulin. As discussed herein, it is to be understood that the
present disclosure also encompasses inter alia versions of these
and other conjugates that include an insulin molecule other than
wild-type human insulin.
[0105] FIG. 56: Plots of serum insulin concentration as a function
of time following administration of RHI or conjugate I-6 in rats
and minipigs.
[0106] FIG. 57: Summary of i.v. half-life results in minipigs for
additional insulin-conjugates.
[0107] FIG. 58: Plot of serum insulin levels after a single
subcutaneous injection of 0.25, 0.5 and 1 U/kg insulin conjugate
II-2 in diabetic and normal minipigs.
[0108] FIG. 59: Plots of serum glucose levels after i.v. injections
of RHI and conjugates I-6, II-3 and II-2 in minipigs with and
without a-MM infusion.
[0109] FIG. 60: Structures of selected insulin conjugates (C3, C4,
and C7) tested in minipigs as controls, along with insulin
conjugates I-6, I-12, II-2, and II-3. As described in the Examples,
these conjugates were each prepared with recombinant wild-type
human insulin (see FIG. 63 for the structure of wild-type human
insulin). The schematic in FIG. 60 is therefore primarily intended
to represent a wild-type human insulin. As discussed herein, it is
to be understood that the present disclosure also encompasses inter
alia versions of these and other conjugates that include an insulin
molecule other than wild-type human insulin.
[0110] FIG. 61: Plots of serum glucose levels after i.v. injections
of RHI and insulin conjugates I-6, I-12, II-2 and C3 in minipigs
with and without a-MM infusion.
[0111] FIG. 62: Plots of serum glucose levels after i.v. injections
of RHI and insulin conjugates C7, C4, II-3 and II-2 in minipigs
with and without a-MM infusion.
[0112] FIG. 63: Structure of wild-type human insulin.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS
[0113] This application refers to a number of documents including
patent and non-patent documents. The entirety of each of these
documents is incorporated herein by reference.
[0114] In one aspect, the disclosure provides methods for
controlling the pharmacokinetic (PK) and/or pharmacodynamic (PD)
profiles of a drug such as insulin in a manner that is responsive
to the systemic concentrations of a saccharide such as glucose. As
discussed in the Examples, the methods are based in part on the
discovery that when certain insulin-conjugates were modified to
include high affinity saccharide ligands they could be made to
exhibit PK/PD profiles that responded to saccharide concentration
changes even in the absence of an exogenous multivalent
saccharide-binding molecule such as Con A. This finding was
unexpected and provides an unprecedented opportunity to generate
simple lectin-free saccharide-responsive drug systems. In another
aspect, the disclosure provides exemplary conjugates and methods
for making these. In general, these conjugates include a drug and
one or more separate ligands that each include a saccharide. In
certain embodiments, the ligands are capable of competing with a
saccharide (e.g., glucose or mannose) for binding to an endogenous
saccharide-binding molecule. In certain embodiments, the ligands
are capable of competing with glucose or mannose for binding to Con
A. As discussed in more detail below, in certain embodiments, the
ligands and drug may be covalently or non-covalently attached to a
conjugate framework. In certain embodiments, the framework is
non-polymeric. In certain embodiments, a conjugate may have a
polydispersity index of one and a MW of less than about 20,000 Da.
In certain embodiments, the conjugate is of formula (I) or (II) as
defined and described herein. In certain embodiments, the conjugate
is long acting (i.e., exhibits a PK profile that is more sustained
than soluble recombinant human insulin or RHI).
[0115] As discussed in more detail below, it is to be understood
that the methods, conjugates and formulations that are described
herein are in no way limited to the delivery of insulin and that
they can be used to deliver any drug. It is also to be understood
that the methods may be used to deliver drugs in response to
saccharides other than glucose. In particular, as discussed in the
Examples, exemplary conjugates have been shown to respond to
exogenous saccharides such as alpha-methyl mannose and L-fucose. In
certain embodiments, this can be used to prepare conjugates that
can be controlled by administration of one of these exogenous
saccharides (i.e., instead of or in addition to being controlled by
fluctuations in endogenous glucose).
Conjugates
[0116] In one aspect, the disclosure provides conjugates that
comprise a drug and a ligand that includes a first saccharide. The
ligand (or ligands when the conjugates include more than one
ligand) are such that when the conjugate is administered to a
mammal at least one pharmacokinetic or pharmacodynamic property of
the conjugate is sensitive to the serum concentration of a second
saccharide. In certain embodiments, the PK and/or PD properties of
the conjugate are sensitive to the serum concentration of an
endogenous saccharide such as glucose. In certain embodiments, the
PK and/or PD properties of the conjugate are sensitive to the serum
concentration of an exogenous saccharide, e.g., without limitation,
mannose, L-fucose, N-acetyl glucosamine and/or alpha-methyl
mannose.
[0117] As discussed in more detail below, in certain embodiments,
the ligand(s) and drug may be covalently or non-covalently attached
to a conjugate framework.
[0118] In certain embodiments, the molecular weight of the
conjugate absent the drug is less than about 10,000 Da. For
example, the molecular weight of the conjugate absent the drug may
be in the range of about 250 to about 5,000 Da, about 450 to about
3,500 Da, about 750 to about 2,500 Da, or about 900 to about 2,000
Da.
[0119] In certain embodiments, the molecular weight of the
conjugate including the drug is less than about 20,000 Da. For
example, the molecular weight of the conjugate including the drug
may be in the range of about 2,000 to about 18,000 Da, about 4,000
to about 15,000 Da, about 5,000 to about 10000 Da, or about 6,500
to about 8,000 Da.
[0120] In certain embodiments, the conjugate has a unique molecular
weight (i.e., has a polydispersity index of one).
PK and PD Properties
[0121] In various embodiments, the pharmacokinetic and/or
pharmacodynamic behavior of a conjugate (i.e., conjugated drug
and/or drug which has been released from a conjugate by chemical or
enzymatic degradation) may be modified by variations in the serum
concentration of a saccharide.
[0122] For example, from a pharmacokinetic (PK) perspective, the
serum concentration curve may shift upward when the serum
concentration of the saccharide (e.g., glucose) increases or when
the serum concentration of the saccharide crosses a threshold
(e.g., is higher than normal glucose levels).
[0123] In certain embodiments, the serum concentration curve of a
conjugate is substantially different when administered to the
mammal under fasted and hyperglycemic conditions. As used herein,
the term "substantially different" means that the two curves are
statistically different as determined by a student t-test
(p<0.05). As used herein, the term "fasted conditions" means
that the serum concentration curve was obtained by combining data
from five or more fasted non-diabetic individuals. In certain
embodiments, a fasted non-diabetic individual is a randomly
selected 18-30 year old human who presents with no diabetic
symptoms at the time blood is drawn and who has not eaten within 12
hours of the time blood is drawn. As used herein, the term
"hyperglycemic conditions" means that the serum concentration curve
was obtained by combining data from five or more fasted
non-diabetic individuals in which hyperglycemic conditions (glucose
C.sub.max at least 100 mg/dL above the mean glucose concentration
observed under fasted conditions) were induced by concurrent
administration of conjugate and glucose. Concurrent administration
of conjugate and glucose simply requires that the glucose C.sub.max
occur during the period when the conjugate is present at a
detectable level in the serum. For example, a glucose injection (or
ingestion) could be timed to occur shortly before, at the same time
or shortly after the conjugate is administered. In certain
embodiments, the conjugate and glucose are administered by
different routes or at different locations. For example, in certain
embodiments, the conjugate is administered subcutaneously while
glucose is administered orally or intravenously.
[0124] In certain embodiments, the serum C.sub.max of the conjugate
is higher under hyperglycemic conditions as compared to fasted
conditions. Additionally or alternatively, in certain embodiments,
the serum area under the curve (AUC) of the conjugate is higher
under hyperglycemic conditions as compared to fasted conditions. In
various embodiments, the serum elimination rate of the conjugate is
slower under hyperglycemic conditions as compared to fasted
conditions. As discussed in the Examples, we have found that in
certain embodiments, the serum concentration curve of the
conjugates can be fit using a two-compartment bi-exponential model
with one short and one long half-life. The long half-life appears
to be particularly sensitive to glucose concentration. Thus, in
certain embodiments, the long half-life is longer under
hyperglycemic conditions as compared to fasted conditions. In
certain embodiments, the fasted conditions involve a glucose
C.sub.max of less than 100 mg/dL (e.g., 80 mg/dL, 70 mg/dL, 60
mg/dL, 50 mg/dL, etc.). In certain embodiments, the hyperglycemic
conditions involve a glucose C.sub.max in excess of 200 mg/dL
(e.g., 300 mg/dL, 400 mg/dL, 500 mg/dL, 600 mg/dL, etc.). It will
be appreciated that other PK parameters such as mean serum
residence time (MRT), mean serum absorption time (MAT), etc. could
be used instead of or in conjunction with any of the aforementioned
parameters.
[0125] The normal range of glucose concentrations in humans, dogs,
cats, and rats is 60 to 200 mg/dL. One skilled in the art will be
able to extrapolate the following values for species with different
normal ranges (e.g., the normal range of glucose concentrations in
miniature pigs is 40 to 150 mg/dl). Glucose concentrations below 60
mg/dL are considered hypoglycemic. Glucose concentrations above 200
mg/dL are considered hyperglycemic. In certain embodiments, the PK
properties of the conjugate may be tested using a glucose clamp
method (see Examples) and the serum concentration curve of the
conjugate may be substantially different when administered at
glucose concentrations of 50 and 200 mg/dL, 50 and 300 mg/dL, 50
and 400 mg/dL, 50 and 500 mg/dL, 50 and 600 mg/dL, 100 and 200
mg/dL, 100 and 300 mg/dL, 100 and 400 mg/dL, 100 and 500 mg/dL, 100
and 600 mg/dL, 200 and 300 mg/dL, 200 and 400 mg/dL, 200 and 500
mg/dL, 200 and 600 mg/dL, etc. Additionally or alternatively, the
serum T.sub.max, serum C.sub.max, mean serum residence time (MRT),
mean serum absorption time (MAT) and/or serum half-life may be
substantially different at the two glucose concentrations. As
discussed below, in certain embodiments, 100 mg/dL and 300 mg/dL
may be used as comparative glucose concentrations. It is to be
understood however that the present disclosure encompasses each of
these embodiments with an alternative pair of comparative glucose
concentrations including, without limitation, any one of the
following pairs: 50 and 200 mg/dL, 50 and 300 mg/dL, 50 and 400
mg/dL, 50 and 500 mg/dL, 50 and 600 mg/dL, 100 and 200 mg/dL, 100
and 400 mg/dL, 100 and 500 mg/dL, 100 and 600 mg/dL, 200 and 300
mg/dL, 200 and 400 mg/dL, 200 and 500 mg/dL, 200 and 600 mg/dL,
etc.
[0126] Thus, in certain embodiments, the C.sub.max of the conjugate
is higher when administered to the mammal at the higher of the two
glucose concentrations (e.g., 300 vs. 100 mg/dL glucose). In
certain embodiments, the C.sub.max of the conjugate is at least 50%
(e.g., at least 100%, at least 200% or at least 400%) higher when
administered to the mammal at the higher of the two glucose
concentrations (e.g., 300 vs. 100 mg/dL glucose).
[0127] In certain embodiments, the AUC of the conjugate is higher
when administered to the mammal at the higher of the two glucose
concentrations (e.g., 300 vs. 100 mg/dL glucose). In certain
embodiments, the AUC of the conjugate is at least 50% (e.g., at
least e.g., at least 100%, at least 200% or at least 400%) higher
when administered to the mammal at the higher of the two glucose
concentrations (e.g., 300 vs. 100 mg/dL glucose).
[0128] In certain embodiments, the serum elimination rate of the
conjugate is slower when administered to the mammal at the higher
of the two glucose concentrations (e.g., 300 vs. 100 mg/dL
glucose). In certain embodiments, the serum elimination rate of the
conjugate is at least 25% (e.g., at least 50%, at least 100%, at
least 200%, or at least 400%) faster when administered to the
mammal at the lower of the two glucose concentrations (e.g., 100
vs. 300 mg/dL glucose).
[0129] As discussed in the Examples, we have found that in certain
embodiments the serum concentration curve of conjugates can be fit
using a two-compartment bi-exponential model with one short and one
long half-life. The long half-life appears to be particularly
sensitive to glucose concentration. Thus, in certain embodiments,
the long half-life is longer when administered to the mammal at the
higher of the two glucose concentrations (e.g., 300 vs. 100 mg/dL
glucose). In certain embodiments, the long half-life is at least
50% (e.g., at least 100%, at least 200% or at least 400%) longer
when administered to the mammal at the higher of the two glucose
concentrations (e.g., 300 vs. 100 mg/dL glucose).
[0130] In certain embodiments, the present disclosure provides a
method in which the serum concentration curve of a conjugate is
obtained at two different glucose concentrations (e.g., 300 vs. 100
mg/dL glucose); the two curves are fit using a two-compartment
bi-exponential model with one short and one long half-life; and the
long half-lives obtained under the two glucose concentrations are
compared. In certain embodiments, this method may be used as an
assay for testing or comparing the glucose sensitivity of one or
more conjugates.
[0131] In certain embodiments, the present disclosure provides a
method in which the serum concentration curves of a conjugated drug
(e.g., an insulin conjugate of the present disclosure) and an
unconjugated version of the drug (e.g., RHI) are obtained under the
same conditions (e.g., fasted conditions); the two curves are fit
using a two-compartment bi-exponential model with one short and one
long half-life; and the long half-lives obtained for the conjugated
and unconjugated drug are compared. In certain embodiments, this
method may be used as an assay for identifying conjugates that are
cleared more rapidly than the unconjugated drug.
[0132] In certain embodiments, the serum concentration curve of a
conjugate is substantially the same as the serum concentration
curve of an unconjugated version of the drug when administered to
the mammal under hyperglycemic conditions. As used herein, the term
"substantially the same" means that there is no statistical
difference between the two curves as determined by a student t-test
(p>0.05). In certain embodiments, the serum concentration curve
of the conjugate is substantially different from the serum
concentration curve of an unconjugated version of the drug when
administered under fasted conditions. In certain embodiments, the
serum concentration curve of the conjugate is substantially the
same as the serum concentration curve of an unconjugated version of
the drug when administered under hyperglycemic conditions and
substantially different when administered under fasted conditions.
In certain embodiments, the hyperglycemic conditions involve a
glucose C.sub.max in excess of 200 mg/dL (e.g., 300 mg/dL, 400
mg/dL, 500 mg/dL, 600 mg/dL, etc.). In certain embodiments, the
fasted conditions involve a glucose C.sub.max of less than 100
mg/dL (e.g., 80 mg/dL, 70 mg/dL, 60 mg/dL, 50 mg/dL, etc.). It will
be appreciated that any of the aforementioned PK parameters such as
serum T.sub.max, serum C.sub.max AUC, mean serum residence time
(MRT), mean serum absorption time (MAT) and/or serum half-life
could be compared.
[0133] From a pharmacodynamic (PD) perspective, the bioactivity of
the conjugate may increase when the glucose concentration increases
or when the glucose concentration crosses a threshold, e.g., is
higher than normal glucose levels. In certain embodiments, the
bioactivity of a conjugate is lower when administered under fasted
conditions as compared to hyperglycemic conditions. In certain
embodiments, the fasted conditions involve a glucose C.sub.max of
less than 100 mg/dL (e.g., 80 mg/dL, 70 mg/dL, 60 mg/dL, 50 mg/dL,
etc.). In certain embodiments, the hyperglycemic conditions involve
a glucose C.sub.max in excess of 200 mg/dL (e.g., 300 mg/dL, 400
mg/dL, 500 mg/dL, 600 mg/dL, etc.).
[0134] In certain embodiments, the PD properties of the conjugate
may be tested by measuring the glucose infusion rate (GIR) required
to maintain a steady glucose concentration. According to such
embodiments, the bioactivity of the conjugate may be substantially
different when administered at glucose concentrations of 50 and 200
mg/dL, 50 and 300 mg/dL, 50 and 400 mg/dL, 50 and 500 mg/dL, 50 and
600 mg/dL, 100 and 200 mg/dL, 100 and 300 mg/dL, 100 and 400 mg/dL,
100 and 500 mg/dL, 100 and 600 mg/dL, 200 and 300 mg/dL, 200 and
400 mg/dL, 200 and 500 mg/dL, 200 and 600 mg/dL, etc. Thus, in
certain embodiments, the bioactivity of the conjugate is higher
when administered to the mammal at the higher of the two glucose
concentrations (e.g., 300 vs. 100 mg/dL glucose). In certain
embodiments, the bioactivity of the conjugate is at least 25%
(e.g., at least 50% or at least 100%) higher when administered to
the mammal at the higher of the two glucose concentrations (e.g.,
300 vs. 100 mg/dL glucose).
[0135] In certain embodiments, the conjugate includes an insulin
molecule as the drug. According to such embodiments, the PD
behavior for insulin 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.
[0136] In general, it will be appreciated that any of the PK and PD
characteristics discussed in this section 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). It is also to be understood that the PK and/or PD
properties may be measured in any mammal (e.g., a human, a rat, a
cat, a minipig, a dog, etc.). In certain embodiments, PK and/or PD
properties are measured in a human. In certain embodiments, PK
and/or PD properties are measured in a rat. In certain embodiments,
PK and/or PD properties are measured in a minipig. In certain
embodiments, PK and/or PD properties are measured in a dog.
[0137] It will also be appreciated that while the foregoing was
described in the context of glucose-responsive conjugates, the same
properties and assays apply to conjugates that are responsive to
other saccharides including exogenous saccharides, e.g., mannose,
L-fucose, N-acetyl glucosamine, alpha-methyl mannose, etc. As
discussed in more detail below and in the Examples, instead of
comparing PK and/or PD properties under fasted and hyperglycemic
conditions, the PK and/or PD properties may be compared under
fasted conditions with and without administration of the exogenous
saccharide. It is to be understood that conjugates can be designed
that respond to different C.sub.max values of a given exogenous
saccharide.
Ligand(s)
[0138] In general, the conjugates include at least one ligand. In
certain embodiments, the conjugates include a single ligand. In
certain embodiments, the conjugates include at least two separate
ligands, e.g., 2, 3, 4, 5 or more ligands. When more than one
ligand is present the ligands may have the same or different
chemical structures.
[0139] In certain embodiments, the ligands are capable of competing
with a saccharide (e.g., glucose or mannose) for binding to an
endogenous saccharide-binding molecule (e.g., without limitation
surfactant proteins A and D or members of the selectin family). In
certain embodiments, the ligands are capable of competing with a
saccharide (e.g., glucose or mannose) for binding to cell-surface
sugar receptor (e.g., without limitation macrophage mannose
receptor, glucose transporter ligands, endothelial cell sugar
receptors, or hepatocyte sugar receptors). In certain embodiments,
the ligands are capable of competing with glucose for binding to an
endogenous glucose-binding molecule (e.g., without limitation
surfactant proteins A and D or members of the selectin family). In
certain embodiments, the ligands are capable of competing with a
saccharide for binding to a non-human lectin (e.g., Con A). In
certain embodiments, the ligands are capable of competing with
glucose or mannose for binding to a non-human lectin (e.g., Con A).
Exemplary glucose-binding lectins include calnexin, calreticulin,
N-acetylglucosamine receptor, selectin, asialoglycoprotein
receptor, collectin (mannose-binding lectin), mannose receptor,
aggrecan, versican, pisum sativum agglutinin (PSA), vicia faba
lectin, lens culinaris lectin, soybean lectin, peanut lectin,
lathyrus ochrus lectin, sainfoin lectin, sophora japonica lectin,
bowringia milbraedii lectin, concanavalin A (Con A), and pokeweed
mitogen.
[0140] In certain embodiments, the ligand is of formula (IIIa) or
(IIIb):
##STR00001##
wherein: [0141] each R.sup.1 is independently hydrogen, --OR.sup.y,
--N(R.sup.y).sub.2, --SR.sup.y, --O--Y, -G-Z, or --CH.sub.2R.sup.x;
[0142] each Rx is independently hydrogen, --OR.sup.y,
--N(R.sup.y).sub.2, --SR.sup.y, or --O--Y; [0143] each R.sup.y is
independently --R.sup.2, --SO.sub.2R.sup.2, --S(O)R.sup.2,
--P(O)(OR.sup.2).sub.2, --C(O)R.sup.2, --CO.sub.2R.sup.2, or
--C(O)N(R.sup.2).sub.2; [0144] each Y is independently a
monosaccharide, disaccharide, or trisaccharide; [0145] each G is
independently a covalent bond or an optionally substituted
C.sub.1-9 alkylene, wherein one or more methylene units of G is
optionally replaced by --O--, --S--, --N(R.sup.2)--, --C(O)--,
--OC(O)--, --C(O)O--, --C(O)N(R.sup.2)--, --N(R.sup.2)C(O)--,
--N(R.sup.2)C(O)N(R.sup.2)--, --SO.sub.2--, --SO.sub.2N(R.sup.2)--,
--N(R.sup.2)SO.sub.2--, or --N(R.sup.2)SO.sub.2N(R.sup.2)--; [0146]
each Z is independently halogen, --N(R.sup.2).sub.2, --OR.sup.2,
--SR.sup.2, --N.sub.3, --C.ident.CR.sup.2, --CO.sub.2R.sup.2,
--C(O)R.sup.2, or --OSO.sub.2R.sup.2; and [0147] each R.sup.2 is
independently hydrogen or an optionally substituted group selected
from C.sub.1-6 aliphatic, phenyl, a 4-7 membered heterocyclic ring
having 1-2 heteroatoms selected from nitrogen, oxygen, or sulfur,
or a 5-6 membered monocyclic heteroaryl ring having 1-4 heteroatoms
selected from nitrogen, oxygen, or sulfur.
[0148] In certain embodiments, the ligand of formula (IIIa) or
(IIIb) is a monosaccharide. In certain embodiments, the ligand is a
disaccharide. In certain embodiments, the ligand is a
trisaccharide. In certain embodiments, the ligand is a
tetrasaccharide. In certain embodiments, the ligand comprises no
more than a total of four monosaccharide moieties.
[0149] As defined generally above, each R.sup.1 is independently
hydrogen, --OR.sup.y, --N(R.sup.y).sub.2, --SR.sup.y, --O--Y, -G-Z,
or --CH.sub.2R.sup.x. In certain embodiments, R.sup.1 is hydrogen.
In certain embodiments, R.sup.1 is --OH. In other embodiments,
R.sup.1 is --NHC(O)CH.sub.3. In certain embodiments, R.sup.1 is
--O--Y. In certain other embodiments, R.sup.1 is -G-Z. In some
embodiments, R.sup.1 is --CH.sub.2OH. In other embodiments, R.sup.1
is --CH.sub.2--O--Y. In yet other embodiments, R.sup.1 is
--NH.sub.2. One of ordinary skill in the art will appreciate that
each R.sup.1 substituent in formula (IIIa) or (IIIb) may be of (R)
or (S) stereochemistry.
[0150] As defined generally above, each R.sup.x is independently
hydrogen, --OR.sup.y, --N(R.sup.y).sub.2, --SR.sup.y, or --O--Y. In
some embodiments, R.sup.x is hydrogen. In certain embodiments,
R.sup.x is --OH. In other embodiments, R.sup.x is --O--Y.
[0151] As defined generally above, each R.sup.y is independently
--R.sup.2, --SO.sub.2R.sup.2, --S(O)R.sup.2,
--P(O)(OR.sup.2).sub.2, --C(O)R.sup.2, --CO.sub.2R.sup.2, or
--C(O)N(R.sup.2).sub.2. In some embodiments, R.sup.y is hydrogen.
In other embodiments, R.sup.y is --R.sup.2. In some embodiments,
R.sup.y is --C(O)R.sup.2. In certain embodiments, R.sup.y is
acetyl. In other embodiments, R.sup.y is --SO.sub.2R.sup.2,
--S(O)R.sup.2, --P(O)(OR.sup.2).sub.2, --CO.sub.2R.sup.2, or
--C(O)N(R.sup.2).sub.2.
[0152] As defined generally above, Y is a monosaccharide,
disaccharide, or trisaccharide. In certain embodiments, Y is a
monosaccharide. In some embodiments, Y is a disaccharide. In other
embodiments, Y is a trisaccharide. In some embodiments, Y is
mannose, glucose, fructose, galactose, rhamnose, or xylopyranose.
In some embodiments, Y is sucrose, maltose, turanose, trehalose,
cellobiose, or lactose. In certain embodiments, Y is mannose. In
certain embodiments, Y is D-mannose. One of ordinary skill in the
art will appreciate that the saccharide Y is attached to the oxygen
group of --O--Y through anomeric carbon to form a glycosidic bond.
The glycosidic bond may be of an alpha or beta configuration.
[0153] As defined generally above, each G is independently a
covalent bond or an optionally substituted C.sub.1-9alkylene,
wherein one or more methylene units of G is optionally replaced by
--O--, --S--, --N(R.sup.2)--, --C(O)--, --OC(O)--, --C(O)O--,
--C(O)N(R.sup.2)--, --N(R.sup.2)C(O)--,
--N(R.sup.2)C(O)N(R.sup.2)--, --SO.sub.2--, --SO.sub.2N(R.sup.2)--,
--N(R.sup.2)SO.sub.2--, or --N(R.sup.2)SO.sub.2N(R.sup.2)--. In
some embodiments, G is a covalent bond. In certain embodiments, G
is --O--C.sub.1-8 alkylene. In certain embodiments, G is
--OCH.sub.2CH.sub.2--.
[0154] As defined generally above, each Z is independently halogen,
--N(R.sup.2).sub.2, --OR.sup.2, --SR.sup.2, --N.sub.3,
--C.ident.CR.sup.2, --CO.sub.2R.sup.2, --C(O)R.sup.2, or
--OSO.sub.2R.sup.2. In some embodiments, Z is a halogen or
--OSO.sub.2R.sup.2. In other embodiments, Z is --N.sub.3 or
--C.ident.CR.sup.2. In certain embodiments, Z is
--N(R.sup.2).sub.2, --OR.sup.2, or --SR.sup.2. In certain
embodiments, Z is --SH. In certain embodiments, Z is --NH.sub.2. In
certain embodiments, -G-Z is --OCH.sub.2CH.sub.2NH.sub.2.
[0155] In some embodiments, the R.sup.1 substituent on the C1
carbon of formula (IIIa) is -G-Z to give a compound of formula
(IIIa-i):
##STR00002##
wherein R.sup.1, G, and Z are as defined and described herein.
[0156] In some embodiments, the ligand is of formula (IIIa-ii):
##STR00003##
wherein R.sup.1, Rx, G, and Z are as defined and described
herein.
[0157] In certain embodiments, the ligand(s) may have the same
chemical structure as glucose or may be a chemically related
species of glucose. In various embodiments it may be advantageous
for the ligand(s) to have a different chemical structure from
glucose, e.g., in order to fine tune the glucose response of the
conjugate. For example, in certain embodiments, one might use a
ligand that includes glucose, mannose, L-fucose or derivatives of
these (e.g., alpha-L-fucopyranoside, mannosamine, beta-linked
N-acetyl mannosamine, methylglucose, methylmannose, ethylglucose,
ethylmannose, propylglucose, propylmannose, etc.) and/or higher
order combinations of these (e.g., a bimannose, linear and/or
branched trimannose, etc.).
[0158] In certain embodiments, the ligand includes a
monosaccharide. In certain embodiments, the ligand includes a
disaccharide. In certain embodiments, the ligand is includes a
trisaccharide. In some embodiments, the ligand comprises a
saccharide and one or more amine groups. In certain embodiments the
saccharide and amine group are separated by a C.sub.1-C.sub.6 alkyl
group, e.g., a C.sub.1-C.sub.3 alkyl group. In some embodiments,
the ligand is aminoethylglucose (AEG). In some embodiments, the
ligand is aminoethylmannose (AEM). In some embodiments, the ligand
is aminoethylbimannose (AEBM). In some embodiments, the ligand is
aminoethyltrimannose (AETM). In some embodiments, the ligand is
3-aminoethyl-N-acetylglucosamine (AEGA). In some embodiments, the
ligand is aminoethylfucose (AEF). In certain embodiments, a
saccharide ligand is of the "D" configuration. In other
embodiments, a saccharide ligand is of the "L" configuration. Below
we show the structures of these exemplary ligands. Other exemplary
ligands will be recognized by those skilled in the art.
##STR00004##
[0159] In general, ligands may be directly or indirectly conjugated
(i.e., via a linker or framework) to the drug. As discussed in more
detail below, the ligands may be naturally present within a
conjugate framework (e.g., as part of a polymer backbone or as a
side group of a monomer). Alternatively (or additionally) ligands
may be artificially incorporated into a conjugate framework (e.g.,
in the form of a chemical group that is synthetically added to a
conjugate framework). In certain embodiments, a conjugate may
include a framework which comprises 5 or more, 10 or more, or 20 or
more ligands. In certain embodiments, a conjugate may comprise as
few as 1, 2, 3, 4 or 5 separate ligands.
[0160] In certain embodiments, at least two separate ligands are
conjugated to the drug via different conjugation points. In certain
embodiments, at least two separate ligands are conjugated to a
single conjugate framework that is also conjugated to the drug. In
some embodiments, at least one ligand, such as AETM, AEG, AEM,
AEBM, AEGA, or AEF, is conjugated to one insulin molecule. In
certain embodiments, at least one AETM ligand is conjugated to one
insulin molecule. In some embodiments, at least two ligands, such
as AETM, AEG, AEM, AEBM, AEGA, or AEF, are conjugated to one
insulin molecule, either through one conjugation point or multiple
conjugation points. In certain embodiments, the at least two
ligands are not the same ligand. In certain embodiments, the at
least two ligands are the same ligand. In certain embodiments, at
least two AETM ligands are conjugated to one insulin molecule,
either through one conjugation point or multiple conjugation
points. As discussed in more detail below in the context of certain
exemplary conjugate frameworks, in certain embodiments the separate
ligands and drug (e.g., an insulin molecule) may each be located on
a separate branch of a branched conjugate framework. For example,
the ligands and drug may be located on termini of these branches.
In certain embodiments a hyperbranched conjugate framework may be
used. Both polymeric and non-polymeric conjugate frameworks are
encompassed.
[0161] Methods for conjugating ligands to a conjugate framework are
discussed in more detail below. In certain embodiments, the
saccharide within the one or more ligands is conjugated (directly
or indirectly by way of a linker) via the C1, C2 or C6 position. In
certain embodiments, the conjugation involves the C1 position. The
C1 position of a saccharide is also referred to as the anomeric
carbon and may be connected to the drug or conjugate framework in
the alpha or beta conformation. In certain embodiments, the C1
position is configured as the alpha anomer. In other embodiments,
the C1 position is configured as the beta anomer.
Drug
[0162] It is to be understood that a conjugate can comprise any
drug. A conjugate can comprise more than one copy of the same drug
and/or can comprise more than one type of drug. The conjugates are
not limited to any particular drug and may include small molecule
drugs or biomolecular drugs. In general, the drug(s) used will
depend on the disease or disorder to be treated. As used herein,
the term "drug" encompasses salt and non-salt forms of the drug.
For example, the term "insulin molecule" encompasses all salt and
non-salt forms of the insulin molecule. It will be appreciated that
the salt form may be anionic or cationic depending on the drug.
[0163] For example, without limitation, in various embodiments a
conjugate can comprise any one of the following drugs: diclofenac,
nifedipine, rivastigmine, methylphenidate, fluoroxetine,
rosiglitazone, prednison, prednisolone, codeine, ethylmorphine,
dextromethorphan, noscapine, pentoxiverine, acetylcysteine,
bromhexine, epinephrine, isoprenaline, orciprenaline, ephedrine,
fenoterol, rimiterol, ipratropium, cholinetheophyllinate,
proxiphylline, bechlomethasone, budesonide, deslanoside, digoxine,
digitoxin, disopyramide, proscillaridin, chinidine, procainamide,
mexiletin, flecainide, alprenolol, proproanolol, nadolol, pindolol,
oxprenolol, labetalol, tirnolol, atenolol,
pentaeritrityltetranitrate, isosorbiddinitrate,
isosorbidmononitrate, niphedipin, phenylamine, verapamil,
diltiazem, cyclandelar, nicotinylalcholhol, inositolnicotinate,
alprostatdil, etilephrine, prenalterol, dobutamine, dopamine,
dihydroergotamine, guanetidine, betanidine, methyldopa, reserpine,
guanfacine, trimethaphan, hydralazine, dihydralazine, prazosine,
diazoxid, captopril, nifedipine, enalapril, nitroprusside,
bendroflumethiazide, hydrochlorthiazide, metychlothiazide,
polythiazide, chlorthalidon, cinetazon, clopamide, mefruside,
metholazone, bumetanide, ethacrynacide, spironolactone, amiloride,
chlofibrate, nicotinic acid, nicheritrol, brompheniramine,
cinnarizine, dexchlorpheniramine, clemastine, antazoline,
cyproheptadine, proethazine, cimetidine, ranitidine, sucralfat,
papaverine, moxaverine, atropin, butylscopolamin, emepron,
glucopyrron, hyoscyamine, mepensolar, methylscopolamine,
oxiphencyclimine, probanteline, terodilin, sennaglycosides,
sagradaextract, dantron, bisachodyl, sodiumpicosulfat, etulos,
diphenolxylate, loperamide, salazosulfapyridine, pyrvin,
mebendazol, dimeticon, ferrofumarate, ferrosuccinate,
ferritetrasemisodium, cyanochobalamine, folid acid heparin, heparin
co-factor, diculmarole, warfarin, streptokinase, urokinase, factor
VIII, factor IX, vitamin K, thiopeta, busulfan, chlorambucil,
cyclophosphamid, melfalan, carmustin, mercatopurin, thioguanin,
azathioprin, cytarabin, vinblastin, vinchristin, vindesin,
procarbazine, dacarbazine, lomustin, estramustin, teniposide,
etoposide, cisplatin, amsachrin, aminogluthetimid, phosphestrol,
medroxiprogresterone, hydroxiprogesterone, megesterol,
noretisteron, tamoxiphen, ciclosporin, sulfosomidine,
bensylpenicillin, phenoxymethylpenicillin, dicloxacillin,
cloxacillin, flucoxacillin, ampicillin, amoxicillin, pivampicillin,
bacampicillin, piperacillin, meziocillin, mecillinam,
pivmecillinam, cephalotin, cephalexin, cephradin, cephadroxil,
cephaclor, cefuroxim, cefotaxim, ceftazidim, cefoxitin, aztreonam,
imipenem, cilastatin, tetracycline, lymecycline, demeclocycline,
metacycline, oxitetracycline, doxycycline, chloramphenicol,
spiramycin, fusidic acid, lincomycin, clindamycin, spectinomycin,
rifampicin, amphotericin B, griseofulvin, nystatin, vancomycin,
metronidazole, tinidazole, trimethoprim, norfloxacin,
salazosulfapyridin, aminosalyl, isoniazid, etambutol,
nitrofurantoin, nalidixic acid, metanamine, chloroquin,
hydroxichloroquin, tinidazol, ketokonazol, acyclovir, interferon
idoxuridin, retinal, tiamin, dexpantenol, pyridoxin, folic acid,
ascorbic acid, tokoferol, phytominadion, phenfluramin,
corticotropin, tetracosactid, tyrotropin, somatotoprin, somatrem,
vasopressin, lypressin, desmopressin, oxytocin,
chloriongonadotropin, cortison, hydrocortisone, fluodrocortison,
prednison, prednisolon, fluoximesteron, mesterolon, nandrolon,
stanozolol, oximetolon, cyproteron, levotyroxin, liotyronin,
propylthiouracil, carbimazol, tiamazol, dihydrotachysterol,
alfacalcidol, calcitirol, insulin, tolbutamid, chlorpropamid,
tolazamid, glipizid, glibenclamid, phenobarbital, methyprylon,
pyrityidion, meprobamat, chlordiazepoxid, diazepam, nitrazepam,
baclofen, oxazepam, dikaliumclorazepat, lorazepam, flunitrazepam,
alprazolam, midazolam, hydroxizin, dantrolene, chlometiazol,
propionmazine, alimemazine, chlorpromazine, levomepromazine,
acetophenazine, fluphenazine, perphenazine, prochlorperazine,
trifluoperazine, dixyrazine, thiodirazine, periciazin,
chloprothixene, tizanidine, zaleplon, zuclopentizol, flupentizol,
thithixen, haloperidol, trimipramin, opipramol, chlomipramin,
desipramin, lofepramin, amitriptylin, nortriptylin, protriptylin,
maptrotilin, caffeine, cinnarizine, cyclizine, dimenhydinate,
meclozine, prometazine, thiethylperazine, metoclopramide,
scopolamine, phenobarbital, phenytoine, ethosuximide, primidone,
carbamazepine, chlonazepam, orphenadrine, atropine, bensatropine,
biperiden, metixene, procylidine, levodopa, bromocriptin,
amantadine, ambenon, pyridostigmine, synstigmine, disulfiram,
morphine, codeine, pentazocine, buprenorphine, pethidine,
phenoperidine, phentanyl, methadone, piritramide,
dextropropoxyphene, ketobemidone, acetylsalicylic acid, celecoxib,
phenazone, phenylbutazone, azapropazone, piroxicam, ergotamine,
dihydroergotamine, cyproheptadine, pizitifen, flumedroxon,
allopurinol, probenecid, sodiummaurothiomalate auronofin,
penicillamine, estradiol, estradiolvalerianate, estriol,
ethinylestradiol, dihydrogesteron, lynestrenol,
medroxiprogresterone, noretisterone, cyclophenile, clomiphene,
levonorgestrel, mestranol, ornidazol, tinidazol, ekonazol,
chlotrimazol, natamycine, miconazole, sulbentin, methylergotamine,
dinoprost, dinoproston, gemeprost, bromocriptine,
phenylpropanolamine, sodiumchromoglicate, azetasolamide,
dichlophenamide, betacarotene, naloxone, calciumfolinate, in
particular clonidine, thephylline, dipyradamol, hydrochlothiazade,
scopolamine, indomethacine, furosemide, potassium chloride,
morphine, ibuprofen, salbutamol, terbutalin, calcitonin, etc. It is
to be understood that this list is intended to be exemplary and
that any drug, whether known or later discovered, may be used in a
conjugate of the present disclosure.
[0164] In various embodiments, a conjugate may include a hormonal
drug which may be peptidic or non-peptidic, e.g., adrenaline,
noradrenaline, angiotensin, atriopeptin, aldosterone,
dehydroepiandrosterone, androstenedione, testosterone,
dihydrotestosterone, calcitonin, calcitriol, calcidiol,
corticotropin, cortisol, dopamine, estradiol, estrone, estriol,
erythropoietin, follicle-stimulating hormone, gastrin, ghrelin,
glucagon, gonadotropin-releasing hormone, growth hormone, growth
hormone-releasing hormone, human chorionic gonadotropin, histamine,
human placental lactogen, insulin, insulin-like growth factor,
inhibin, leptin, a leukotriene, lipotropin, melatonin, orexin,
oxytocin, parathyroid hormone, progesterone, prolactin,
prolactin-releasing hormone, a prostglandin, renin, serotonin,
secretin, somatostatin, thrombopoietin, thyroid-stimulating
hormone, thyrotropin-releasing hormone (or thyrotropin),
thyrotropin-releasing hormone, thyroxine, triiodothyronine,
vasopressin, etc. In certain embodiments, the hormone may be
selected from glucagon, insulin, insulin-like growth factor,
leptin, thyroid-stimulating hormone, thyrotropin-releasing hormone
(or thyrotropin), thyrotropin-releasing hormone, thyroxine, and
triiodothyronine. In certain embodiments, the drug is insulin-like
growth factor 1 (IGF-1). It is to be understood that this list is
intended to be exemplary and that any hormonal drug, whether known
or later discovered, may be used in a conjugate of the present
disclosure.
[0165] In various embodiments, a conjugate may include a thyroid
hormone.
[0166] In various embodiments, a conjugate may include an
anti-diabetic drug (i.e., a drug which has a beneficial effect on
patients suffering from diabetes).
[0167] In various embodiments, a conjugate may include an insulin
molecule. As used herein, the term "insulin" or "insulin molecule"
encompasses all salt and non-salt forms of the insulin molecule. It
will be appreciated that the salt form may be anionic or cationic
depending on the insulin molecule. By "insulin" or "an insulin
molecule" we intend to encompass both wild-type and modified forms
of insulin as long as they are bioactive (i.e., capable of causing
a detectable reduction in glucose when administered in vivo).
Wild-type insulin includes insulin from any species whether in
purified, synthetic or recombinant form (e.g., human insulin,
porcine insulin, bovine insulin, rabbit insulin, sheep insulin,
etc.). A number of these are available commercially, e.g., from
Sigma-Aldrich (St. Louis, Mo.). A variety of modified forms of
insulin are known in the art (e.g. see Crotty and Reynolds,
Pediatr. Emerg. Care. 23:903-905, 2007 and Gerich, Am. J. Med.
113:308-16, 2002 and references cited therein). Modified forms of
insulin may be chemically modified (e.g., by addition of a chemical
moiety such as a PEG group or a fatty acyl chain as described
below) and/or mutated (i.e., by addition, deletion or substitution
of one or more amino acids).
[0168] In certain embodiments, an insulin molecule of the present
disclosure will differ from a wild-type insulin by 1-10 (e.g., 1-9,
1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4,
2-3, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9,
5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, 8-9, 9, 8, 7, 6, 5, 4, 3, 2
or 1) amino acid substitutions, additions and/or deletions. In
certain embodiments, an insulin molecule of the present disclosure
will differ from a wild-type insulin by amino acid substitutions
only. In certain embodiments, an insulin molecule of the present
disclosure will differ from a wild-type insulin by amino acid
additions only. In certain embodiments, an insulin molecule of the
present disclosure will differ from a wild-type insulin by both
amino acid substitutions and additions. In certain embodiments, an
insulin molecule of the present disclosure will differ from a
wild-type insulin by both amino acid substitutions and
deletions.
[0169] In certain embodiments, amino acid substitutions may be made
on the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. In certain embodiments, a substitution may
be conservative, that is, one amino acid is replaced with one of
similar shape and charge. Conservative substitutions are well known
in the art and typically include substitutions within the following
groups: glycine, alanine; valine, isoleucine, leucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and tyrosine, phenylalanine. In certain
embodiments, the hydrophobic index of amino acids may be considered
in choosing suitable mutations. The importance of the hydrophobic
amino acid index in conferring interactive biological function on a
polypeptide is generally understood in the art. Alternatively, the
substitution of like amino acids can be made effectively on the
basis of hydrophilicity. The importance of hydrophilicity in
conferring interactive biological function of a polypeptide is
generally understood in the art. The use of the hydrophobic index
or hydrophilicity in designing polypeptides is further discussed in
U.S. Pat. No. 5,691,198.
[0170] The wild-type sequence of human insulin (A-chain and
B-chain) is shown below and in FIG. 63.
TABLE-US-00001 A-Chain (SEQ ID NO: 1): GIVEQCCTSICSLYQLENYCN
B-Chain (SEQ ID NO: 2): FVNQHLCGSHLVEALYLVCGERGFFYTPKT
[0171] Human insulin differs from rabbit, porcine, bovine, and
sheep insulin only in amino acids A8, A9, A10, and B30 (see table
below).
TABLE-US-00002 Amino Acid Position Insulin A8 A9 A10 B30 human Thr
Ser Ile Thr rabbit Thr Ser Ile Ser porcine Thr Ser Ile Ala bovine
Ala Ser Val Ala sheep Ala Gly Val Ala
[0172] In various embodiments, an insulin molecule of the present
disclosure is mutated at the B28 and/or B29 positions of the
B-peptide sequence. For example, insulin lispro (HUMALOG.RTM.) is a
rapid acting insulin mutant in which the penultimate lysine and
proline residues on the C-terminal end of the B-peptide have been
reversed (Lys.sup.B28Pro.sup.B29-human insulin). This modification
blocks the formation of insulin multimers. Insulin aspart
(NOVOLOG.RTM.) is another rapid acting insulin mutant in which
proline at position B28 has been substituted with aspartic acid
(Asp.sup.B28-human insulin). This mutant also prevents the
formation of multimers. In some embodiments, mutation at positions
B28 and/or B29 is accompanied by one or more mutations elsewhere in
the insulin polypeptide. For example, insulin glulisine
(APIDRA.RTM.) is yet another rapid acting insulin mutant in which
aspartic acid at position B3 has been replaced by a lysine residue
and lysine at position B29 has been replaced with a glutamic acid
residue (Lys.sup.B3Glu.sup.B29-human insulin).
[0173] In various embodiments, an insulin molecule of the present
disclosure has an isoelectric point that is shifted relative to
human insulin. In some embodiments, the shift in isoelectric point
is achieved by adding one or more arginine residues to the
N-terminus of the insulin A-peptide and/or the C-terminus of the
insulin B-peptide. Examples of such insulin polypeptides include
Arg.sup.A0-human insulin, Arg.sup.B31Arg.sup.B32-human insulin,
Gly.sup.A21Arg.sup.B31Arg.sup.B32-human insulin,
Arg.sup.A0Arg.sup.B31Arg.sup.B32-human insulin, and
Arg.sup.A0Gly.sup.A21Arg.sup.B31Arg.sup.B32-human insulin. By way
of further example, insulin glargine (LANTUS.RTM.) is an exemplary
long acting insulin mutant in which Asp.sup.A21 has been replaced
by glycine, and two arginine residues have been added to the
C-terminus of the B-peptide. The effect of these changes is to
shift the isoelectric point, producing a solution that is
completely soluble at pH 4. Thus, in some embodiments, an insulin
molecule of the present disclosure comprises an A-peptide sequence
wherein A21 is Gly and B-peptide sequence wherein B31 and B32 are
Arg-Arg. It is to be understood that the present disclosure
encompasses all single and multiple combinations of these mutations
and any other mutations that are described herein (e.g.,
Gly.sup.A21-human insulin, Gly.sup.A21Arg.sup.B31-human insulin,
Arg.sup.B31Arg.sup.B32-human insulin, Arg.sup.B31 human
insulin).
[0174] In various embodiments, an insulin molecule of the present
disclosure is truncated. For example, in certain embodiments, a
B-peptide sequence of an insulin polypeptide of the present
disclosure is missing B1, B2, B3, B26, B27, B28, B29 and/or B30. In
certain embodiments, combinations of residues are missing from the
B-peptide sequence of an insulin polypeptide of the present
disclosure. For example, the B-peptide sequence may be missing
residues B(1-2), B(1-3), B(29-30), B(28-30), B(27-30) and/or
B(26-30). In some embodiments, these deletions and/or truncations
apply to any of the aforementioned insulin molecules (e.g., without
limitation to produce des(B30)-insulin lispro, des(B30)-insulin
aspart, des(B30)-insulin glulisine, des(B30)-insulin glargine,
etc.).
[0175] In some embodiments, an insulin molecule contains additional
amino acid residues on the N- or C-terminus of the A or B-peptide
sequences. In some embodiments, one or more amino acid residues are
located at positions A0, A21, B0 and/or B31. In some embodiments,
one or more amino acid residues are located at position A0. In some
embodiments, one or more amino acid residues are located at
position A21. In some embodiments, one or more amino acid residues
are located at position B0. In some embodiments, one or more amino
acid residues are located at position B31. In certain embodiments,
an insulin molecule does not include any additional amino acid
residues at positions A0, A21, B0 or B31.
[0176] In certain embodiments, an insulin molecule of the present
disclosure is mutated such that one or more amidated amino acids
are replaced with acidic forms. For example, asparagine may be
replaced with aspartic acid or glutamic acid. Likewise, glutamine
may be replaced with aspartic acid or glutamic acid. In particular,
Asn.sup.A18, Asn.sup.A21, or Asn.sup.B3, or any combination of
those residues, may be replaced by aspartic acid or glutamic acid.
Gln.sup.A15 or Gln.sup.B4, or both, may be replaced by aspartic
acid or glutamic acid. In certain embodiments, an insulin molecule
has aspartic acid at position A21 or aspartic acid at position B3,
or both.
[0177] One skilled in the art will recognize that it is possible to
mutate yet other amino acids in the insulin molecule while
retaining biological activity. For example, without limitation, the
following modifications are also widely accepted in the art:
replacement of the histidine residue of position B10 with aspartic
acid (His.sup.B10.fwdarw.Asp.sup.B10); replacement of the
phenylalanine residue at position B1 with aspartic acid
(Phe.sup.B1.fwdarw.Asp.sup.B1); replacement of the threonine
residue at position B30 with alanine
(Thr.sup.B30.fwdarw.Ala.sup.B30); replacement of the tyrosine
residue at position B26 with alanine
(Tyr.sup.B26.fwdarw.Ala.sup.B26); and replacement of the serine
residue at position B9 with aspartic acid
(Ser.sup.B9.fwdarw.Asp.sup.B9).
[0178] In various embodiments, an insulin molecule of the present
disclosure has a protracted profile of action. Thus, in certain
embodiments, an insulin molecule of the present disclosure may be
acylated with a fatty acid. That is, an amide bond is formed
between an amino group on the insulin molecule and the carboxylic
acid group of the fatty acid. The amino group may be the
alpha-amino group of an N-terminal amino acid of the insulin
molecule, or may be the epsilon-amino group of a lysine residue of
the insulin molecule. An insulin molecule of the present disclosure
may be acylated at one or more of the three amino groups that are
present in wild-type human insulin or may be acylated on lysine
residue that has been introduced into the wild-type human insulin
sequence. In certain embodiments, an insulin molecule may be
acylated at position B1. In certain embodiments, an insulin
molecule may be acylated at position B29. In certain embodiments,
the fatty acid is selected from myristic acid (C14), pentadecylic
acid (C15), palmitic acid (C16), heptadecylic acid (C17) and
stearic acid (C18). For example, insulin detemir (LEVEMIR.RTM.) is
a long acting insulin mutant in which Thr.sup.B30 has been deleted,
and a C14 fatty acid chain (myristic acid) has been attached to
Lys.sup.B29.
[0179] In some embodiments, the N-terminus of the A-peptide, the
N-terminus of the B-peptide, the epsilon-amino group of Lys at
position B29 or any other available amino group in an insulin
molecule of the present disclosure is covalently linked to a fatty
acid moiety of general formula:
##STR00005##
wherein RE is hydrogen or a C.sub.1-30 alkyl group. In some
embodiments, R is a C.sub.1-20 alkyl group, a C.sub.3-19 alkyl
group, a C.sub.5-18 alkyl group, a C.sub.6-17 alkyl group, a
C.sub.8-16 alkyl group, a C.sub.10-15 alkyl group, or a C.sub.12-14
alkyl group. In certain embodiments, the insulin polypeptide is
conjugated to the moiety at the A1 position. In certain
embodiments, the insulin polypeptide is conjugated to the moiety at
the B1 position. In certain embodiments, the insulin polypeptide is
conjugated to the moiety at the epsilon-amino group of Lys at
position B29. In certain embodiments, position B28 of the insulin
molecule is Lys and the epsilon-amino group of Lys.sup.B28 is
conjugated to the fatty acid moiety. In certain embodiments,
position B3 of the insulin molecule is Lys and the epsilon-amino
group of Lys.sup.B3 is conjugated to the fatty acid moiety. In some
embodiments, the fatty acid chain is 8-20 carbons long. In some
embodiments, the fatty acid is octanoic acid (C8), nonanoic acid
(C9), decanoic acid (C10), undecanoic acid (C11), dodecanoic acid
(C12), or tridecanoic acid (C13). In certain embodiments, the fatty
acid is myristic acid (C14), pentadecanoic acid (C15), palmitic
acid (C16), heptadecanoic acid (C17), stearic acid (C18),
nonadecanoic acid (C19), or arachidic acid (C20).
[0180] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules: Lys.sup.B28Pro.sup.B29
human insulin (insulin lispro), Asp.sup.B28-human insulin (insulin
aspart), Lys.sup.B3Glu.sup.B29-human insulin (insulin glulisine),
Arg.sup.B31Arg.sup.B32-human insulin (insulin glargine),
N.sup..epsilon.B29-myristoyl-des(B30)-human insulin (insulin
detemir), Ala.sup.B26-human insulin, Asp.sup.B1-human insulin,
Arg.sup.A0-human insulin, Asp.sup.B1Glu.sup.B13-human insulin,
Gly.sup.A21-human insulin, Gly.sup.A21Arg.sup.B31Arg.sup.B32-human
insulin, Arg.sup.A0Arg.sup.B31Arg.sup.B32-human insulin,
Arg.sup.A0Gly.sup.A21Arg.sup.B31Arg.sup.B32-human insulin,
des(B30)-human insulin, des(B27)-human insulin, des(B28-B30)-human
insulin, des(B1)-human insulin, des(B1-B3)-human insulin.
[0181] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-palmitoyl-human insulin,
N.sup..epsilon.B29-myrisotyl-human insulin,
N.sup..epsilon.B28-palmitoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-myristoyl-Lys.sup.B28Pro.sup.B29-human
insulin.
[0182] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-palmitoyl-des(B30)-human insulin,
N.sup..epsilon.B30-myristoyl-Thr.sup.B29Lys.sup.B30-human insulin,
N.sup..epsilon.B30-palmitoyl-Thr.sup.B29Lys.sup.B30-human insulin,
N.sup..epsilon.B29--(N-palmitoyl-.gamma.-glutamyl)-des(B30)-human
insulin,
N.sup..epsilon.B29--(N-lithocolyl-.gamma.-glutamyl)-des(B30)-hum-
an insulin,
N.sup..epsilon.B29-(co-carboxyheptadecanoyl)-des(B30)-human
insulin, N.sup..epsilon.B29-(.omega.-carboxyheptadecanoyl)-human
insulin.
[0183] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-octanoyl-human insulin,
N.sup..epsilon.B29-myristoyl-Gly.sup.A21Arg.sup.B31Arg.sup.B31-h-
uman insulin,
N.sup..epsilon.B29-myristoyl-Gly.sup.A21Gln.sup.B3Arg.sup.B31Arg.sup.B32--
human insulin,
N.sup..epsilon.B29-myristoyl-Arg.sup.A0Gly.sup.A21Arg.sup.B31Arg.sup.B32--
human insulin,
N.sup..epsilon.B29-Arg.sup.A0Gly.sup.A21Gln.sup.B3Arg.sup.B31Arg.sup.B32--
human insulin,
N.sup..epsilon.B29-myristoyl-Arg.sup.A0Gly.sup.A21Asp.sup.B3Arg.sup.B31Ar-
g.sup.B32-human insulin,
N.sup..epsilon.B29-myristoyl-Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B29-myristoyl-Arg.sup.A0Arg.sup.B31Arg.sup.B32-human
insulin,
N.sup..epsilon.B29-octanoyl-Gly.sup.A21Arg.sup.B31Arg.sup.B32-hu-
man insulin,
N.sup..epsilon.B29-octanoyl-Gly.sup.A21Gln.sup.B3Arg.sup.B31Arg.sup.B32-h-
uman insulin,
N.sup..epsilon.B29-octanoyl-Arg.sup.A0Gly.sup.A21Arg.sup.B31Arg.sup.B32-h-
uman insulin,
N.sup..epsilon.B29-octanoyl-Arg.sup.A0Gly.sup.A21Gln.sup.B3Arg.sup.B3
1Arg.sup.B32-human insulin,
N.sup..epsilon.B29-octanoyl-Arg.sup.B0Gly.sup.A21Asp.sup.B3Arg.sup.B31Arg-
.sup.B32-human insulin,
N.sup..epsilon.B29-octanoyl-Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B29-octanoyl-Arg.sup.A0Arg.sup.B31Arg.sup.B32-human
insulin.
[0184] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin polypeptides:
N.sup..epsilon.B28-myristoyl-Gly.sup.A21Lys.sup.B28Pro.sup.B29Arg.sup.B31-
Arg.sup.B32-human insulin,
N.sup..epsilon.B28-myristoyl-Gly.sup.A21Gln.sup.B3Lys.sup.B28Pro.sup.B30A-
rg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-myristoyl-Arg.sup.A0Gly.sup.A21Lys.sup.B28Pro.sup.B29A-
rg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-myristoyl-Arg.sup.A0Gly.sup.A21Gln.sup.B3Lys.sup.B28Pr-
o.sup.B29Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-myristoyl-Arg.sup.A0Gly.sup.A21Asp.sup.B3Lys.sup.B28Pr-
o.sup.B29Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-myristoyl-Lys.sup.B28Pro.sup.B29Arg.sup.B31Arg.sup.B32-
-human insulin,
N.sup..epsilon.B28-myristoyl-arg.sup.A0Lys.sup.B28Pro.sup.B29Arg.sup.B31A-
rg.sup.B32-human insulin,
N.sup..epsilon.B28-octanoyl-Gly.sup.A21Lys.sup.B28Pro.sup.B29Arg.sup.B31A-
rg.sup.B32-human insulin.
[0185] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-octanoyl-Gly.sup.A21Gln.sup.B3Lys.sup.B28
Pro.sup.B29Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-octanoyl-Arg.sup.A0Gly.sup.A21Lys.sup.B28Pro.sup.B29Ar-
g.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-octanoyl-Arg.sup.A0Gly.sup.A21Gln.sup.B3Lys.sup.B28Pro-
.sup.B29Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-octanoyl-Arg.sup.A0Gly.sup.A21Asp.sup.B3Lys.sup.B28Pro-
.sup.B29Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B28-octanoyl-Lys.sup.B28Pro.sup.B29Arg.sup.B31Arg.sup.B32--
human insulin,
N.sup..epsilon.B28-octanoyl-Arg.sup.A0Lys.sup.B28Pro.sup.B29Arg.sup.B31Ar-
g.sup.B32-human insulin.
[0186] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-des(B30)-human insulin,
N.sup..epsilon.B29-tetradecanoyl-des(B30)-human insulin,
N.sup..epsilon.B29-decanoyl-des(B30)-human insulin,
N.sup..epsilon.B29-dodecanoyl-des(B30)-human insulin,
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21-des(B30)-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21-des(B30)-human
insulin, N.sup..epsilon.B29-decanoyl-Gly.sup.A21-des(B30)-human
insulin, N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21-des(B30)-human
insulin,
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21Gln.sup.B3-des(B30)-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21Gln.sup.B3-des(B30)--
human insulin,
N.sup..epsilon.B29-decanoyl-Gly.sup.A21-Gln.sup.B3-des(B30)-human
insulin,
N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21-Gln.sup.B3-des(B30)-hu-
man insulin,
N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21-des(B30)-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21-des(B30)-human
insulin, N.sup..epsilon.B29-decanoyl-Ala.sup.A21-des(B30)-human
insulin, N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21-des(B30)-human
insulin,
N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21-Gln.sup.B3-des(B30)-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21Gln.sup.B3-des(B30)--
human insulin,
N.sup..epsilon.B29-decanoyl-Ala.sup.A21Gln.sup.B3-des(B30)-human
insulin,
N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21Gln.sup.B3-des(B30)-human
insulin, N.sup..epsilon.B29-tridecanoyl-Gln.sup.B3-des(B30)-human
insulin, N.sup..epsilon.B29-tetradecanoyl-Gln.sup.B3-des(B30)-human
insulin, N.sup..epsilon.B29-decanoyl-Gln.sup.B3-des(B30)-human
insulin, N.sup..epsilon.B29-dodecanoyl-Gln.sup.B3-des(B30)-human
insulin.
[0187] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21-human insulin,
N.sup..epsilon.B29-decanoyl-Gly.sup.A21-human insulin,
N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21-human insulin,
N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21-human insulin,
N.sup..epsilon.B29-decanoyl-Ala.sup.A21-human insulin,
N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21-human insulin.
[0188] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21Gln.sup.B3-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21Gln.sup.B3-human
insulin, N.sup..epsilon.B29-decanoyl-Gly.sup.A21Gln.sup.B3-human
insulin, N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21Gln.sup.B3-human
insulin, N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21Gln.sup.B3-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21Gln.sup.B3-human
insulin, N.sup..epsilon.B29-decanoyl-Ala.sup.A21Gln.sup.B3-human
insulin, N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21Gln.sup.B3-human
insulin.
[0189] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gln.sup.B3-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Gln.sup.B3-human insulin,
N.sup..epsilon.B29-decanoyl-Gln.sup.B3-human insulin,
N.sup..epsilon.B29-dodecanoyl-Gln.sup.B3-human insulin.
[0190] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Glu.sup.B30-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Glu.sup.B30-human insulin,
N.sup..epsilon.B29-decanoyl-Glu.sup.B30-human insulin,
N.sup..epsilon.B29-dodecanoyl-Glu.sup.B30-human insulin.
[0191] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21Glu.sup.B30-human
insulin, N.sup..epsilon.B29-decanoyl-Gly.sup.A21Glu.sup.B30-human
insulin, N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21Glu.sup.B30-human
insulin.
[0192] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gly.sup.A21Gln.sup.B3Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Gly.sup.A21Gln.sup.B3Glu.sup.B3-
0-human insulin,
N.sup..epsilon.B29-decanoyl-Gly.sup.A21Gln.sup.B3Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-dodecanoyl-Gly.sup.A21Gln.sup.B3Glu.sup.B30-h-
uman insulin,
N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21Glu.sup.B30-human
insulin, N.sup..epsilon.B29-decanoyl-Ala.sup.A21Glu.sup.B30-human
insulin, N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-tridecanoyl-Ala.sup.A21Gln.sup.B3Glu.sup.B30--
human insulin,
N.sup..epsilon.B29-tetradecanoyl-Ala.sup.A21Gln.sup.B3Glu.sup.B30-human
insulin,
N.sup..epsilon.B29-decanoyl-Ala.sup.A21Gln.sup.B3Glu.sup.B30-hum-
an insulin,
N.sup..epsilon.B29-dodecanoyl-Ala.sup.A21Gln.sup.B3Glu.sup.B30-human
insulin.
[0193] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-tridecanoyl-Gln.sup.B3Glu.sup.B30-human insulin,
N.sup..epsilon.B29-tetradecanoyl-Gln.sup.B3Glu.sup.B30-human
insulin, N.sup..epsilon.B29-decanoyl-Gln.sup.B3Glu.sup.B30-human
insulin, N.sup..epsilon.B29-dodecanoyl-Gln.sup.B3Glu.sup.B30-human
insulin.
[0194] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-formyl-human insulin,
N.sup..alpha.B1-formyl-human insulin, N.sup..alpha.A1-formyl-human
insulin, N.sup..epsilon.B29-formyl-N.sup..alpha.B1-formyl-human
insulin, N.sup..epsilon.B29-formyl-N.sup..alpha.A1-formyl-human
insulin, N.sup..alpha.A1-formyl-N.sup..epsilon.B1-formyl-human
insulin,
N.sup..epsilon.B29-formyl-N.sup..alpha.A1-formyl-N.sup..alpha.B1-formyl-h-
uman insulin.
[0195] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-acetyl-human insulin,
N.sup..alpha.B1-acetyl-human insulin, N.sup..alpha.A1-acetyl-human
insulin, N.sup..epsilon.B29-acetyl-N.sup..alpha.B1-acetyl-human
insulin, N.sup..epsilon.B29-acetyl-N.sup..alpha.A1-acetyl-human
insulin, N.sup..alpha.A1-acetyl-N.sup..alpha.B1-acetyl-human
insulin, N.sup..epsilon.B29
acetyl-N.sup..alpha.A1-acetyl-N.sup..alpha.B1-acetyl-human
insulin.
[0196] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-propionyl-human insulin,
N.sup..alpha.B1-propionyl-human insulin,
N.sup..alpha.A1-propionyl-human insulin,
N.sup..epsilon.B29-acetyl-N.sup..alpha.B1-propionyl-human insulin,
N.sup..epsilon.B29-propionyl-N.sup..alpha.A1-propionyl-human
insulin, N.sup..alpha.A1-propionyl-N.sup..alpha.B1-propionyl-human
insulin,
N.sup..epsilon.B29-propionyl-N.sup..alpha.A1-propionyl-N.sup..alpha.B1-pr-
opionyl-human insulin.
[0197] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-butyryl-human insulin,
N.sup..alpha.B1-butyryl-human insulin,
N.sup..alpha.A1-butyryl-human insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.B1-butyryl-human insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.A1-butyryl-human insulin,
N.sup..alpha.A1-butyryl-N.sup..alpha.B1-butyryl-human insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.A1-butyryl-N.sup..alpha.B1-butyry-
l-human insulin.
[0198] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-pentanoyl-human insulin,
N.sup..alpha.B1-pentanoyl-human insulin,
N.sup..alpha.A1-pentanoyl-human insulin,
N.sup..epsilon.B29-pentanoyl-N.sup..alpha.B1-pentanoyl-human
insulin,
N.sup..epsilon.B29-pentanoyl-N.sup..alpha.A1-pentanoyl-human
insulin, N.sup..alpha.A1-pentanoyl-N.sup..alpha.B1-pentanoyl-human
insulin,
N.sup..epsilon.B29-pentanoyl-N.sup..alpha.A1-pentanoyl-N.sup..alpha.B1-pe-
ntanoyl-human insulin.
[0199] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-hexanoyl-human insulin,
N.sup..alpha.B1-hexanoyl-human insulin,
N.sup..alpha.A1-hexanoyl-human insulin,
N.sup..epsilon.B29-hexanoyl-N.sup..alpha.B1-hexanoyl-human insulin,
N.sup..epsilon.B29-hexanoyl-N.sup..alpha.A1-hexanoyl-human insulin,
N.sup..alpha.A1-hexanoyl-N.sup..alpha.B1-hexanoyl-human insulin,
N.sup..epsilon.B29-hexanoyl-N.sup..alpha.A1-hexanoyl-N.sup..alpha.B1-hexa-
noyl-human insulin.
[0200] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-heptanoyl-human insulin,
N.sup..alpha.B1-heptanoyl-human insulin,
N.sup..alpha.A1-heptanoyl-human insulin,
N.sup..epsilon.B29-heptanoyl-N.sup..alpha.B1-heptanoyl-human
insulin,
N.sup..epsilon.B29-heptanoyl-N.sup..alpha.A1-heptanoyl-human
insulin, N.sup..alpha.A1-heptanoyl-N.sup..alpha.B1-heptanoyl-human
insulin,
N.sup..epsilon.B29-heptanoyl-N.sup..alpha.A1-heptanoyl-N.sup..alpha.B1-he-
ptanoyl-human insulin.
[0201] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..alpha.B1-octanoyl-human insulin,
N.sup..alpha.A1-octanoyl-human insulin,
N.sup..epsilon.B29-Octanoyl-N.sup..alpha.B1-octanoyl-human insulin,
N.sup..epsilon.B29-octanoyl-N.sup..alpha.A1-octanoyl-human insulin,
N.sup..alpha.A1-octanoyl-N.sup..alpha.B1-octanoyl-human insulin,
N.sup..epsilon.B29-octanoyl-N.sup..alpha.A1-octanoyl-N.sup..alpha.B1-octa-
noyl-human insulin.
[0202] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-nonanoyl-human insulin,
N.sup..alpha.B1-nonanoyl-human insulin,
N.sup..alpha.A1-nonanoyl-human insulin,
N.sup..epsilon.B29-nonanoyl-N.sup..alpha.B1-nonanoyl-human insulin,
N.sup..epsilon.B29-nonanoyl-N.sup..alpha.A1-nonanoyl-human insulin,
N.sup..alpha.A1-nonanoyl-N.sup..alpha.B1-nonanoyl-human insulin,
N.sup..epsilon.B29-nonanoyl-N.sup..alpha.A1-nonanoyl-N.sup..alpha.B1-nona-
noyl-human insulin.
[0203] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-decanoyl-human insulin,
N.sup..alpha.B1-decanoyl-human insulin,
N.sup..alpha.A1-decanoyl-human insulin,
N.sup..epsilon.B29-decanoyl-N.sup..alpha.B1-decanoyl-human insulin,
N.sup..epsilon.B29-decanoyl-N.sup..alpha.A1-decanoyl-human insulin,
N.sup..alpha.A1-decanoyl-N.sup..alpha.B1-decanoyl-human insulin,
N.sup..epsilon.B29-decanoyl-N.sup..alpha.A1-decanoyl-N.sup..alpha.B1-deca-
noyl-human insulin.
[0204] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-formyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-formyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-formyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-formyl-N.sup..alpha.B1-formyl-Lys.sup.B28Pro.sup.B29-h-
uman insulin,
N.sup..epsilon.B28-formyl-N.sup..alpha.A1-formyl-Lys.sup.B28Pro.sup.B29-h-
uman insulin,
N.sup..alpha.A1-formyl-N.sup..alpha.B1-formyl-Lys.sup.B28Pro.sup.B29-huma-
n insulin,
N.sup..epsilon.B28-formyl-N.sup..alpha.A1-formyl-N.sup..alpha.B-
1-formyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B29-acetyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-acetyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-acetyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-acetyl-N.sup..alpha.B1-acetyl-Lys.sup.B28Pro.sup.B29-h-
uman insulin.
[0205] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-acetyl-N.sup..alpha.A1-acetyl-Lys.sup.B28Pro.sup.B29-h-
uman insulin,
N.sup..alpha.A1-acetyl-N.sup..alpha.B1-acetyl-Lys.sup.B28Pro.sup.B29-huma-
n insulin,
N.sup..epsilon.B28-acetyl-N.sup..alpha.A1-acetyl-N.sup..alpha.B-
1-acetyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0206] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-propionyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-propionyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-propionyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-propionyl-N.sup..alpha.B1-propionyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..epsilon.B28-propionyl-N.sup..alpha.A1-propionyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..alpha.A1-propionyl-N.sup..alpha.B1-propionyl-Lys.sup.B28Pro.sup.B2-
9-human insulin,
N.sup..epsilon.B28-propionyl-N.sup..alpha.A1-propionyl-N.sup..alpha.B1-pr-
opionyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0207] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-butyryl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-butyryl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-butyryl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-butyryl-N.sup..alpha.B1-butyryl-Lys.sup.B28Pro.sup.B29-
-human insulin,
N.sup..epsilon.B28-butyryl-N.sup..alpha.A1-butyryl-Lys.sup.B28Pro.sup.B29-
-human insulin,
N.sup..alpha.A1-butyryl-N.sup..alpha.B1-butyryl-Lys.sup.B28Pro.sup.B29-hu-
man insulin,
N.sup..epsilon.B28-butyryl-N.sup..alpha.A1-butyryl-N.sup..alpha.A1-butyry-
l-Lys.sup.B28Pro.sup.B29-human insulin.
[0208] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-pentanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-pentanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-pentanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-pentanoyl-N.sup..alpha.B1-pentanoyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..epsilon.B28-pentanoyl-N.sup..alpha.A1-pentanoyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..alpha.A1-pentanoyl-N.sup..alpha.B1-pentanoyl-Lys.sup.B28Pro.sup.B2-
9-human insulin,
N.sup..epsilon.B28-pentanoyl-N.sup..alpha.A1-pentanoyl-N.sup..alpha.B1-pe-
ntanoyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0209] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-hexanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-hexanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-hexanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-hexanoyl-N.sup..alpha.B1-hexanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..epsilon.B28-hexanoyl-N.sup..alpha.A1-hexanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..alpha.A1-hexanoyl-N.sup..alpha.B1-hexanoyl-Lys.sup.B28Pro.sup.B29--
human insulin,
N.sup..epsilon.B28-hexanoyl-N.sup..alpha.A1-hexanoyl-N.sup..alpha.B1-hexa-
noyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0210] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-heptanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-heptanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-heptanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-heptanoyl-N.sup..alpha.B1-heptanoyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..epsilon.B28-heptanoyl-N.sup..alpha.A1-heptanoyl-Lys.sup.B28Pro.sup-
.B29-human insulin,
N.sup..alpha.A1-heptanoyl-N.sup..alpha.B1-heptanoyl-Lys.sup.B28Pro.sup.B2-
9-human insulin,
N.sup..epsilon.B28-heptanoyl-N.sup..alpha.A1-heptanoyl-N.sup..alpha.B1-he-
ptanoyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0211] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-octanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-octanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-octanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-octanoyl-N.sup..alpha.B1-octanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..epsilon.B28-octanoyl-N.sup..alpha.A1-octanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..alpha.A1-octanoyl-N.sup..alpha.B1-octanoyl-Lys.sup.B28Pro.sup.B29--
human insulin,
N.sup..epsilon.B28-octanoyl-N.sup..alpha.A1-octanoyl-N.sup..alpha.B1-octa-
noyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0212] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-nonanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-nonanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-nonanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-nonanoyl-N.sup..alpha.B1-nonanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..epsilon.B28-nonanoyl-N.sup..alpha.A1-nonanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..alpha.A1-nonanoyl-N.sup..alpha.B1-nonanoyl-Lys.sup.B28Pro.sup.B29--
human insulin,
N.sup..epsilon.B28-nonanoyl-N.sup..alpha.A1-nonanoyl-N.sup..alpha.B1-nona-
noyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0213] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B28-decanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.B1-decanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..alpha.A1-decanoyl-Lys.sup.B28Pro.sup.B29-human insulin,
N.sup..epsilon.B28-decanoyl-N.sup..alpha.B1-decanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..epsilon.B28-decanoyl-N.sup..alpha.A1-decanoyl-Lys.sup.B28Pro.sup.B-
29-human insulin,
N.sup..alpha.A1-decanoyl-N.sup..alpha.B1-decanoyl-Lys.sup.B28Pro.sup.B29--
human insulin,
N.sup..epsilon.B28-decanoyl-N.sup..alpha.A1-decanoyl-N.sup..alpha.B1-deca-
noyl-Lys.sup.B28Pro.sup.B29-human insulin.
[0214] In certain embodiments, an insulin molecule of the present
disclosure comprises the mutations and/or chemical modifications of
one of the following insulin molecules:
N.sup..epsilon.B29-pentanoyl-Gly.sup.A21Arg.sup.B31Arg.sup.B32-human
insulin,
N.sup..alpha.B1-hexanoyl-Gly.sup.A21Arg.sup.B31Arg.sup.B32-human
insulin,
N.sup..alpha.A1-heptanoyl-Gly.sup.A21Arg.sup.B31Arg.sup.B32-huma- n
insulin,
N.sup..epsilon.B29-octanoyl-N.sup..alpha.B1-octanoyl-Gly.sup.A2-
1Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B29-propionyl-N.sup..alpha.A1-propionyl-Gly.sup.A21Arg.sup-
.B31Arg.sup.B32-human insulin,
N.sup..alpha.A1-acetyl-N.sup..alpha.B1-acetyl-Gly.sup.A21Arg.sup.B31Arg.s-
up.B32-human insulin,
N.sup..epsilon.B29-formyl-N.sup..alpha.A1-formyl-N.sup..alpha.B1-formyl-G-
ly.sup.A21Arg.sup.B31Arg.sup.B32-human insulin,
N.sup..epsilon.B29-formyl-des(B26)-human insulin,
N.sup..alpha.B1-acetyl-Asp.sup.B28-human insulin,
N.sup..epsilon.B29-propionyl-N.sup..alpha.A1-propionyl-N.sup..alpha.B1-pr-
opionyl-Asp.sup.B1Asp.sup.B3Asp.sup.B21-human insulin,
N.sup..epsilon.B29-pentanoyl-Gly.sup.A21-human insulin,
N.sup..alpha.B1-hexanoyl-Gly.sup.A21-human insulin,
N.sup..alpha.A1-heptanoyl-Gly.sup.A21-human insulin,
N.sup..epsilon.B29-octanoyl-N.sup..alpha.B1-octanoyl-Gly.sup.A21-human
insulin,
N.sup..epsilon.B29-propionyl-N.sup..alpha.A1-propionyl-Gly.sup.A-
21-human insulin,
N.sup..alpha.A1-acetyl-N.sup..alpha.B1-acetyl-Gly.sup.A21-human
insulin,
N.sup..epsilon.B29-formyl-N.sup..alpha.A1-formyl-N.sup..alpha.B1-formyl-G-
ly.sup.A21-human insulin, N.sup..epsilon.B29-butyryl-des(B30)-human
insulin, N.sup..alpha.B1-butyryl-des(B30)-human insulin,
N.sup..alpha.A1-butyryl-des(B30)-human insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.B1-butyryl-des(B30)-human
insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.A1-butyryl-des(B30)-huma- n
insulin,
N.sup..alpha.A1-butyryl-N.sup..alpha.B1-butyryl-des(B30)-human
insulin,
N.sup..epsilon.B29-butyryl-N.sup..alpha.A1-butyryl-N.sup..alpha.-
B1-butyryl-des(B30)-human insulin.
[0215] The present disclosure also encompasses modified forms of
non-human insulins (e.g., porcine insulin, bovine insulin, rabbit
insulin, sheep insulin, etc.) that comprise any one of the
aforementioned mutations and/or chemical modifications.
[0216] These and other modified insulin molecules are described in
detail in U.S. Pat. Nos. 6,906,028; 6,551,992; 6,465,426;
6,444,641; 6,335,316; 6,268,335; 6,051,551; 6,034,054; 5,952,297;
5,922,675; 5,747,642; 5,693,609; 5,650,486; 5,547,929; 5,504,188;
5,474,978; 5,461,031; and 4,421,685; and in U.S. Pat. Nos.
7,387,996; 6,869,930; 6,174,856; 6,011,007; 5,866,538; and
5,750,497, the entire disclosures of which are hereby incorporated
by reference.
[0217] In various embodiments, an insulin molecule of the present
disclosure includes the three wild-type disulfide bridges (i.e.,
one between position 7 of the A-chain and position 7 of the
B-chain, a second between position 20 of the A-chain and position
19 of the B-chain, and a third between positions 6 and 11 of the
A-chain).
[0218] In some embodiments, an insulin molecule is modified and/or
mutated to reduce its affinity for the insulin receptor. Without
wishing to be bound to a particular theory, it is believed that
attenuating the receptor affinity of an insulin molecule through
modification (e.g., acylation) or mutation may decrease the rate at
which the insulin molecule is eliminated from serum. In some
embodiments, a decreased insulin receptor affinity in vitro
translates into a superior in vivo activity for an insulin
conjugate. In certain embodiments, an insulin molecule is mutated
such that the site of mutation is used as a conjugation point, and
conjugation at the mutated site reduces binding to the insulin
receptor (e.g., Lys.sup.A3). In certain other embodiments,
conjugation at an existing wild-type amino acid or terminus reduces
binding to the insulin receptor (e.g., Gly.sup.A1). In some
embodiments, an insulin molecule is conjugated at position A4, A5,
A8, A9, or B30. In certain embodiments, the conjugation at position
A4, A5, A8, A9, or B30 takes place via a wild-type amino acid side
chain (e.g., Glu.sup.A4). In certain other embodiments, an insulin
molecule is mutated at position A4, A5, A8, A9, or B30 to provide a
site for conjugation (e.g., Lys.sup.A4, Lys.sup.A5, Lys.sup.A8,
Lys.sup.A9, or Lys.sup.B30).
[0219] Methods for conjugating drugs including insulin molecules
are described below. In certain embodiments, an insulin molecule is
conjugated to a ligand or conjugate framework via the A1 amino acid
residue. In certain embodiments the A1 amino acid residue is
glycine. It is to be understood however, that the present
disclosure is not limited to N-terminal conjugation and that in
certain embodiments an insulin molecule may be conjugated via a
non-terminal A-chain amino acid residue. In particular, the present
disclosure encompasses conjugation via the epsilon-amine group of a
lysine residue present at any position in the A-chain (wild-type or
introduced by site-directed mutagenesis). It will be appreciated
that different conjugation positions on the A-chain may lead to
different reductions in insulin activity. In certain embodiments,
an insulin molecule is conjugated to the conjugate framework via
the B1 amino acid residue. In certain embodiments the B1 amino acid
residue is phenylalanine. It is to be understood however, that the
present disclosure is not limited to N-terminal conjugation and
that in certain embodiments an insulin molecule may be conjugated
via a non-terminal B-chain amino acid residue. In particular, the
present disclosure encompasses conjugation via the epsilon-amine
group of a lysine residue present at any position in the B-chain
(wild-type or introduced by site-directed mutagenesis). For
example, in certain embodiments an insulin molecule may be
conjugated via the B29 lysine residue. In the case of insulin
glulisine, conjugation to the at least one ligand via the B3 lysine
residue may be employed. It will be appreciated that different
conjugation positions on the B-chain may lead to different
reductions in insulin activity.
[0220] In certain embodiments, the ligands are conjugated to more
than one conjugation point on a drug such as an insulin molecule.
For example, an insulin molecule can be conjugated at both the A1
N-terminus and the B29 lysine. In some embodiments, amide
conjugation takes place in carbonate buffer to conjugate at the B29
and A1 positions, but not at the B1 position. In other embodiments,
an insulin molecule can be conjugated at the A1 N-terminus, the B1
N-terminus, and the B29 lysine. In yet other embodiments,
protecting groups are used such that conjugation takes place at the
B1 and B29 or B1 and A1 positions. It will be appreciated that any
combination of conjugation points on an insulin molecule may be
employed. In some embodiments, at least one of the conjugation
points is a mutated lysine residue, e.g., Lys.sup.A3.
[0221] In various embodiments, a conjugate may include an insulin
sensitizer (i.e., a drug which potentiates the action of insulin).
Drugs which potentiate the effects of insulin include biguanides
(e.g., metformin) and glitazones. The first glitazone drug was
troglitazone which turned out to have severe side effects. Second
generation glitazones include pioglitazone and rosiglitazone which
are better tolerated although rosiglitazone has been associated
with adverse cardiovascular events in certain trials.
[0222] In various embodiments, a conjugate may include an insulin
secretagogue (i.e., a drug which stimulates insulin secretion by
beta cells of the pancreas). For example, in various embodiments, a
conjugate may include a sulfonylurea. Sulfonylureas stimulate
insulin secretion by beta cells of the pancreas by sensitizing them
to the action of glucose. Sulfonylureas can, moreover, inhibit
glucagon secretion and sensitize target tissues to the action of
insulin. First generation sulfonylureas include tolbutamide,
chlorpropamide and carbutamide. Second generation sulfonylureas
which are active at lower doses include glipizide, glibenclamide,
gliclazide, glibornuride and glimepiride. In various embodiments, a
conjugate may include a meglitinide. Suitable meglitinides include
nateglinide, mitiglinide and repaglinide. Their hypoglycemic action
is faster and shorter than that of sulfonylureas. Other insulin
secretagogues include glucagon-like peptide 1 (GLP-1) and GLP-1
analogs (i.e., a peptide with GLP-1 like bioactivity that differs
from GLP-1 by 1-10 amino acid substitutions, additions or deletions
and/or by a chemical modification). GLP-1 reduces food intake by
inhibiting gastric emptying, increasing satiety through central
actions and by suppressing glucagon release. GLP-1 lowers plasma
glucose levels by increasing pancreas islet cell proliferation and
increases insulin production following food consumption. GLP-1 may
be chemically modified, e.g., by lipid conjugation as in
liraglutide to extend its in vivo half-life. Yet other insulin
secretagogues include exendin-4 and exendin-4 analogs (i.e., a
peptide with exendin-4 like bioactivity that differs from exendin-4
by 1-10 amino acid substitutions, additions or deletions and/or by
a chemical modification). Exendin-4, found in the venom of the Gila
Monster, exhibits GLP-1 like bioactivity. It has a much longer
half-life than GLP-1 and, unlike GLP-1, it can be truncated by 8
amino acid residues at its N-terminus without losing bioactivity.
The N-terminal region of GLP-1 and exendin-4 are almost identical,
a significant difference being the second amino acid residue,
alanine in GLP-1 and glycine in exendin-4, which gives exendin-4
its resistance to in vivo digestion. Exendin-4 also has an extra 9
amino acid residues at its C-terminus as compared to GLP-1. Mann et
al. Biochem. Soc. Trans. 35:713-716, 2007 and Runge et al.,
Biochemistry 46:5830-5840, 2007 describe a variety of GLP-1 and
exendin-4 analogs which may be used in a conjugate of the present
disclosure. The short half-life of GLP-1 results from enzymatic
digestion by dipeptidyl peptidase IV (DPP-IV). In certain
embodiments, the effects of endogenous GLP-1 may be enhanced by
administration of a DPP-IV inhibitor (e.g., vildagliptin,
sitagliptin, saxagliptin, linagliptin or alogliptin).
[0223] In various embodiments, a conjugate may include amylin or an
amylin analog (i.e., a peptide with amylin like bioactivity that
differs from amylin by 1-10 amino acid substitutions, additions or
deletions and/or by a chemical modification). Amylin plays an
important role in glucose regulation (e.g., see Edelman and Weyer,
Diabetes Technol. Ther. 4:175-189, 2002). Amylin is a
neuroendocrine hormone that is co-secreted with insulin by the beta
cells of the pancreas in response to food intake. While insulin
works to regulate glucose disappearance from the bloodstream,
amylin works to help regulate glucose appearance in the bloodstream
from the stomach and liver. Pramlintide acetate (SYMLIN.RTM.) is an
exemplary amylin analog. Since native human amylin is
amyloidogenic, the strategy for designing pramlintide involved
substituting certain residues with those from rat amylin, which is
not amyloidogenic. In particular, proline residues are known to be
structure-breaking residues, so these were directly grafted from
the rat sequence into the human sequence. Glu-10 was also
substituted with an asparagine.
[0224] In various embodiments, a pre-conjugated drug may contain
one or more reactive moieties (e.g., carboxyl or reactive ester,
amine, hydroxyl, aldehyde, sulfhydryl, maleimidyl, alkynyl, azido,
etc. moieties). As discussed below, these reactive moieties may, in
certain embodiments, facilitate the conjugation process. Specific
examples include peptidic drugs bearing alpha-terminal amine and/or
epsilon-amine lysine groups. It will be appreciated that any of
these reactive moieties may be artificially added to a known drug
if not already present. For example, in the case of peptidic drugs
a suitable amino acid (e.g., a lysine) may be added or substituted
into the amino acid sequence. In addition, as discussed in more
detail below, it will be appreciated that the conjugation process
may be controlled by selectively blocking certain reactive moieties
prior to conjugation.
[0225] In various embodiments, a conjugate of the present
disclosure may be exploited to manipulate a natural feedback
mechanism between glucose and insulin (where the level of insulin
increases as the level of glucose increases and the level of
glucose decreases as the level of insulin increases).
Alternatively, in various embodiments, the drug can be a molecule
that (a) has the same function as insulin (e.g., reduces glucose
levels), (b) stimulates the production of the insulin and/or (c)
potentiates the effect(s) of insulin. For example, as discussed
above one could use an insulin secretagogue or an insulin
sensitizer instead of insulin as the drug.
[0226] In various embodiments, a conjugate can be used which
includes a drug with a function that is not directly related to
glucose. Without limitation, a conjugate which responds to glucose
may be used to provide long-term, mealtime dosing of a drug. Any
drug which needs to be dosed periodically and/or with food would
benefit from such a delivery system. As is well known in the art,
many traditional drugs need to be administered with food or at
mealtimes. For example, drugs which inhibit the absorption of fats
(e.g., orlistat) are advantageously present during mealtime.
Similarly, drugs which lower lipid levels, e.g., lovastatin,
atorvastatin, or simvastatin, or triglyceride levels, e.g.,
gemfibrozil, may also be advantageously released at mealtimes.
Exemplary Insulin Conjugates
[0227] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to one or more
ligands that are independently selected from the group consisting
of aminoethylglucose (AEG), aminoethylmannose (AEM),
aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),
.beta.-aminoethyl-N-acetylglucosamine (AEGA), and aminoethylfucose
(AEF). In certain embodiments, the insulin molecule is conjugated
via the A1 amino acid residue. In certain embodiments, the insulin
molecule is conjugated via the B1 amino acid residue. In certain
embodiments, the insulin molecule is conjugated via the
epsilon-amino group of Lys.sup.B29. In certain embodiments, the
insulin molecule is insulin glulisine conjugated via the
epsilon-amino group of Lys.sup.B3.
[0228] In certain embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to one or more
aminoethylglucose (AEG) ligands. In certain embodiments, a
conjugate of the present disclosure comprises an insulin molecule
conjugated to one or more aminoethylmannose (AEM) ligands. In
certain embodiments, a conjugate of the present disclosure
comprises an insulin molecule conjugated to one or more
aminoethylbimannose (AEBM) ligands. In certain embodiments, a
conjugate of the present disclosure comprises an insulin molecule
conjugated to one or more aminoethyltrimannose (AETM) ligands. In
certain embodiments, a conjugate of the present disclosure
comprises an insulin molecule conjugated to one or more
.beta.-aminoethyl-N-acetylglucosamine (AEGA) ligands. In certain
embodiments, a conjugate of the present disclosure comprises an
insulin molecule conjugated to one or more aminoethylfucose (AEF)
ligands.
[0229] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to two or more
separate ligands. In some embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to two separate
ligands. In other embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to three
separate ligands. In certain embodiments, a conjugate of the
present disclosure comprises an insulin molecule conjugated to four
separate ligands. In certain embodiments, the two or more separate
ligands of such a conjugate are aminoethylglucose (AEG). In certain
embodiments, the two or more separate ligands of such a conjugate
are aminoethylmannose (AEM). In certain embodiments, the two or
more separate ligands of such a conjugate are aminoethylbimannose
(AEBM). In certain embodiments, the two or more separate ligands of
such a conjugate are aminoethyltrimannose (AETM). In certain
embodiments, the two or more separate ligands of such a conjugate
are 3-aminoethyl-N-acetylglucosamine (AEGA). In certain
embodiments, the two or more separate ligands of such a conjugate
are aminoethylfucose (AEF).
[0230] In various embodiments, a conjugate of the present
disclosure comprises two or more separate ligands conjugated to a
single conjugate framework that is also conjugated to an insulin
molecule. In some embodiments, the two or more separate ligands and
insulin molecule of such a conjugate are each located on a separate
branch of a single branched conjugate framework. In some
embodiments, the two or more separate ligands and insulin molecule
of such a conjugate are each located on termini of separate
branches of a single branched conjugate framework. In some
embodiments, the two or more separate ligands of such a conjugate
are conjugated to the insulin molecule via two or more different
conjugation points. In certain such embodiments, the insulin
molecule is conjugated via the A1 amino acid residue and the
epsilon-amino group of Lys.sup.B29. In certain such embodiments,
the insulin molecule is conjugated to two separate conjugate
frameworks that are each conjugated to one or more separate
ligands. In other such embodiments, the insulin molecule is
conjugated to two separate conjugate frameworks that are each
conjugated to one ligand. In yet other such embodiments, the
insulin molecule is conjugated to two separate branched conjugate
frameworks that are each conjugated to two ligands. In certain such
embodiments, the ligands are located on separate branches of the
branched conjugate frameworks. In other such embodiments, the
ligands are located on termini of separate branches of the branched
conjugate frameworks.
[0231] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
aminoethylglucose (AEG). In certain such embodiments, the insulin
molecule is conjugated via the A1 amino acid residue. In certain
such embodiments, the insulin molecule is conjugated via the B1
amino acid residue. In certain such embodiments, the insulin
molecule is conjugated via the epsilon-amino group of Lys.sup.B29.
In certain such embodiments, the insulin molecule is insulin
glulisine conjugated via the epsilon-amino group of Lys.sup.B3. In
certain such embodiments, the insulin molecule is conjugated via
the A1 amino acid residue and the epsilon-amino group of
Lys.sup.B29.
[0232] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
aminoethylmannose (AEM). In certain such embodiments, the insulin
molecule is conjugated via the A1 amino acid residue. In certain
such embodiments, the insulin molecule is conjugated via the B1
amino acid residue. In certain such embodiments, the insulin
molecule is conjugated via the epsilon-amino group of Lys.sup.B29.
In certain such embodiments, the insulin molecule is insulin
glulisine conjugated via the epsilon-amino group of Lys.sup.B3. In
certain such embodiments, the insulin molecule is conjugated via
the A1 amino acid residue and the epsilon-amino group of
Lys.sup.B29.
[0233] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
aminoethylbimannose (AEBM). In certain such embodiments, the
insulin molecule is conjugated via the A1 amino acid residue. In
certain such embodiments, the insulin molecule is conjugated via
the B1 amino acid residue. In certain such embodiments, the insulin
molecule is conjugated via the epsilon-amino group of Lys.sup.B29.
In certain such embodiments, the insulin molecule is insulin
glulisine conjugated via the epsilon-amino group of Lys.sup.B3. In
certain such embodiments, the insulin molecule is conjugated via
the A1 amino acid residue and the epsilon-amino group of
Lys.sup.B29.
[0234] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
aminoethyltrimannose (AETM). In certain such embodiments, the
insulin molecule is conjugated via the A1 amino acid residue. In
certain such embodiments, the insulin molecule is conjugated via
the B1 amino acid residue. In certain such embodiments, the insulin
molecule is conjugated via the epsilon-amino group of Lys.sup.B29.
In certain such embodiments, the insulin molecule is insulin
glulisine conjugated via the epsilon-amino group of Lys.sup.B3. In
certain such embodiments, the insulin molecule is conjugated via
the A1 amino acid residue and the epsilon-amino group of
Lys.sup.B29.
[0235] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
.beta.-aminoethyl-N-acetylglucosamine (AEGA). In certain such
embodiments, the insulin molecule is conjugated via the A1 amino
acid residue. In certain such embodiments, the insulin molecule is
conjugated via the B1 amino acid residue. In certain such
embodiments, the insulin molecule is conjugated via the
epsilon-amino group of Lys.sup.B29. In certain such embodiments,
the insulin molecule is insulin glulisine conjugated via the
epsilon-amino group of Lys.sup.B3. In certain such embodiments, the
insulin molecule is conjugated via the A1 amino acid residue and
the epsilon-amino group of Lys.sup.B29.
[0236] In various embodiments, a conjugate of the present
disclosure comprises an insulin molecule conjugated to
aminoethylfucose (AEF). In certain such embodiments, the insulin
molecule is conjugated via the A1 amino acid residue. In certain
such embodiments, the insulin molecule is conjugated via the B1
amino acid residue. In certain such embodiments, the insulin
molecule is conjugated via the epsilon-amino group of Lys.sup.B29.
In certain such embodiments, the insulin molecule is insulin
glulisine conjugated via the epsilon-amino group of Lys.sup.B3. In
certain such embodiments, the insulin molecule is conjugated via
the A1 amino acid residue and the epsilon-amino group of
Lys.sup.B29.
Conjugate Frameworks
[0237] This section describes some exemplary conjugate frameworks.
In various embodiments, a conjugate of the present disclosure may
have the general formula (I):
##STR00006##
[0238] wherein: [0239] each occurrence of
[0239] ##STR00007## represents a potential branch within the
conjugate; [0240] each occurrence of
[0240] ##STR00008## represents a potential repeat within a branch
of the conjugate; [0241] each occurrence of is independently a
covalent bond, a carbon atom, a heteroatom, or an optionally
substituted group selected from the group consisting of acyl,
aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic;
[0242] each occurrence of T is independently a covalent bond or a
bivalent, straight or branched, saturated or unsaturated,
optionally substituted C.sub.1-30 hydrocarbon chain wherein one or
more methylene units of T are optionally and independently replaced
by --O--, --S--, --N(R)--, --C(O)--, --C(O)O--, --OC(O)--,
--N(R)C(O)--, --C(O)N(R)--, --S(O)--, --S(O).sub.2--,
--N(R)SO.sub.2--, --SO.sub.2N(R)--, a heterocyclic group, an aryl
group, or a heteroaryl group; [0243] each occurrence of R is
independently hydrogen, a suitable protecting group, or an acyl
moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl
moiety, or heteroaliphatic moiety; [0244] --B is -T-L.sup.B-X;
[0245] each occurrence of X is independently a ligand; [0246] each
occurrence of L.sup.B is independently a covalent bond or a group
derived from the covalent conjugation of a T with an X; [0247] -D
is -T-L.sup.D-W; [0248] each occurrence of W is independently a
drug; [0249] each occurrence of L.sup.D is independently a covalent
bond or a group derived from the covalent conjugation of a T with a
W; [0250] k is an integer from 1 to 12, inclusive; [0251] q is an
integer from 1 to 4, inclusive; [0252] each occurrence of p is
independently an integer from 1 to 5, inclusive; and [0253] each
occurrence of n is independently an integer from 0 to 5, inclusive;
and [0254] each occurrence of m is independently an integer from 1
to 5, inclusive; and [0255] each occurrence of v is independently
an integer from 0 to 5, inclusive, with the proviso that within
each k-branch at least one occurrence of n is .gtoreq.1 and at
least one occurrence of v is .gtoreq.1.
[0256] It is to be understood that general formula (I) (and other
formulas herein) does not expressly list every hydrogen. For
example, if the central is a C.sub.6 aryl group and k+q<6 it
will be appreciated that the open position(s) on the C.sub.6 aryl
ring include a hydrogen.
[0257] In general, it will be appreciated that each occurrence of
represents a potential branching node and that the number of
branches at each node are determined by the values of k for the
central and n for non-central occurrences of . One of ordinary
skill will appreciate that because each occurrence of n may be an
integer from 0 to 5, the present disclosure contemplates linear,
branched, and hyperbranched (e.g., dendrimer-like) embodiments of
these conjugates. The proviso which requires that within each
k-branch at least one occurrence of n is .gtoreq.1 and at least one
occurrence of v is .gtoreq.1 ensures that every conjugate includes
at least one occurrence of B (i.e., a ligand).
[0258] In certain embodiments, each occurrence of in a p-bracketed
moiety is substituted by a number of n-bracketed moieties
corresponding to a value of n.gtoreq.1. For example, when k=2 and
p=2 in both k-branches, the conjugate may be of the formula
(Ia):
##STR00009##
[0259] In other embodiments, only terminal occurrences of in a
p-bracketed moiety are substituted by a number of n-bracketed
moieties corresponding to a value of n.gtoreq.1. For example, when
k=2 and p=2 in both k-branches (and n=0 for the first p-bracketed
moiety in both k-branches), the conjugate may be of the formula
(Ib):
##STR00010##
[0260] In certain embodiments, each occurrence of in an m-bracketed
moiety is substituted by a number of B moieties corresponding to
the value of v.gtoreq.1. For example, when k=2, each occurrence of
p=1, and each occurrence of m=2, the conjugate may be of the
formula (Ic):
##STR00011##
[0261] In other embodiments, only terminal occurrences of in an
m-bracketed moiety are substituted by a number of B moieties
corresponding to a value of v.gtoreq.1. For example, when k=2, each
occurrence of p=1, and each occurrence of m=2 (and v=0 for the
first m-bracketed moiety in each n-branch), the conjugate may be of
the formula (Id):
##STR00012##
[0262] By way of further example, when q=1 and n=1 in both
k-branches of the previous formula, the conjugate may be of the
formula (Ie):
##STR00013##
[0263] Alternatively, when q=1 and n=2 in both k-branches of the
previous formula, the conjugate may be of the formula (If):
##STR00014##
[0264] In various embodiments, the present disclosure also provides
conjugates which include ligands and/or a drug which is
non-covalently bound to a conjugate framework.
[0265] For example, in some embodiments, the present disclosure
provides conjugates of any of the foregoing formulas, wherein:
[0266] each of , T, D, k, q, p, n, m and v is defined as described
above and herein; [0267] --B is -T-LRP.sup.B-X; [0268] each
occurrence of X is independently a ligand; and [0269] each
occurrence of LRP.sup.B is independently a ligand-receptor pair
which forms a non-covalent bond between T and X with a dissociation
constant in human serum of less than 1 pmol/L.
[0270] In yet other embodiments, the present disclosure provides
conjugates of any of the foregoing formulas, wherein: [0271] each
of , T, B, k, q, p, n, m and v is defined as described above and
herein; [0272] -D is -T-LRP.sup.D-W; [0273] each occurrence of W is
independently a drug; and [0274] each occurrence of LRP.sup.D is
independently a ligand-receptor pair which forms a non-covalent
bond between T and W with a dissociation constant in human serum of
less than 1 pmol/L.
[0275] In other embodiments, the present disclosure provides
conjugates of any of the foregoing formulas wherein: [0276] each of
, T, k, q, p, n, m and v is defined as described above and herein;
[0277] --B is -T-LRP.sup.B-X; [0278] each occurrence of X is
independently a ligand; [0279] each occurrence of LRP.sup.B is
independently a ligand-receptor pair which forms a non-covalent
bond between T and X with a dissociation constant in human serum of
less than 1 pmol/L; [0280] -D is -T-LRP.sup.D-W; [0281] each
occurrence of W is independently a drug; and [0282] each occurrence
of LRP.sup.D is independently a ligand-receptor pair which forms a
non-covalent bond between T and W with a dissociation constant in
human serum of less than 1 pmol/L.
[0283] In another aspect, a conjugate of the present disclosure may
have the general formula (II):
##STR00015##
wherein , B, T, D, v, m, n, p, and k are as defined and described
herein, and j is an integer from 1 to 4 inclusive. Conjugates of
formula (II) may have multiple sites of conjugation of ligand to
drug (i.e., where two or more ligands are conjugated to a single
drug molecule via different sites on the drug, e.g., different
amino acids in a biomolecular drug). It will be appreciated that,
when q is 1, the subgenera described above (i.e., formulae
(Ia)-(If)) apply to conjugates of formula (II) when j is 1.
Likewise, similar subgenera can be contemplated by one skilled in
the art for conjugates wherein j is 2, 3, or 4.
[0284] For purposes of exemplification and for the avoidance of
confusion it is to be understood that an occurrence of:
##STR00016##
in a conjugate of formula (II) (i.e., when j is 2) could be
represented as:
##STR00017##
(when the drug is covalently bound to the conjugate framework)
or
##STR00018##
(when the drug is non-covalently bound to the conjugate
framework).
Description of Exemplary Groups
[0285] (Node)
[0286] In certain embodiments, each occurrence of is independently
an optionally substituted group selected from the group consisting
of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and
heterocyclic. In some embodiments, each occurrence of is the same.
In some embodiments, the central is different from all other
occurrences of . In certain embodiments, all occurrences of are the
same except for the central .
[0287] In some embodiments, is an optionally substituted aryl or
heteroaryl group. In some embodiments, is 6-membered aryl. In
certain embodiments, is phenyl.
[0288] In certain embodiments, is a heteroatom selected from N, O,
or S. In some embodiments, is nitrogen atom. In some embodiments,
is an oxygen atom. In some embodiments, is sulfur atom. In some
embodiments, is a carbon atom.
T (Spacer)
[0289] In certain embodiments, each occurrence of T is
independently a bivalent, straight or branched, saturated or
unsaturated, optionally substituted C.sub.1-20 hydrocarbon chain
wherein one or more methylene units of T are optionally and
independently replaced by --O--, --S--, --N(R)--, --C(O)--,
--C(O)O--, --OC(O)--, --N(R)C(O)--, --C(O)N(R)--, --S(O)--,
--S(O).sub.2--, --N(R)SO.sub.2--, --SO.sub.2N(R)--, a heterocyclic
group, an aryl group, or a heteroaryl group. In certain
embodiments, one, two, three, four, or five methylene units of T
are optionally and independently replaced. In certain embodiments,
T is constructed from a C.sub.1-10, C.sub.1-8, C.sub.1-6,
C.sub.1-4, C.sub.2-12, C.sub.4-12, C.sub.6-12, C.sub.8-12, or
C.sub.10-12 hydrocarbon chain wherein one or more methylene units
of T are optionally and independently replaced by --O--, --S--,
--N(R)--, --C(O)--, --C(O)O--, --OC(O)--, --N(R)C(O)--,
--C(O)N(R)--, --S(O)--, --S(O).sub.2--, --N(R)SO.sub.2--,
--SO.sub.2N(R)--, a heterocyclic group, an aryl group, or a
heteroaryl group. In some embodiments, one or more methylene units
of T is replaced by a heterocyclic group. In some embodiments, one
or more methylene units of T is replaced by a triazole moiety. In
certain embodiments, one or more methylene units of T is replaced
by --C(O)--. In certain embodiments, one or more methylene units of
T is replaced by --C(O)N(R)--. In certain embodiments, one or more
methylene units of T is replaced by --O--.
[0290] In some embodiments, T is
##STR00019##
[0291] In some embodiments, T is
##STR00020##
[0292] In some embodiments, T is
##STR00021##
[0293] In some embodiments, T is
##STR00022##
[0294] In some embodiments, T is
##STR00023##
[0295] In some embodiments, T is
##STR00024##
[0296] In certain embodiments, each occurrence of T is the
same.
[0297] In certain embodiments, each occurrence of T (outside groups
B and D) is a covalent bond and the conjugate is of the general
formula (IV) or (V):
##STR00025##
wherein , B, D, v, m, n, p, k, and j are as defined and described
herein.
[0298] In certain embodiments of formulae (IV) and (V), each
occurrence of except for the central is a covalent bond, each
occurrence of v=1, and the conjugate is of the formula (VI) or
(VII):
##STR00026##
wherein , B, D, q, k, and j are as defined and described
herein.
[0299] In certain such embodiments for formula (VI), k=1 and
q=1.
[0300] In other embodiments, k=2 and q=1.
[0301] In other embodiments, k=3 and q=1.
[0302] In other embodiments, k=1 and q=2.
[0303] In other embodiments, k=2 and q=2.
[0304] In certain such embodiments for formula (VII), k=1 and
j=2.
[0305] In other embodiments, k=2 and j=2.
[0306] In other embodiments, k=3 and j=2.
[0307] In other embodiments, k=1 and j=1.
[0308] In other embodiments, k=2 and j=1.
[0309] In other embodiments, k=3 and j=1.
[0310] In other embodiments, k=1 and j=3.
[0311] In other embodiments, k=2 and j=3.
[0312] In other embodiments, k=3 and j=3.
[0313] In some embodiments, the present disclosure provides
conjugates of general formula (VIa):
##STR00027##
wherein B and D are as defined and described herein.
[0314] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00028##
wherein W and X are as defined and described herein.
[0315] In some embodiments, the present disclosure provides
conjugates of general formula (VIb):
##STR00029##
wherein B and D are as defined and described herein.
[0316] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00030##
wherein W and X are as defined and described herein.
[0317] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00031##
wherein W and X are as defined and described herein.
[0318] In some embodiments, the present disclosure provides
conjugates of general formula (VIc):
##STR00032##
wherein B and D are as defined and described herein.
[0319] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00033##
wherein W and X are as defined and described herein.
[0320] In some embodiments, the present disclosure provides
conjugates of general formula (VId) or (VIe):
##STR00034##
wherein B and D are as defined and described herein.
[0321] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00035##
wherein W and X are as defined and described herein.
[0322] It will be appreciated that subgenera of formulae (VIa),
(VIb), (VIc), (VId), and (VIe) and species thereof, apply to
conjugates of formula (VII) wherein j is 1. Likewise, similar
subgenera and species can be contemplated by one skilled in the art
for conjugates of formula (VII) wherein j is 2, 3, or 4. For
example, when j is 2, the present disclosure provides conjugates of
formula:
##STR00036##
wherein B and D are as defined and described herein.
[0323] In certain embodiments, the present disclosure provides
conjugates of formula:
##STR00037##
wherein W, X, and j are as defined and described herein.
[0324] In another aspect, a conjugate of the present disclosure may
have the general formula (VIII):
##STR00038##
wherein , T, D, v, m, and n are as defined and described for
conjugates of formula (I), and B is -T-L.sup.B-X, wherein each
occurrence of X is independently a ligand that includes a
saccharide.
[0325] For example, in some embodiments, the present disclosure
provides conjugates of formula:
##STR00039##
wherein W and X are as defined and described for conjugates of
formula (I).
[0326] In yet another aspect, the conjugate scaffold is not of
formula (I) or (II), but instead is a macrocycle. In some
embodiments, the macrocycle is a polyamine macrocycle. For example,
in various embodiments, a conjugate of the present disclosure may
have the general formula (IX):
##STR00040##
wherein D is as defined and described for conjugates of formula
(I), and B is -T-L.sup.B-X, wherein each occurrence of X is
independently a ligand that includes a saccharide.
B (Ligand)
[0327] In certain embodiments, --B is -T-L.sup.B-X where X is a
ligand and L.sup.B is a covalent bond or a group derived from the
covalent conjugation of an X with a T. Exemplary ligands and their
saccharide components were described above.
D (Drug)
[0328] In certain embodiments, -D is -T-L.sup.D-W where W is a drug
and L.sup.D is a covalent bond or a group derived from the covalent
conjugation of a W with a T. Exemplary drugs were described
above.
L.sup.B and L.sup.D (Covalent Conjugation)
[0329] One of ordinary skill will appreciate that a variety of
conjugation chemistries may be used to covalently conjugate an X
with a T and/or a W with a T (generally "components"). Such
techniques are widely known in the art, and exemplary techniques
are discussed below. Components can be directly bonded (i.e., with
no intervening chemical groups) or indirectly bonded through a
spacer (e.g., a coupling agent or covalent chain that provides some
physical separation between the conjugated element and the
remainder of the conjugate framework). It is to be understood that
components may be covalently bound to a conjugate framework through
any number of chemical bonds, including but not limited to amide,
amine, ester, ether, thioether, isourea, imine, etc. bonds. In
certain embodiments, L.sup.B and/or L.sup.D (generally "L" for the
purposes of this section) is a covalent bond. In some embodiments,
L is an optionally substituted moiety derived from conjugating an
optionally substituted carbonyl-reactive, thiol-reactive,
amine-reactive, or hydroxyl-reactive moiety of T with a carboxyl,
thiol, amine, or hydroxyl group of X or W. In some embodiments, L
is an optionally substituted moiety derived from conjugating an
optionally substituted carboxyl-reactive, thiol-reactive,
amine-reactive, or hydroxyl-reactive moiety of X or W with a
carboxyl, thiol, amine, or hydroxyl group of T. In some
embodiments, L is
##STR00041##
In some embodiments, L is a succinimide moiety.
[0330] In various embodiments, components may be covalently bound
to a conjugate framework using "click chemistry" reactions as is
known in the art. These include, for example, cycloaddition
reactions, nucleophilic ring-opening reactions, and additions to
carbon-carbon multiple bonds (e.g., see Kolb and Sharpless, Drug
Discovery Today 8:1128-1137, 2003 and references cited therein as
well as Dondoni, Chem. Asian J. 2:700-708, 2007 and references
cited therein). As discussed above, in various embodiments, the
components may be bound to a conjugate framework via natural or
chemically added pendant groups. In general, it will be appreciated
that the first and second members of a pair of reactive groups
(e.g., a carboxyl group and an amine group which react to produce
an amide bond) can be present on either one of the component and
framework (i.e., the relative location of the two members is
irrelevant as long as they react to produce a conjugate). Exemplary
linkages are discussed in more detail below.
[0331] In various embodiments, carboxyl (or reactive ester) bearing
components can be conjugated to --OH bearing frameworks (OBFs)
using the procedure outlined by Kim et al., Biomaterials
24:4843-4851 (2003). Briefly, the OBF is dissolved in DMSO along
with the carboxyl bearing component and reacted by means of
N',N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine
(DMAP) as catalysts under a dry atmosphere. Carboxyl bearing
components can be conjugated to --NH.sub.2 bearing frameworks
(NBFs) using a carbodiimide (EDAC) coupling procedure. Using this
procedure, the carboxyl bearing component is functionalized by
reaction with EDAC in a pH 5 buffer followed by the addition of the
NBF. In either of these cases (and in any of the following cases),
the resulting products may be purified by any number of means
available to those skilled in the art including, but not limited
to, size exclusion chromatography, reversed phase chromatography,
silica gel chromatography, ion exchange chromatography,
ultrafiltration, and selective precipitation.
[0332] In various embodiments, amine bearing components can be
coupled to --COOH bearing frameworks (CBFs). CBFs using activated
ester moieties (e.g., see Hermanson in Bioconjugate Techniques,
2.sup.nd edition, Academic Press, 2008 and references cited
therein). Briefly, a CBF with terminal activated carboxylic acid
esters such as --NHS, --SSC, --NPC, etc. is dissolved in an
anhydrous organic solvent such as DMSO or DMF. The desired number
of equivalents of amine bearing component are then added and mixed
for several hours at room temperature. Amine bearing components can
also be conjugated to CBFs 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 CBF and an amine bearing
component in dimethylsulfoxide (DMSO) under anhydrous conditions.
Amine bearing components can alternatively be coupled to OBFs
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. CDAP reactions
are carried out in a mixture of DMSO and water at mildly basic pH
using triethylamine (TEA) as a catalyst. In certain embodiments,
amine bearing components can be conjugated to NBFs, e.g., through
glutaraldehyde coupling in aqueous buffered solutions containing
pyridine followed by quenching with glycine. In certain
embodiments, amine bearing components can be conjugated to aldehyde
bearing frameworks using a Schiff Base coupling procedure followed
by reduction (e.g., see Hermanson in Bioconjugate Techniques,
2.sup.nd edition, Academic Press, 2008 and references cited therein
as well as Mei et al. in Pharm. Res. 16: 1680-1686, 1999 and
references cited therein). Briefly, a framework with terminal
activated aldehydes (e.g., acetaldehyde, propionaldehyde,
butyraldehyde, etc.) is dissolved in an aqueous buffer with the pH
at or below neutral to prevent unwanted aldehyde hydrolysis. The
desired number of equivalents of an amine bearing component are
then added and mixed at room temperature followed by addition of an
excess of suitable reducing agent (e.g., sodium borohydride, sodium
cyanobrohydride, sodium triacetoxyborohydride pyridine borane,
triethylamine borane, etc.).
[0333] In various embodiments, hydroxyl bearing components can be
conjugated to OBFs according to the divinylsulfone (DVS) procedure.
Using this procedure, the OBF is added to a pH 11.4 bicarbonate
buffer and activated with DVS followed by addition of a hydroxyl
bearing component after which glycine is added to neutralize and
quench the reaction. Hydroxyl bearing components may also be
coupled to OBFs using activated ester moieties as described above
to produce ester bonds.
[0334] In various embodiments, sulfhydryl bearing components can be
coupled to maleimide bearing frameworks (MBFs) using a relatively
mild procedure to produce thioether bonds (e.g., see Hermanson in
Bioconjugate Techniques, 2.sup.nd edition, Academic Press, 2008 and
references cited therein). Because the maleimide group is much less
susceptible to hydrolysis than activated esters, the reaction can
be carried out under aqueous conditions. Briefly, an MBF is
dissolved in a buffered aqueous solution at pH 6.5-7.5 followed by
the desired number of equivalents of sulfhydryl bearing component.
After mixing at room temperature for several hours, the thioether
coupled conjugate may be purified. Sulfhydryl bearing components
can also be conjugated to NBFs according to a method described by
Thoma et al., J. Am. Chem. Soc. 121:5919-5929, 1999. This reaction
involves suspending the NBF in anhydrous dimethylformamide (DMF)
followed by the addition of 2,6-lutidine and acid anhydride and
subsequent purification of the reactive intermediate. A sulfhydryl
bearing component is then added to a solution of the intermediate
in DMF with triethylamine.
[0335] In various embodiments, azide bearing components can be
coupled to an alkyne bearing framework (ABF) using the
copper(I)-catalyzed modern version of the Huisgen-type azide-alkyne
cycloaddition to give a 1,4-di-substituted 1,2,3-triazole (e.g.,
see Dondoni, Chem. Asian J. 2:700-708, 2007 and references cited
therein as well as Dedola et al., Org. Biomol. Chem. 5: 1006-1017,
2007). This reaction, commonly referred to as a "click" reaction,
may be carried out for example in neat THF using
N,N-diisopropylethylamine and Cu(PPh.sub.3).sub.3Br as the catalyst
system (e.g., see Wu et al., Chem. Commun. 5775-5777, 2005). The
reaction may also be carried out in a 3:1 (THF:water) mixture using
sodium ascorbate and CuSO.sub.4.5H.sub.2O as the catalyst system
(e.g., see Wu et al., supra). In either case, the azide bearing
component is added to the ABF at the desired number of equivalents
followed by mixing for 12-48 hours at room temperature.
Alternatively, alkyne bearing components may be conjugated to an
azide bearing framework using exactly the same conditions described
above.
[0336] Certain components may naturally possess more than one of
the same chemically reactive moiety. In some examples, it is
possible to choose the chemical reaction type and conditions to
selectively react the component at only one of those sites. For
example, in the case where insulin is conjugated through reactive
amines, in certain embodiments, the N-terminal .alpha.-Phe-B1 is a
preferred site of attachment over the N-terminal .alpha.-Gly-A1 and
.epsilon.-Lys-B29 to preserve insulin bioactivity (e.g., see Mei et
al., Pharm. Res. 16: 1680-1686, 1999 and references cited therein
as well as Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997). In an
exemplary reaction between insulin with hexadecenal (an
aldehyde-terminated molecule), researchers found that mixing the
two components overnight in a 1.5M pH 6.8 sodium salicylate aqueous
solution containing 54% isopropanol at a ratio of 1:6
(insulin:aldehyde mol/mol) in the presence of sodium
cyanoborohydride resulted in over 80% conversion to the
single-substituted Phe-B1 secondary amine-conjugated product (Mei
et al., Pharm. Res. 16:1680-1686, 1999). Their studies showed that
the choice of solvent, pH, and insulin:aldehyde ratio all affected
the selectivity and yield of the reaction. In most cases, however,
achieving selectivity through choice of chemical reaction
conditions is difficult. Therefore, in certain embodiments it may
be advantageous to selectively protect the component (e.g.,
insulin) at all sites other than the one desired for reaction
followed by a deprotection step after the material has been reacted
and purified. For example, there are numerous examples of selective
protection of insulin amine groups available in the literature
including those that may be deprotected under acidic (BOC),
slightly acidic (citraconic anhydride), and basic (MSC) conditions
(e.g., see Tsai et al., J. Pharm. Sci. 86: 1264-1268, 1997; Dixon
et al., Biochem. J. 109: 312-314, 1968; and Schuettler et al., D.
Brandenburg Hoppe Seyler's Z. Physiol. Chem. 360: 1721, 1979). In
one example, the Gly-A1 and Lys-B29 amines may be selectively
protected with tert-butoxycarbonyl (BOC) groups which are then
removed after conjugation by incubation for one hour at 4 C in a
90% trifluoroacetic acid (TFA)/10% anisole solution. In one
embodiment, a dry powder of insulin is dissolved in anhydrous DMSO
followed by an excess of triethylamine. To this solution,
approximately two equivalents of di-tert-butyl dicarbonate solution
in THF are added slowly and the solution allowed to mix for 30-60
minutes. After reaction, the crude solution is poured in an excess
of acetone followed by dropwise addition of dilute HCl to
precipitate the reacted insulin. The precipitated material is
centrifuged, washed with acetone and dried completely under vacuum.
The desired di-BOC protected product may be separated from
unreacted insulin, undesired di-BOC isomers, and mono-BOC and
tri-BOC byproducts using preparative reverse phase HPLC or ion
exchange chromatography (e.g., see Tsai et al., J. Pharm. Sci. 86:
1264-1268, 1997). In the case of reverse phase HPLC, a solution of
the crude product in 70% water/30% acetonitrile containing 0.1% TFA
is loaded onto a C8 column and eluted with an increasing
acetonitrile gradient. The desired di-BOC peak is collected,
rotovapped to remove acetonitrile, and lyophilized to obtain the
pure product.
LRP.sup.B and LRP.sup.D (Non-Covalent Conjugation)
[0337] One of ordinary skill will appreciate that a variety of
conjugation chemistries may be used to non-covalently conjugate an
X with a T and/or W with a T (generally "components"). Such
techniques are widely known in the art, and exemplary techniques
are discussed below. In certain embodiments, the dissociation
constant (K.sub.d) of the non-covalent linkage in human serum is
less than 1 pmol/L. For example, a component may be non-covalently
bound to a conjugate framework via a non-covalent ligand-receptor
pair as is well known in the art (e.g., without limitation a
biotin-avidin based pair). In such an embodiment, one member of the
ligand receptor-pair is covalently bound to the component while the
other member of the pair is covalently bound to the conjugate
framework. When the component and conjugate framework are combined,
the strong non-covalent interaction between the ligand and its
receptor causes the component to become non-covalently bound to the
conjugate framework. Typical ligand/receptor pairs include
protein/co-factor and enzyme/substrate pairs. Besides the commonly
used biotin/avidin pair, these include without limitation,
biotin/streptavidin, digoxigenin/anti-digoxigenin,
FK506/FK506-binding protein (FKBP), rapamycin/FKBP,
cyclophilin/cyclosporin and glutathione/glutathione transferase
pairs. Other suitable ligand/receptor pairs would be recognized by
those skilled in the art, e.g., monoclonal antibodies paired with a
epitope tag such as, without limitation, glutathione-S-transferase
(GST), c-myc, FLAG.RTM. and further those described in Kessler pp.
105-152 of Advances in Mutagenesis" Ed. by Kessler,
Springer-Verlag, 1990; "Affinity Chromatography: Methods and
Protocols (Methods in Molecular Biology)" Ed. by Pascal Baillon,
Humana Press, 2000; and "Immobilized Affinity Ligand Techniques" by
Hermanson et al., Academic Press, 1992.
k and q
[0338] k is an integer from 1 to 12, inclusive. In certain
embodiments, k=1 to 6, e.g., 1, 2, or 3. q is an integer from 1 to
4, inclusive, and defines the number of D groups which are bound to
the central group. In certain embodiments, q=1. In some
embodiments, q=2. In certain embodiments, k+q is an integer from 2
to 6, inclusive. In certain embodiments, k+q=2, 3 or 4.
p and m
[0339] Each occurrence of p is independently an integer from 1 to
5, inclusive. In certain embodiments, each occurrence of p is the
same. In certain embodiments, p=1, 2 or 3. In certain embodiments,
p=1.
[0340] Each occurrence of m is independently an integer from 1 to
5, inclusive. In certain embodiments, each occurrence of m is the
same. In certain embodiments, m=1, 2 or 3. In certain embodiments,
m=1.
n and v
[0341] Each occurrence of n is independently an integer from 0 to
5, inclusive, with the proviso that within each k-branch at least
one occurrence of n is .gtoreq.1. Branches within a given k-branch
are referred to herein as n-branches.
[0342] In certain embodiments, each occurrence of in a p-bracketed
moiety is substituted by a number of n-bracketed moieties
corresponding to a value of n.gtoreq.1, e.g., see formula (Ia)
above. In some such embodiments, each occurrence of n in the
conjugate is the same. In some of these embodiments, n=1 or 2.
[0343] In other embodiments, only terminal occurrences of in a
p-bracketed moiety are substituted by a number of n-bracketed
moieties corresponding to a value of n.gtoreq.1, e.g., see formula
(Ib) above. In certain embodiments, each k-branch includes just one
occurrence of n.gtoreq.1 (i.e., all other occurrences of n=0). In
some such embodiments, each occurrence of n in the conjugate is the
same. In some of these embodiments, n=1 or 2.
[0344] Each occurrence of v is independently an integer from 0 to
5, inclusive, with the proviso that within each k-branch at least
one occurrence of v is .gtoreq.1.
[0345] In certain embodiments, each occurrence of in an m-bracketed
moiety is substituted by a number of B moieties corresponding to
the value of v.gtoreq.1, e.g., see formula (Ic) above. In some such
embodiments, each occurrence of v in the conjugate is the same. In
some of these embodiments, v=1 or 2.
[0346] In other embodiments, only terminal occurrences of in an
m-bracketed moiety are substituted by a number of B moieties
corresponding to a value of v.gtoreq.1, e.g., see formula (Id)
above. In certain embodiments, each k-branch includes just one
occurrence of v.gtoreq.1 (i.e., all other occurrences of v=0). In
some such embodiments, each occurrence of v in the conjugate is the
same. In some of these embodiments, v=1 or 2. In certain
embodiments, each n-branch includes at least one occurrence of
v.gtoreq.1. In certain embodiment, each n-branch includes just one
occurrence of v.gtoreq.1 (i.e., all other occurrences of v=0). In
some such embodiments, each occurrence of v in the conjugate is the
same. In some of these embodiments, v=1 or 2.
j
[0347] j of formula (II) is an integer from 1 to 4, inclusive, and
defines the number of conjugations to the D group. In certain
embodiments, j=1. In certain embodiments, j=2. In some embodiments,
j=3. In other embodiments, j=4.
Drug Loading
[0348] In general, the amount of drug that is loaded onto a
conjugate can be controlled by adjusting the molecular weight of
the conjugate framework and/or the level of chemical activation
(i.e., when pendant groups are added to the framework). In various
embodiments, the drug loading level may be in the range of 5 to 99%
w/w of drug to conjugate. In various embodiments, loading levels
within the narrower range of 50 to 99% may be used, e.g., in the
range of 80 to 99%.
Other
[0349] It is to be understood that while the preceding sections
describe components of the conjugates (e.g., ligand, drug,
framework) under separate headings, the present disclosure
encompasses conjugates that are comprised of any and all of the
disclosed ligands, drugs and frameworks.
Intermediates for Preparing Conjugates
[0350] In one aspect, the present disclosure provides reagents for
preparing conjugates. Thus, in various embodiments, a compound of
general formula (I) is provided wherein: [0351] each of , T, D, k,
q, k+q, p, n, m and v is defined as described above and herein;
[0352] --B is -T-L.sup.B'; and [0353] each occurrence of L.sup.B'
is independently hydrogen, an alkyne-containing moiety, an
azide-containing moiety, or an optionally substituted
carbonyl-reactive, thiol-reactive, amine-reactive, or
hydroxyl-reactive moiety.
[0354] In other embodiments, a compound of general formula (I) is
provided wherein: [0355] each of , T, B, k, q, k+q, p, n, m and v
is defined as described above and herein; [0356] -D is -T-L.sup.D';
and [0357] each occurrence of L.sup.D' is independently hydrogen,
an alkyne-containing moiety, an azide-containing moiety, or an
optionally substituted carbonyl-reactive, thiol-reactive,
amine-reactive, or hydroxyl-reactive moiety.
Methods for Preparing Conjugates
[0358] As described in the Examples, we have exemplified methods
for preparing the aforementioned conjugates using insulin as an
exemplary drug and aminoethylglucose (AEG), aminoethylmannose
(AEM), aminoethylbimannose (AEBM), aminoethyltrimannose (AETM),
aminoethylfucose (AEF), and/or
.beta.-aminoethyl-N-acetylglucosamine (AEGA) as exemplary ligands.
Without limitation, conjugates with two ligands per conjugation
site and with short distances between all framework components may
be prepared using tris(hydroxymethyl) aminomethane (Tris),
tris-succinimidyl aminotriacetate (TSAT),
tris-succinimidyl-1,3,5-benzenetricarboxylate (TSB), and benzene-1,
3, 5-tricarboxy-(N-4-butyric-NHS-ester)amide (TSB-C4) as conjugate
frameworks. If more space between framework components is desired,
then succinimidyl (6-aminocaproyl)aminotriacetate (TSAT-C6),
succinimidyl (6-amino(PEO-6))aminotriacetate (TSAT-PEO-6),
benzene-1, 3, 5-tricarboxy-(N-6-aminocaproic-NHS ester)amide
(TSB-C6), and benzene-1, 3, 5-tricarboxy-(N-10-aminodecanoic-NHS
ester)amide (TSB-C10) may be used. The TSAT-C6 spacer arm chemistry
imparts more hydrophobic character to the conjugate as compared to
TSAT-PEO-6.
[0359] For example, for purposes of illustration, in one
embodiment, both the ligand (e.g., AEG, AEM, AEMB and AETM) and
insulin may be reacted to a TSAT-C6 framework through the terminal
activated esters to produce insulin-TSAT-C6-AEG-2,
insulin-TSAT-C6-AEM-2, insulin-TSAT-C6-AEMB-2, and
insulin-TSAT-C6-AETM-2 conjugates. The various ligands are
synthesized ahead of time as discussed in the Examples. In some
embodiments, the A1 and B29 amino groups of insulin are
BOC-protected as described in the Examples so that each insulin can
only react at the Phe-B1 .alpha.-amino group. In some embodiments,
the B1 and B29 amino groups of insulin are BOC-protected as
described in the Examples so that each insulin can only react at
the Gly-A1 .alpha.-amino group. Approximately one equivalent of
BOC-insulin as a 40-50 mg/ml solution in DMSO is added at room
temperature to a 50 mg/ml solution of TSAT-C6 in DMSO containing
excess triethylamine and allowed to react for approximately one
hour. Next, an excess of AEG, AEM, AEBM, and/or AETM (2-10
equivalents) as a 100 mg/ml solution in DMSO is added and allowed
to react for an additional 2 hours. After reaction, the DMSO
solution is superdiluted by 10.times. into a pH 5 saline buffer
after which the pH is adjusted to 8.0 and the solution passed
through a Biogel P2 column to remove low molecular reactants and
salts. The material eluting in the void fraction is concentrated
using a 3K ultrafiltration apparatus after which it is injected on
a prep scale reverse phase HPLC column (C8, acetonitrile/water
mobile phase containing 0.1% TFA) to purify the desired product
from unreacted BOC2-insulin. The desired elution peak is collected
pooled and rotovapped to remove acetonitrile followed by
lyophilization to obtain a dry powder. Finally, the BOC protecting
groups are removed by dissolving the lyophilized powder in 90%
TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in HEPES pH 8.2 buffer containing 0.150 M NaCl. The
pH is adjusted to between 7.0 and 8.0 using NaOH solution after
which the material is passed through a Biogel P2 column to remove
anisole, BOC, and any other contaminating salts. The deprotected,
purified aqueous conjugate solution is then concentrated to the
desired level and stored at 4 C until needed.
[0360] In another aspect, reaction may take place at the B29
epsilon-amino group using unprotected insulin in carbonate buffer,
since under those conditions the B29 amino group is the most
reactive of the three amino groups present in wild-type insulin. In
an exemplary synthesis, the framework containing N-terminal
activated esters is dissolved at 60 mM in anhydrous DMSO followed
by the addition of triethylamine (TEA). The solution is stirred
rapidly for 10 minutes at room temperature. In parallel, a 448 mM
solution of ligand is prepared in an appropriate volume of
anhydrous DMSO. Once dissolved, enough ligand solution is added
dropwise over the course of ten minutes to provide a number of
reactive equivalents equal to 1.5 times the number of activated
ester groups on the framework, N, minus one. For example, if there
are N=3 initial activated ester groups per framework, then
(3.times.(3-1).times.60 mM/370 mM)=0.973 ml of ligand solution are
added. If there are N=4 initial activated ester groups per
framework, then (3.times.(4-1).times.60 mM/370 mM)=1.46 ml of
ligand solution are added, and so on.
[0361] After the ligand solution is added, the solution is stirred
for one hour at room temperature.
[0362] The amine-bearing drug is then dissolved separately at 17.2
mM in sodium carbonate buffer (0.1 M, pH 11) and the pH
subsequently adjusted to 10.8 with 1.0 N sodium hydroxide. Once
dissolved, the entire framework/DMSO/ligand/TEA solution is added
dropwise over the course of 75 minutes to the drug/carbonate buffer
solution. During the addition, the pH of the resulting mixture is
adjusted every 5 minutes to 10.8 if necessary using dilute HCl or
NaOH. The solution is allowed to stir for an additional 15 minutes
after the dropwise addition to ensure complete reaction.
[0363] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 40 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC. Once collected, the solution is rotovapped to
remove acetonitrile and lyophilized to obtain pure conjugate.
[0364] Furthermore, under the carbonate buffer conditions, the A1
amino group is the second most reactive amino group of wild-type
insulin. Thus, in certain embodiments, A1,B29-disubstituted
insulin-conjugates are synthesized using the conditions described
above with approximately ten times the amount of multivalent active
ester framework and ligand per insulin molecule compared to the
B29-monosubstituted insulin-conjugate synthesis.
[0365] In another aspect, B29-monosubstituted insulin-conjugates
are synthesized using N-terminal protecting amino acid sequences
using similar methods to those reported in U.S. Pat. No. 7,402,565.
Specifically, N-terminal peptide sequences are engineered onto the
insulin A-chain and B-chain such that the protecting amino acid
sequences contain Arg.sup.A0 and Arg.sup.B0 to give an insulin
intermediate. Conjugation takes places at Lys.sup.B29 on the
insulin intermediate, while the N-termini are protected from
conjugation side-products. The conjugated insulin intermediate is
treated with trypsin to cleave the N-terminal protecting amino acid
sequences to give an insulin-conjugate wherein solely Lys.sup.B29
is conjugated. In some embodiments, the insulin intermediate is
derived from a single chain insulin precursor as described in U.S.
Pat. No. 7,402,565. In some embodiments, the insulin intermediate
is a mutant that contains a conjugation site other than Lys.sup.B29
and an analogous synthesis to the one described for Lys.sup.B29 is
performed.
[0366] It will be appreciated that these exemplary procedures may
be used to produce other conjugates with different ligands and
drugs, different conjugation chemistries, different separations
between framework components, and/or different valencies by
substituting the TSAT-C6 framework with a different framework as
described below.
[0367] For example, if yet more distance is required between
framework components and/or a preserved charge is required at the
site of conjugation, then an appropriately-sized amine-bearing
diethyl acetal (e.g., aminopropionaldehyde diethyl acetal (APDA) or
aminobutyraldehyde diethyl acetal (ABDA)) may be conjugated to one
of the reactive groups on the frameworks listed here followed by
complete reaction of the remaining reactive groups with the ligand
of interest (e.g., AEM, AEBM, or AETM). A reactive aldehyde group
can then be revealed from the diethyl acetal under acidic
conditions followed by a reductive amination with insulin to
complete the drug conjugation step then ABDA-TSAT, ABDA-LCTSAT,
etc. may be employed.
[0368] In yet another example, tetrakis-(N-succinimidyl
carboxypropyl)pentaerythritol (TSPE), may be used to attach three
ligands per conjugation site for increased multivalency. It will
also be appreciated by those skilled in the art that any of the
above teachings may be used to produce hyperbranched (e.g.,
dendrimer-like) conjugates with even higher order valencies. For
example, Rockendorf and Lindhorst provide a comprehensive review of
current approaches for producing hyperbranched structures in Topics
in Current Chemistry. 217: 202-238, 2001.
[0369] Furthermore, ligands already containing a predetermined
degree of multivalency may again be reacted according to the
procedures described above to produce even higher orders of ligand
multiplicity. For example, a divalent AEM-2, AEBM-2, or AETM-2
molecule containing a terminal reactive amine may be prepared by
conjugating two of each ligand to a suitable framework to which a
reactive amine is also conjugated. A trivalent AEM-3, AEBM-3, or
AETM-3 molecule containing a terminal reactive amine may be
prepared by conjugating three of each ligand to a suitable
framework to which a reactive amine is also conjugated. The
NH.sub.2-divalent saccharides may be reacted with the same
frameworks described above to produce drug conjugates with 4 and 6
ligands per drug molecule. The NH.sub.2-trivalent saccharides may
be reacted with the same frameworks described above to produce drug
conjugates with 6 and 9 ligands per drug molecule.
[0370] In all cases, it should be recognized that a mixture of
different ligands may be conjugated to the same drug via a
multivalent framework by adjusting the framework chemistry,
valency, and the ligand:framework stoichiometry. For example,
Insulin-AEM-1-AEBM-1, Insulin-AEBM-1-AETM-1, Insulin-AEM-2-AETM-2,
and Insulin-AEM-1-AETM-2 may all be synthesized according to this
mixed ligand method.
[0371] In some cases, it may be desirable to conjugate the ligand
to the framework through a different means than the drug. For
example, a divalent maleimide/monovalent activate ester
functionalized framework (e.g., succinimidyl-3,5-dimaleimidophenyl
benzoate (SDMB)) may be used to conjugate two sulfhydryl
functionalized ligands and one amine-functionalized drug in
separate steps. For example, insulin or another amine-containing
drug may be conjugated to the activated ester portion of the
framework using methods described herein. In a separate step, the
aminoethyl saccharide (AEM, AEBM, AETM) may be converted to a
terminal sulfhydryl-bearing ligand by reaction with
4-iminothiolane. Finally, the framework-di-maleimide-insulin
conjugate may be mixed with an excess of sulfhydryl-functionalized
saccharide to produce the resulting divalent-ligand-insulin
conjugate.
Sustained Release Formulations
[0372] As discussed in the Examples, in certain embodiments it may
be advantageous to administer a conjugate in a sustained fashion
(i.e., in a form that exhibits an absorption profile that is more
sustained than soluble recombinant human insulin). This will
provide a sustained level of conjugate that can respond to
fluctuations in glucose on a timescale that it more closely related
to the typical glucose fluctuation timescale (i.e., hours rather
than minutes). In certain embodiments, the sustained release
formulation may exhibit a zero-order release of the conjugate when
administered to a mammal under non-hyperglycemic conditions (i.e.,
fasted conditions).
[0373] It will be appreciated that any formulation that provides a
sustained absorption profile may be used. In certain embodiments
this may be achieved by combining the conjugate with other
ingredients that slow its release properties into systemic
circulation.
[0374] For example, PZI (protamine zinc insulin) formulations may
be used for this purpose. As described in the Examples, we have
found that in certain embodiments the absorption profile and
stability of PZI formulations prepared with conjugates of the
present disclosure are sensitive to the absolute and relative
amounts of protamine and zinc included in the formulation. For
example, whereas commercial PZI and NPH (neutral protamine
Hagedorn) insulin formulations require only about 0.05 to about 0.2
mg protamine/mg insulin, some PZI-conjugate preparations required
about 1 to about 5 mg protamine/mg conjugate in order to
effectively sustain the absorption profile. Furthermore, while
commercial protamine insulin preparations contain about 0.006 mg
zinc/mg insulin, we have found that increasing the zinc
concentration along with the protamine concentration can, in
certain embodiments, lead to more stable, easily dispersible
formulations. In some cases, the zinc content is in the range of
about 0.05 to about 0.5 mg zinc/mg conjugate. Furthermore, we have
also unexpectedly found that in certain embodiments, insulin
conjugates substituted at the B1-amine group require more protamine
and zinc to effectively sustain the release profile versus an
insulin conjugate substituted at the B29-amine group. The present
disclosure encompasses amorphous and crystalline forms of these PZI
formulations.
[0375] Thus, in certain embodiments, a formulation of the present
disclosure includes from about 0.05 to about 10 mg protamine/mg
conjugate. For example, from about 0.2 to about 10 mg protamine/mg
conjugate, e.g., about 1 to about 5 mg protamine/mg conjugate.
[0376] In certain embodiments, a formulation of the present
disclosure includes from about 0.006 to about 0.5 mg zinc/mg
conjugate. For example, from about 0.05 to about 0.5 mg zinc/mg
conjugate, e.g., about 0.1 to about 0.25 mg zinc/mg conjugate.
[0377] In certain embodiments, a formulation of the present
disclosure includes protamine and zinc in a ratio (w/w) in the
range of about 100:1 to about 5:1, for example, from about 50:1 to
about 5:1, e.g., about 40:1 to about 10:1. In certain embodiments,
a PZI formulation of the present disclosure includes protamine and
zinc in a ratio (w/w) in the range of about 20:1 to about 5:1, for
example, about 20:1 to about 10:1, about 20:1 to about 15:1, about
15:1 to about 5:1, about 10:1 to about 5:1, about 10:1 to about
15:1.
[0378] The Examples also describe the benefits of including one or
more of the following components in a PZI formulation: an
antimicrobial preservative, an isotonic agent, and/or an
unconjugated insulin molecule.
[0379] In certain embodiments a formulation of the present
disclosure includes an antimicrobial preservative (e.g., m-cresol,
phenol, methylparaben, or propylparaben). In certain embodiments
the antimicrobial preservative is m-cresol. For example, in certain
embodiments, a formulation may include from about 0.1 to about 1.0%
v/v m-cresol. For example, from about 0.1 to about 0.5% v/v
m-cresol, e.g., about 0.15 to about 0.35% v/v m-cresol.
[0380] In certain embodiments a formulation of the present
disclosure includes a polyol as isotonic agent (e.g., mannitol,
propylene glycol or glycerol). In certain embodiments the isotonic
agent is glycerol. In certain embodiments, the isotonic agent is a
salt, e.g., NaCl. For example, a formulation may comprise from
about 0.05 to about 0.5 M NaCl, e.g., from about 0.05 to about 0.25
M NaCl or from about 0.1 to about 0.2 M NaCl.
[0381] In certain embodiments a formulation of the present
disclosure includes an amount of unconjugated insulin molecule. In
certain embodiments, a formulation includes a molar ratio of
conjugated insulin molecule to unconjugated insulin molecule in the
range of about 100:1 to 1:1, e.g., about 50:1 to 2:1 or about 25:1
to 2:1.
[0382] The present disclosure also encompasses the use of standard
sustained (also called extended) release formulations that are well
known in the art of small molecule formulation (e.g., see
Remington's Pharmaceutical Sciences, 19.sup.th ed., Mack Publishing
Co., Easton, Pa., 1995). The present disclosure also encompasses
the use of devices that rely on pumps or hindered diffusion to
deliver a conjugate on a gradual basis. In certain embodiments, a
long acting formulation may (additionally or alternatively) be
provided by using a modified insulin molecule. For example, one
could use insulin glargine (LANTUS.RTM.) or insulin detemir
(LEVEMIR.RTM.) instead of wild-type human insulin in preparing the
conjugate. Insulin glargine is an exemplary long acting insulin
analog in which Asp-A21 has been replaced by glycine, and two
arginines have been added to the C-terminus of the B-chain. The
effect of these changes is to shift the isoelectric point,
producing a solution that is completely soluble at pH 4. Insulin
detemir is another long acting insulin analog in which Thr-B30 has
been deleted, and a C14 fatty acid chain has been attached to
Lys-B29.
Uses of Conjugates
[0383] In another aspect, the present disclosure provides methods
of using conjugates. In general, the conjugates can be used to
controllably provide a bioactive drug in response to a saccharide
(e.g., glucose or an exogenous saccharide such as mannose,
alpha-methyl mannose, L-fucose, etc. as described herein). The
disclosure encompasses treating a disease or condition by
administering a conjugate of the present disclosure. Although the
conjugates can be used to treat any patient (e.g., dogs, cats,
cows, horses, sheep, pigs, mice, etc.), they are most preferably
used in the treatment of humans. A conjugate can be administered to
a patient by any route. In general the most appropriate route of
administration will depend upon a variety of factors including the
nature of the disease or condition being treated, the nature of the
drug, the condition of the patient, etc. In general, the present
disclosure encompasses administration by 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. General considerations in the formulation and
manufacture of pharmaceutical compositions for these different
routes may be found, for example, in Remington's Pharmaceutical
Sciences, 19.sup.th ed., Mack Publishing Co., Easton, Pa., 1995. In
various embodiments, the conjugate may be administered
subcutaneously, e.g., by injection. The conjugate can be dissolved
in a carrier for ease of delivery. For example, the carrier can be
an aqueous solution including, but not limited to, sterile water,
saline or buffered saline.
[0384] In general, a therapeutically effective amount of a drug in
the form of a conjugate will be administered. By a "therapeutically
effective amount" of a drug is meant a sufficient amount of the
drug to treat the disease or condition at a reasonable benefit/risk
ratio, which involves a balancing of the efficacy and toxicity of
the drug. 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 drug. Although in general drugs 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 or condition, 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.
[0385] In various embodiments, the drug is insulin and the average
daily dose of insulin is in the range of 10 to 200 U, e.g., 25 to
100 U (where 1 Unit of insulin is .about.0.04 mg). In certain
embodiments, an amount of conjugate with these insulin doses is
administered on a daily basis. In certain embodiments, an amount of
conjugate with 5 to 10 times these insulin doses is administered on
a weekly basis. In certain embodiments, an amount of conjugate with
10 to 20 times these insulin doses is administered on a bi-weekly
basis. In certain embodiments, an amount of conjugate with 20 to 40
times these insulin doses is administered on a monthly basis. Those
skilled in the art will recognize that this same approach may be
extrapolated to other approved drugs with known dose ranges, e.g.,
any of the approved insulin sensitizers and insulin secretagogues
described herein. Typically, the dose of conjugated drug will be
higher than the normal dose of unconjugated drug.
[0386] In certain embodiments, a conjugate of the present
disclosure may be used to treat hyperglycemia in a patient (e.g., a
mammalian patient). In certain embodiments, the patient is
diabetic. However, the present methods are not limited to treating
diabetic patients. For example, in certain embodiments, a conjugate
may be used to treat hyperglycemia in a patient with an infection
associated with impaired glycemic control. In certain embodiments,
a conjugate may be used to treat diabetes.
[0387] In certain embodiments, when a conjugate or formulation of
the present disclosure (with an insulin molecule as the drug) is
administered to a patient (e.g., a mammalian patient) it induces
less hypoglycemia than an unconjugated version of the insulin
molecule. In certain embodiments, a formulation of the present
disclosure (with a conjugate that includes an insulin molecule as
the drug) induces a lower HbA1c value in a patient (e.g., a
mammalian patient) than a formulation comprising an unconjugated
version of the insulin molecule. In certain embodiments, the
formulation leads to an HbA1c value that is at least 10% lower
(e.g., at least 20% lower, at least 30% lower, at least 40% lower,
at least 50% lower) than a formulation comprising an unconjugated
version of the insulin molecule. In certain embodiments, the
formulation leads to an HbA1c value of less than 7%, e.g., in the
range of about 4 to about 6%. In certain embodiments, a formulation
comprising an unconjugated version of the insulin molecule leads to
an HbA1c value in excess of 7%, e.g., about 8 to about 12%.
[0388] It will be understood that the total daily usage of a drug
for any given patient will be decided by the attending physician
within the scope of sound medical judgment. The specific
therapeutically effective amount for any particular patient will
depend upon a variety of factors including the disease or condition
being treated; 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. In various embodiments, a conjugate
of the present disclosure may be administered on more than one
occasion. For example, the present disclosure specifically
encompasses methods in which a conjugate is administered by
subcutaneous injection to a patient on a continuous schedule (e.g.,
once a day, once every two days, once a week, once every two weeks,
once a month, etc.).
[0389] In various embodiments, a conjugate of the present
disclosure may be administered to a patient who is receiving at
least one additional therapy. In various embodiments, the at least
one additional therapy is intended to treat the same disease or
disorder as the administered conjugate. In various embodiments, the
at least one additional therapy is intended to treat a side-effect
of the primary drug. The two or more therapies may be administered
within the same, overlapping or non-overlapping timeframes as long
as there is a period when the patient is receiving a benefit from
both therapies. The two or more therapies may be administered on
the same or different schedules as long as there is a period when
the patient is receiving a benefit from both therapies. The two or
more therapies may be administered within the same or different
formulations as long as there is a period when the patient is
receiving a benefit from both therapies. In certain embodiments, a
single conjugate of the present disclosure may include more than
one drug for treating the same disease or disorder. In certain
embodiments, two or more separate conjugates of the present
disclosure may be administered (as a mixture or separately) that
include different drugs for treating the same disease or disorder.
In certain embodiments, an unconjugated secondary drug may be
admixed with a conjugate of the present disclosure (i.e., a drug
which is simply mixed with the conjugate formulation and not
covalently bound to the conjugate). For example, in certain
embodiments, any of these approaches may be used to administer more
than one anti-diabetic drug to a subject. Certain exemplary
embodiments of this approach are described in more detail below in
the context of insulin-related therapies; however, it will be
appreciated from the foregoing that other therapies will benefit
from such combination approaches.
[0390] Insulin sensitizers (e.g., biguanides such as metformin,
glitazones) act by increasing a patient's response to a given
amount of insulin. A patient receiving an insulin sensitizer will
therefore require a lower dose of an insulin conjugate of the
present disclosure than an otherwise identical patient would. Thus,
in certain embodiments, an insulin conjugate may be administered to
a patient who is also being treated with an insulin sensitizer. In
various embodiments, the conjugate of the present disclosure may be
administered at up to 75% of the normal dose required in the
absence of the insulin sensitizer. In various embodiments, up to
50, 40, 30 or 20% of the normal dose may be administered.
[0391] Insulin resistance is a disorder in which normal amounts of
insulin are inadequate to produce a normal insulin response. For
example, insulin-resistant patients may require high doses of
insulin in order to overcome their resistance and provide a
sufficient glucose-lowering effect. In these cases, insulin doses
that would normally induce hypoglycemia in less resistant patients
fail to even exert a glucose-lowering effect in highly resistant
patients. Similarly, the conjugates of the present disclosure are
only effective for this subclass of patients when they provide high
levels of bioactive insulin in a suitable timeframe. In certain
embodiments, the treatment of this subclass of patients may be
facilitated by combining the two approaches. Thus in certain
embodiments, a traditional insulin-based therapy is used to provide
a baseline level of insulin and a conjugate of the present
invention is administered to provide a controlled supplement of
bioactive insulin when needed by the patient. Thus, in certain
embodiments, insulin conjugates may be administered to a patient
who is also being treated with insulin. In various embodiments, the
insulin may be administered at up to 75% of the normal dose
required in the absence of a conjugate of the present disclosure.
In various embodiments, up to 50, 40, 30 or 20% of the normal dose
may be administered. It will be appreciated that this combination
approach may also be used with insulin resistant patients who are
receiving an insulin secretagogue (e.g., a sulfonylurea, GLP-1,
exendin-4, etc.) and/or an insulin sensitizer (e.g., a biguanide
such as metformin, a glitazone).
Exogenous Trigger
[0392] As mentioned previously, the methods, conjugates and
compositions that are described herein are not limited to glucose
responsive-conjugates. As demonstrated in the Examples, several
exemplary glucose-responsive conjugates were also responsive to
exogenous saccharides such as alpha-methyl mannose and L-fucose. It
will therefore be appreciated that in certain embodiments a
conjugate may be triggered by exogenous administration of a
saccharide other than glucose such as alpha-methyl mannose and
L-fucose or any other saccharide that can alter the PK or PD
properties of the conjugate.
[0393] Once a conjugate has been administered as described above
(e.g., as a sustained release formulation) it can be triggered by
administration of a suitable exogenous saccharide. In a certain
embodiment, a triggering amount of the exogenous saccharide is
administered. As used herein, a "triggering amount" of exogenous
saccharide is an amount sufficient to cause a change in at least
one PK and/or PD property of the conjugate (e.g., C.sub.max, AUC,
half-life, etc. as discussed previously). It is to be understood
that any of the aforementioned methods of administration for the
conjugate apply equally to the exogenous saccharide. It is also be
to be understood that the methods of administration for the
conjugate and exogenous saccharide may be the same or different. In
various embodiments, the methods of administration are different
(e.g., for purposes of illustration the conjugate may be
administered by subcutaneous injection on a weekly basis while the
exogenous saccharide is administered orally on a daily basis). The
oral administration of an exogenous saccharide is of particular
value since it facilitates patient compliance. In general, it will
be appreciated that the PK and PD properties of the conjugate will
be related to the PK profile of the exogenous saccharide. Thus, the
conjugate PK and PD properties can be tailored by controlling the
PK profile of the exogenous saccharide. As is well known in the
art, the PK profile of the exogenous saccharide can be tailored
based on the dose, route, frequency and formulation used. For
example, if a short and intense activation of the conjugate is
desired then an oral immediate release formulation might be used.
In contrast, if a longer less intense activation of conjugate is
desired then an oral extended release formulation might be used
instead. General considerations in the formulation and manufacture
of immediate and extended release formulation may be found, for
example, in Remington's Pharmaceutical Sciences, 19.sup.th ed.,
Mack Publishing Co., Easton, Pa., 1995.
[0394] It will also be appreciated that the relative frequency of
administration of a conjugate of the present disclosure and an
exogenous saccharide may be the same or different. In certain
embodiments, the exogenous saccharide is administered more
frequently than the conjugate. For example, in certain embodiment,
the conjugate may be administered daily while the exogenous
saccharide is administered more than once a day. In certain
embodiment, the conjugate may be administered twice weekly, weekly,
biweekly or monthly while the exogenous saccharide is administered
daily. In certain embodiments, the conjugate is administered
monthly and the exogenous saccharide is administered twice weekly,
weekly, or biweekly. Other variations on these schemes will be
recognized by those skilled in the art and will vary depending on
the nature of the conjugate and formulation used.
Examples
[0395] The structures of exemplary conjugates I-1 to I-17 and II-1
to II-7 that are described and used in the Examples are shown in
FIG. 45.
I. Methods of Making Exemplary Conjugates
[0396] 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
Synthesis of Azidoethylglucose (AzEG)
[0397] a. Synthesis of Bromoethyleglucose
[0398] DOWEX 50W.times.4 resin (Alfa Aesar, Ward Hill, Mass.) was
washed with deionized water to remove color. A mixture of 225 gm
D-glucose (1.25 mol; 1 equiv., Alfa Aesar) and 140 gm DOWEX
50W.times.4 was treated with 2.2 L 2-bromoethanol (30.5 mol, 25
equiv.; 124.97 gm/mol; 1.762 gm/mL; BP=150 C; Alfa Aesar) and the
stirred mixture heated to 80 C for 4 hours. The reaction was
monitored by TLC (20% methanol/dichloromethane (DCM)). Reaction was
complete after about four hours, and it was allowed to cool to room
temperature. The solution was filtered to remove the resin, and the
resin washed with ethyl acetate and DCM. The resulting filtrate was
stripped to an amber oil in a rotory evaporator. A total of 400 gm
after stripping.
[0399] The amber oil was purified on silica gel (4 kg silica packed
in DCM) in the following manner. The crude was dissolved in DCM and
loaded onto the column, and then eluted with 2.times.4 L 10%
methanol/DCM; 2.times.4 L 15% methanol/DCM; and 3.times.4 L 20%
methanol/DCM. Product containing fractions (on the basis of TLC)
were pooled and stripped to dryness to afford 152 gm of
1-.alpha.-bromoethyl-glucose (42%).
[0400] b. Conversion of Bromoethylglucose to Azidoethylglucose
(AzEM)
[0401] A 5 L round bottom three-necked flask, equipped with a
heating mantle, an overhead stirrer, and a thermometer, was charged
with 150 gm bromoethylglucose (525 mmol). The oil was dissolved in
2 L water and treated with 68.3 gm sodium azide (1.05 mol, 2
equiv.; 65 gm/mol; Alfa-Aesar) followed by 7.9 gm sodium iodide
(52.5 mmol, 0.08 equiv.; 149.89 gm/mol; Alfa-Aesar) and the
solution warmed to 50 C and stirred overnight. The solution was
cooled to room temperature and concentrated to dryness on the
rotovap. The solid residue was digested with 3.times.500 mL of 5:1
vol. CHCl.sub.3:MeOH at 40 C. The combined organic portions were
filtered and evaporated to dryness to afford azidoethylglucose (86
gm) as an off-white solid. TLC (20% MeOH/DCM; char with
H.sub.2SO.sub.4): single spot, indistinguishable from the starting
material.
[0402] c. Repurification of Azidoethylglucose
[0403] 32 gm of azidoethylglucose was taken into 100 mL water. The
turbid solution was filtered through a glass microfibre filter
(Whatman GF/B). The golden filtrate was evaporated to a solid on a
rotovapor. The solid was taken into methanol (100 mL) and the
turbid solution was again filtered through a glass microfibre
filter. The resulting pale yellow filtrate was stripped to a solid
under vacuum.
[0404] The solid was taken into a minimum of methanol (50 mL) and
ethyl acetate (150 mL) was added slowly with stirring. The heavy
slurry was cooled and filtered. The solid was air dried
(hygroscopic) and put in a 60 C oven overnight. TLC has very little
origin material. Yield 15.4 gm. The Mother Liquor was evaporated
under vacuum to a yellow gum. No attempt was made to further purify
this material at this time.
Example 2
Synthesis of Azidoethylmannose (AzEM)
[0405] a. Synthesis of Bromoethylmannose
[0406] DOWEX 50W.times.4 resin (Alfa Aesar, Ward Hill, Mass.) is
washed with deionized water to remove color. A mixture of 225 gm
D-mannose (1.25 mol; 1 equiv., Alfa Aesar) and 140 gm DOWEX
50W.times.4 is treated with 2.2 L 2-bromoethanol (30.5 mol, 25
equiv.; 124.97 gm/mol; 1.762 gm/mL; BP=150 C; Alfa Aesar) and the
stirred mixture heated to 80 C for 4 hours. The reaction is
monitored by TLC (20% methanol/dichloromethane (DCM)). Reaction is
complete after about four hours, and then allowed to cool to room
temperature. The solution is filtered to remove the resin, and the
resin washed with ethyl acetate and DCM. The resulting filtrate is
stripped to an amber oil in a rotory evaporator.
[0407] The amber oil is purified on silica gel (4 kg silica packed
in DCM) in the following manner. The crude is dissolved in DCM and
loaded onto the column, and then eluted with 2.times.4 L 10%
methanol/DCM; 2.times.4 L 15% methanol/DCM; and 3.times.4 L 20%
methanol/DCM. Product containing fractions (on the basis of TLC)
are pooled and stripped to dryness to afford 152 gm of
1-.alpha.-bromoethyl-mannose (42%).
[0408] b. Conversion of Bromoethylmannose to Azidoethylmannose
(AzEM)
[0409] A 5 L round bottom three-necked flask, equipped with a
heating mantle, an overhead stirrer, and a thermometer, is charged
with 150 gm bromoethylmannose (525 mmol). The oil is dissolved in 2
L water and treated with 68.3 gm sodium azide (1.05 mol, 2 equiv.;
65 gm/mol; Alfa-Aesar) followed by 7.9 gm sodium iodide (52.5 mmol,
0.08 equiv.; 149.89 gm/mol; Alfa-Aesar) and the solution warmed to
50 C and stirred overnight. The solution is cooled to room
temperature and concentrated to dryness on the rotovap. The solid
residue is digested with 3.times.500 mL of 5:1 vol. CHCl.sub.3:MeOH
at 40 C. The combined organic portions are filtered and evaporated
to dryness to afford azidoethylmannose as an off-white solid.
[0410] c. Repurification of Azidoethylmannose
[0411] 32 gm of azidoethylmannose is taken into 100 mL water. The
turbid solution is filtered through a glass microfibre filter
(Whatman GF/B). The filtrate is evaporated to a solid on a
rotovapor. The solid is taken into Methanol (100 mL) and the turbid
solution is again filtered through a glass microfibre filter. The
resulting pale yellow filtrate is stripped to a solid under
vacuum.
[0412] The solid is taken into a minimum of methanol (50 mL) and
ethyl acetate (150 mL) is added slowly with stirring. The heavy
slurry is cooled and filtered. The solid is air dried (hygroscopic)
and put in a 60 C oven overnight. The Mother Liquor is evaporated
under vacuum to a yellow gum.
Example 3
Synthesis of Azidoethylmannobiose (AzEBM)
[0413] The AzEM compound from Example 2 is selectively protected
using benzene dimethyl ether, purified by column chromatography and
subsequently reacted with benzyl bromide to give
1-.alpha.-(2-azidoethyl)-4,6-benzaldehyde
diacetal-3-benzyl-mannopyranoside. The product is subsequently
glycosylated with
1-.alpha.-bromo-2,3,4,6-tetrabenzoylmannopyranoside using silver
triflate chemistry under rigorously anhydrous conditions to give
the protected-azidoethylmannobiose product. The intermediate
product is then deprotected to remove the benzoyl groups to give
AzEBM.
Example 4
Synthesis of Azidoethylmannotriose (AzETM)
a. 1-.alpha.-bromo-2,3,4,6-tetrabenzoyl-mannose
[0414] To a 500 mL 3-neck flask containing a stir bar and nitrogen
inlet was added 40 gm (60.9 mmole) of pentabenzoylmannose and 80 mL
methylene chloride. The resulting solution was cooled in an ice
bath to <5 C, and 80 mL 33% HBr-acetic acid solution was added
via an addition funnel at such a rate to maintain the reaction
temperature <10 C. Upon complete addition (.about.30 min.) the
ice bath was removed and stirring was continued for 3 hours.
[0415] The reaction solution was diluted with an equal volume (160
mL) of DCM and extracted successively with water (2.times.500 mL),
saturated bicarbonate (2.times.50 mL) and Brine (1.times.50 mL),
dried over magnesium sulfate and the solvent evaporated to give 41
gm of solid foam. (Theoretical yield 40.1 gm) and was stored under
N.sub.2 in a freezer. This material was used without further
purification. The reaction was monitored by TLC: silica gel
(Hexane/Ethyl Acetate, 7/3) starting material R.sub.f 0.65, product
R.sub.f 0.8 UV visualization. .sup.1H NMR (CDCl.sub.3) .delta. 8.11
(d, 2H), 8.01 (m, 4H), 7.84 (d, 2H), 7.58 (m, 4H), 7.41 (m, 6H),
7.28 (t, 2H), 6.58 (s, 1H), 6.28 (m, 2H), 5.8 (m, 1H), 4.75 (dd,
1H) 4.68 (dd, 1H) 4.5 (dd, 1H).
b. 1-Azidoethyl-2,4-dibenzoylmannose
[0416] To a 1.0 L, 3-neck flask containing a stir bar, nitrogen
inlet and 300 mL of anhydrous acetonitrile was added 25 gm
1-azidoethylmannose (100.4 mmole), and 50 mL triethyl orthobenzoate
(220 mmole, 2.2 equiv.). The resulting slurry was stirred at room
temperature and 0.8 mL (10 mmole) trifluoroacetic acid (TFA) was
added neat. The solution cleared within 10 minutes and stirring was
continued for an additional two hours, then 25 mL of 10% aqueous
TFA was added and stirring was continued for an additional 2 hours
to hydrolyze the intermediate to the ester isomers. The solvent was
evaporated under vacuum to a viscous oil, which was triturated with
50 mL DCM and again evaporated to a viscous oil.
[0417] Toluene (70 mL) was added to the residue and the viscous
solution was seeded with 2,4-dibenzoylazidoethylmannose. A fine
precipitate formed within 15 minutes and stirring was continued
overnight at room temperature. The resulting heavy suspension was
set in the freezer for 2-4 hours, then filtered and the solid
washed with ice cold toluene (2.times.10 mL). The solid was air
dried to a constant weight to give 21 gm (TY 22.85 gm @50% isomeric
purity) of .about.95% isomeric purity. The product was taken into
40 mL toluene, stirred for 1 hour and then set in the freezer for
an additional 2 hours. The solid was filtered and washed
(2.times.10 mL) with ice cold toluene and air dried to a constant
weight to give 18.5 gm of the single isomer product
2,4-dibenzoylazidoethylmannose in 83% yield. The mother liquors
contained the undesired isomer and a small amount of the desired
isomer. The reaction was monitored by TLC: SG (Hexane/Ethyl Acetate
7/3) Starting Material R.sub.f 0.0, orthoester intermediate R.sub.f
0.9. (Hexane/Ethyl Acetate: 8/2) SM R.sub.f 0.8, desired isomer
R.sub.f 0.4, un-desired isomer R.sub.f 0.2
[0418] .sup.1H NMR 300 MHz (CDCl.sub.3) .delta. 8.12 (t, 4H), 7.66
(t, 2H), 7.5 (m, 4H), 5.56 (t, 1H), 5.48 (m, 1H), 5.14 (m, 1H), 4.5
(dd, 1H), 4.0 (m, 2H), 3.8 (m, 3H), 3.56 (m, 1H), 3.44 (m, 1H).
c.
Perbenzoylated-man(.alpha.-1,3)-man(.alpha.-1.6)-.alpha.-1-azidoethylma-
nnopyranoside
[0419] To a 1.0 L 3-neck flask with a stir bar, nitrogen inlet was
added 41 gm crude 1-bromo-tetrabenzoymannose (60.9 mmole,
.about.2.5 equiv.) in 185 mL DCM. To this was added 11.2 gm
2,4-dibenzoylazidoethylmannose (24.5 mmole) followed by 11.2 gm 4 A
sieves. The slurry was stirred a room temperature for 10 minutes
and cooled to -15.degree. C. in a methanol/ice bath.
[0420] In a separate dark vessel was added 190 mL toluene followed
by 15.1 gm silver-trifluoromethanesulfonate (AgOTf) (58.8 mmole,
2.4 equiv.) and was stirred into solution in the dark. This
solution was transferred to a large addition funnel, and added
drop-wise to the stirring suspension while protecting the reaction
from light. The reaction temperature was maintained <-10 C by
adjusting the AgOTf addition rate. Upon complete addition
(.about.30 minutes) the cold bath was removed and the reaction
stirred for an additional 2 hours until a single product remained
by TLC (SG, Hexane/Ethyl Acetate: 7/3, Bromo R.sub.f 0.9, azido
R.sub.f 0.4, trios product R.sub.f 0.5, uv visualization).
[0421] Triethylamine (7 mL, 5.0 equiv.) was added followed by 200
mL DCM. The resulting slurry was filtered through a pad of silica
gel and celite and washed with 2.times.75 mL DCM. The solvent was
evaporated under vacuum and the residue taken into ethyl acetate
and washed sequentially with water (2.times.100 mL), bicarb
(2.times.50 mL), brine (1.times.75 mL) and dried over magnesium
sulfate. The solvent was evaporated under vacuum to give 39 gm of
solid foam (TY 39.5 gm). .sup.1H NMR 300 MHz (CDCl.sub.3) .delta.
8.3 (d, 2H), 8.2 (m, 8H), 7.85 (d, 4H), 7.75 (dd, 4H), 7.3-7.65 (m,
30H), 7.2 (t, 2H), 6.05 (m, 4H), 5.9 (t, 2H), 5.63 (m, 2H), 5.38
(s, 2H), 5.18 (d, 1H), 4.65 (m, 4H), 4.5 (m, 2H), 4.35 (m, 4H), 3.8
(m, 2H), 3.54 (m, 2H).
d.
Man(.alpha.-1,3)-man(.alpha.-1.6)-.alpha.-1-azidoethylmannopyranoside
[0422] To a stirring suspension of 3.0 gm perbenzoylated-man
(.alpha.-1,3)-man(.alpha.-1.6)-.alpha.-1-azidoethylmannopyranoside
(1.86 mmole) in 40 mL methanol was added 0.2 mL 4.28M sodium
methoxide in methanol. The resulting suspension was stirred 20
hours at room temperature giving a clear solution. The completion
of the reaction was monitored by TLC, (SG, hexane/ethyl acetate:
8/2 SM R.sub.f 0.4, product R.sub.f 0.0).
[0423] The methanol was evaporated under vacuum giving an oily
semi-solid. The residue was taken into ethyl acetate (50 mL) and
stirred for 3 hours. The solid was filtered, washed with fresh
ethyl acetate (2.times.20 mL) and air dried to a constant weight to
give 1.09 gm (TY 1.07 gm) of product.
[0424] The mother liquors contained residual methyl benzoate, the
de-protection by-product.
Example 5
Synthesis of Aminoethyl-Saccharides (AEG, AEM, AEBM, AETM) from
Azidoethyl-Saccharides (AzEG, AzEM, AzEBM, AzETM)
[0425] The azido-terminated compounds from Examples 1-4 are readily
hydrogenated at room temperature by using palladium/carbon
catalyst, a small amount of acetic acid, and ethanol as a solvent
to give the corresponding amine-terminated compounds. FIG. 10 shows
the chemical structures of AEG, AEM, AEBM, AETM. The process is
identical to the one described for AETM below, except that those
skilled in the art will understand that the amounts of reagents,
solvents, etc. should be scaled to the number of moles of
saccharide-ligand to be hydrogenated.
a. Man
(.alpha.-1,3)-Man(.alpha.-1.6)-.alpha.-1-aminoethylmannopyranoside
("aminoethyltrimannose", AETM)
[0426] To a solution of 5.3 gm (9.25 mmole)
man(.alpha.-1,3)-man(.alpha.-1.6)-.alpha.-1-azidoethylmannopyranoside
in 100 mL water and 50 mL ethanol was added 0.8 gm 5% Pd/C. The
vigorously stirring suspension was hydrogenated at 30-40 psi for 48
hours or until no starting material was apparent by TLC (SG,
Methanol, SM R.sub.f 0.75, Pdt R.sub.f 0.0, PMA vis.). The
suspension was filtered over celite, which was rinsed with ethanol
(2.times.50 mL) and the filtrate concentrated under vacuum.
[0427] HPLC of this material (C18, 3% Acetonitrile/97% 0.1%
H.sub.3PO.sub.4, 220 nm, 2 ml/min) gave uv adsorption of the
injection column void material, Rt 2.5 minutes, indicative of
benzoate ester.
[0428] The filtrate was diluted with 70 mL water and 12 mL of 1N
NaOH and the solution stirred overnight at room temperature (HPLC:
no uv material at column void Rt 2.5 min., uv material at Rt 10.5
minutes co-eluting with benzoic acid). 2 gm of decolorizing
charcoal were added and the stirring suspension heated to 80 C,
cooled to room temperature and filtered over celite. The filtrate
pH was adjusted to 8.0 with 2N HCl and the colorless solution
concentrated under vacuum to about 50% volume.
[0429] The solution was loaded onto a resin column (Dowex 50W, 50
gm) and washed with water until eluting fractions were neutral to
pH (6.times.75 mL) removing any residual acid by-products. The
amine product was washed off the column with 0.25N ammonium
hydroxide (6.times.75 mL) and the fractions containing the amine
product-ninhydrin detection were combined and concentrated to 25-30
mL under vacuum. This concentrated solution was added drop-wise to
300 mL stirring ethanol and stirring continued for an additional 2
hours. The product was filtered, washed with fresh ethanol
(2.times.50 mL) and air dried to a constant weight. The resulting
white amorphous solid was dried further in a vacuum oven at 80 C
for 5 hours to give 4.1 gm of a white granular solid (TY 5.1 gm).
The NMR was clean of any aromatic protons. .sup.1H NMR 300 MHz
(D.sub.2O) .delta. 5.08 (s, 1H), 4.87 (s, 1H), 4.81 (s, 1H),
4.8-3.6 (m, 18H), 2.9 (m, 2H).
Example 6
Dipropargyl Saccharide Synthesis and Production of AE-Ligand
a. Synthesis of Diethyl Diproparglymalonate
[0430] Diethylmalonate (122.5 g, 0.7648 mol) was added to absolute
ethanol (800 ml) containing sodium ethoxide (prepared from sodium
metal, 38.5 g, 1.67 mol). After 30 min, propargyl bromide (200 g,
1.68 mol) was slowly added to the stirred suspension, keeping the
temperature under 60 C. The mixture was refluxed overnight (15
hours). The precipitated salts were removed by filtration and
washed with ethanol. Solvent was removed in vacuo, and the residue
diluted with water and extracted with ethanol (2.times.200 ml). The
combined extracts were dried over MgSO4, filtered, washed with Et2O
and the solvent removed in vacuo to afford a golden colored oil.
The oil was placed on high vacuum (40 C) for 3 hours and allowed to
stand. Solids began to crystallize forming an oily solid. Let stand
overnight (16 hours). Cyclohexane was charged to flask, solids
broken-up, filtered, and washed with cyclohexane to afford white
crystalline product (81 gm, 44.8% yield). Reaction was followed by
GC.
b. Synthesis of Dipropargylmalonic Acid
[0431] Diethyl dipropargyl malonate (80 gm, 0.339 mol) was refluxed
in 600 ml of 10% alcoholic potassium hydroxide overnight (15
hours). Solvent was removed in vacuo and the residue was acidified
with 3N HCl. The residue was extracted with Et2O (2.times.300 ml).
The combined extracts were dried over MgSO4, filtered, washed with
Et2O and concentrated in vacuo to an oil. Placed on high vac (40 C)
for 2 hours and let stand to afford dipropargylmalonic acid as an
oil (46 gm, 75.4% yield). Reaction was followed by GC.
c. Synthesis of Dipropargylacetic Acid
[0432] The dipropargylmalonic acid (26 gm, 0.443 mol) was heated
neat at 135 C until CO.sub.2 stopped evolving. It was then allowed
to cool to an oil. The oil was distilled at 0.5 psi. The remaining
oily residue in the distillation flask and solid were combined
(15.7 gm, 79.9% yield) and was used as is in the next step.
d. Synthesis of [2-(3-prop-2-ynyl-hex-5-ynoylamino)-ethyl]-carbamic
acid t-butyl ester
[0433] N-boc-ethylenediamine (18.3 gm, 0.1143 mol) in 50 ml of
CH.sub.3CN was added slowly via an addition funnel to a stirred
solution containing dipropargylacetic acid (15.56 gm, 0.1143 mol),
TBTU (36.74 gm, 0.114 mol) and DIPEA (29.6 gm, 0.229 mol) in 300 ml
of CH.sub.3CN at 0 C. Precipitation occurred. The ice bath was
removed and the product was stirred at ambient temperature
overnight (16 hours). The reaction was now totally homogeneous. The
solution was concentrated in vacuo and the residue was diluted with
800 ml of water. The resulting solids were filtered, washed
copiously with water, and vacuum dried to give 14.3 gm of crude
product. Re-crystallization (2.times.) from DCM, filtration and
washing with hexanes affords the product (9.85 gm, 31% yield, 98%
purity by HPLC (214 nm)).
e. Click reaction of azidosaccharide to
[2-(3-prop-2-ynyl-hex-5-ynoylamino)-ethyl]-carbamic acid t-butyl
ester
[0434] To 1,1 dipropargyl-acetyl-(-1N,
2N--BOC-1,2-diaminoethyl)amide (DP, 418 mg, 1.5 mmole) in DCM (20
mL) was added drop-wise TFA (4 mL) over 5 minutes at 0 C. The
darkening solution was stirred at room temperature overnight. The
volatiles were evaporated under reduced pressure. Toluene (20 mL)
was added to the residue and stripped under reduced pressure two
times. The resulting dark oil was used without further
purification.
[0435] To this residue was added THF (20 mL) and water (20 mL) with
stirring for 15 minutes. Copper Sulfate (225 mg, 0.9 mmole) was
added followed by sodium ascorbate (180 mg, 0.9 mmole). The
resulting mixture was heated to 55-60 C for 6 hours and then
stirred at room temperature for 18 hours. The solution was
evaporated under reduced pressure to approx. half volume and
filtered through a microfibre glass filter. The resulting clear
solution was placed on a resin column (Dowex 50X-2) which was
washed with water (6.times.75 mL) until neutral pH, and then washed
with 10% NH.sub.4OH (8.times.75 mL). The fractions staining
positive with Ninhydrin were combined and evaporated under reduced
pressure to a glassy solid. The glass residue was taken into water
(250 mL) and treated with 0.5 gm charcoal and heated to reflux. The
cooled slurry was filtered over celite and a microfibre filter. The
resulting pale yellow solution was evaporated to a glassy solid
under reduced pressure and methanol was added and evaporated
(2.times.) to give a off white foam (0.9 gm, TY 1.0 gm).
Example 7
Tripropargyl Saccharide Synthesis and Production of AE-Ligand
a.
2-(2-BOC-aminoethyl)thioacetamide-tris[(propargyloxy)methyl]aminomethan-
e
[0436] To a solution of t-butyl N-(2-mercaptoethyl)carbamate
(Frontrun Organix, Ipswich, Mass.; 177.26 mg, 1 mmole) in ethanol
(5 mL) was added NaOH (1.1 mmole) with stirring at room
temperature. To this solution was added
2-bromoacetamide-tris[(propargyloxy)methyl]aminomethane (356 mg,
1.0 mmole, see J. Org. Chem. 73, 5602, 2008) and stirring was
continued for 20 hours (TLC SG 8/2 hexane/ethyl acetate, pdt
R.sub.f 0.4). The solvent was evaporated under vacuum and the
residue was taken into ethyl acetate (40 mL) and washed
successively with water (25 mL), 0.5 N NaOH (25 mL) and Brine (25
mL), dried over Na.sub.2SO.sub.4 filtered and concentrated to an
oil (360 mg, TY 452.3 mg). NMR CDCl.sub.3, (ppm): 7.05 (s, 1H,
N--H); 5.25 ((s, 1H, N--H); 4.85 (s, 6H); 3.85 (s, 6H); 3.3 (m,
2H); 3.15 (s, 2H); 2.7 (m, 2H); 2.42 (s, 3H); 1.22 (s, 9H).
b. 2-(2-aminoethyl)thioacetamide-tris[(triazolo-1-(2-ethylmannose)
4-methoxy)methyl]aminomethane
[0437] To a stirring solution of
2-(2-BOC-aminoethyl)thioacetamide-tris[(propargyloxy)methyl]aminomethane
(1 gm, 2.21 mmole) in DCM (40 mL) at room temperature was added TFA
(4 mL) dropwise. The resulting solution was stirred overnight. The
solvents were removed under vacuum and the residue taken into
toluene (15 mL) and evaporated to dryness.
[0438] The residue was taken into THF (40 mL), water (40 mL) and
stirred into solution. Azidoethylmannose (3.75 eq., 2.0 gm, 8.3
mmole) was added followed by copper sulfate (500 mg, 2.0 mmole) and
sodium ascorbate (400 mg, 2.0 mmole) and the resultant mixture
stirred at 55-60 C (oil bath) for 6 hours, cooled to room
temperature and stirred overnight. The resulting mixture was
concentrated under vacuum to one half volume and filtered thru a
micro-glass filter. The filtrate was loaded on a resin column
(Dowex 50w 50.times.4-100) and eluted with water (6.times.75 mL)
until neutral. The column was then eluted with 15% Ammonium
Hydroxide (10.times.75 mL) and the fractions positive to ninhydrin
were pooled and concentrated to a glassy foam (1.29 gm, TY (MW 1099
g/mol), 53% over two steps).
Example 8
Synthesis of NH.sub.2--B1-BOC2(A1,B29)-insulin
[0439] In a typical synthesis, 4 g of powdered insulin (Sigma
Aldrich, St. Louis, Mo.) is dissolved in 100 ml of anhydrous DMSO
at room temperature followed by the addition of 4 ml of
triethylamine (TEA). The solution is stirred for 30 minutes at room
temperature. Next, 1.79 ml (2.6 equivalents) of
di-tert-butyl-dicarbonate/THF solution (Sigma Aldrich, St. Louis,
Mo.) is slowly added to the insulin-TEA solution and mixed for
approximately one hour. The reaction is quenched via the addition
of 4 ml of a stock solution containing 250 ul of ethanolamine in 5
ml of DMSO followed by mixing for five minutes. After quenching,
the entire solution is poured into 1600 ml of acetone and mixed
briefly with a spatula. Next, 8.times.400 .mu.l aliquots of a 18.9%
HCl:water solution are added dropwise over the surface of the
mixture to precipitate the reacted insulin. The precipitated
material is then centrifuged and the supernatant decanted into a
second beaker while the precipitate cake is set aside. To the
supernatant solution, another 8.times.400 .mu.l aliquots of a 18.9%
HCl:water solution are added dropwise over the surface of the
mixture to obtain a second precipitate of reacted insulin. This
second precipitate is centrifuged and the supernatant is discarded.
The combined centrifuge cakes from the two precipitation steps are
washed once with acetone followed by drying under vacuum at room
temperature to yield the crude powder which typically contains 60%
of the desired BOC2 product and 40% of the BOC3 material.
[0440] A preparative reverse phase HPLC method is used to isolate
the pure BOC2-insulin from the crude powder. Buffer A is deionized
water containing 0.1% TFA and Buffer B is acetonitrile containing
0.1% TFA. The crude powder is dissolved at 25 mg/ml in a 70% A/30%
B mixture and syringe filtered prior to injection on the column.
Before purification, the column (Waters SymmetryPrep C18, 7 um,
19.times.150 mm) is equilibrated at 15 ml/minutes with a 70% A/30%
B mobile phase using a Waters DeltraPrep 600 system. Approximately
5 ml of the crude powder solution is injected onto the column at a
flow rate of 15 ml/minutes over the course of 5 minutes after which
a linear gradient is employed from 70% A/30% B to 62% A/38% B over
the course of the next 3.5 minutes and held there for an additional
2.5 minutes. Using this method, the desired BOC2 peak elutes at
approximately 10.6 minutes followed closely by the BOC3 peak. Once
collected, the solution is rotovapped to remove acetonitrile and
lyophilized to obtain pure BOC2-insulin powder. Identity is
verified by LC-MS (HT Laboratories, San Diego, Calif.) and site of
conjugation determined by N-terminal sequencing (Western
Analytical, St. Louis, Mo.).
Example 9
Synthesis of benzene-1,3,5-tricarboxy-(N-.omega.-aminoacid-NHS
ester) amide frameworks
[0441] A solution of 1,3,5-benzenetricarbonyl chloride (1 gm, 3.8
mmole) in dichloromethane (DCM) (5 mL) is added drop-wise to a
vigorously stirring solution of an .omega.-aminoacid (3.1
equivalents) in 1N NaOH (25 mL) in an ice bath. The ice bath is
removed and stirring is continued for 4 hours at room temperature.
2N HCl (.about.15 mL) is added dropwise to approximately pH 2 and
the resulting slurry is stirred for an additional 2 hours. The
precipitate is filtered, washed with cold water (2.times.20 mL) and
dried in air under vacuum and then in a 60 C oven overnight. The
resulting white solid is used without further purification. Yield
for each .omega.-aminoacid (4-aminobutyric acid: yield 1.6 gm, 91%;
6-aminocaproic acid: yield 1.9 gm, 92%)
[0442] The above material is taken into DMSO (5 mL) containing
N-hydroxysuccinimide (3.1 mmole, 3.1 equiv.) and
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDCI, 3.6 mmole,
3.6 equiv.) is added at room temperature. The resulting solution is
stirred for 24 hours, diluted with water (125 mL) and extracted
with ethyl acetate (3.times.50 mL). The combined organic phase is
washed with water (2.times.50 mL), brine (1.times.50 mL) and dried
over MgSO.sub.4. The solvent is evaporated and the semi-solid
residue triturated with acetonitrile (10 mL). The solid is filtered
and washed with cold solvent, dried in air under vacuum and then in
a 60 C oven overnight. The product is free of urea bi-product.
Benzene-1,3,5-tricarboxy-(N-6-aminocaproic-NHS ester)amide
(TSB-C6): 304 mg, 36%, mp 140-142 C. Benzene-1, 3,
5-tricarboxy-(N-4-butyric-NHS-ester)amide (TSB-C4): 245 mg, 45%, mp
182-184 C.
Example 10
Dendritic Framework Synthesis
a. Hydrogenation of Nitro-Group Containing, Alkyne-Terminally
Functionalized Dendrons
[0443] Dendrons containing either n=2, 4, or 8 terminal alkynes and
a nitropropionic acid core are obtained (e.g., from Polymer
Factory, Sweden) and used without further purification. The dendron
is dissolved in 100 mL a 50:50 vol. mixture of DCM and ethanol, and
0.8 gm of 5% Pd/C is added. The vigorously stirring suspension is
hydrogenated at 30-40 psi for 48 hours or until no starting
material is apparent by TLC. The suspension is filtered over
celite, which is rinsed with ethanol (2.times.50 mL) and the
filtrate concentrated under vacuum.
[0444] The filtrate is diluted with 70 mL water and 12 mL of 1N
NaOH and the solution stirred overnight at room temperature. 2 gm
of decolorizing charcoal are added and the stirring suspension
heated to 80 C, cooled to room temperature and filtered over
celite. The filtrate pH is adjusted to 8.0 with 2N HCl and the
colorless solution concentrated under vacuum to about 50%
volume.
[0445] The solution is loaded onto a resin column (Dowex 50W, 50
gm) and washed with water until eluting fractions are neutral to pH
(6.times.75 mL) removing any residual acid by-products. The amine
product is washed off the column with 0.25N ammonium hydroxide
(6.times.75 mL) and the fractions containing the amine product
(ninhydrin detection) are combined and evaporated to vacuum using a
rotary evaporator.
b. Reaction of Dendron (Amine, Alkyne-4) with Azidoethyl
Mannose
[0446] The dendron product containing the amino core and four
terminal alkyne groups obtained after hydrogenation (8.3 mmol) is
taken into THF (40 mL), water (40 mL) and stirred into solution.
Azidoethylmannose (4.75 eq., 2.53 gm, 10.51 mmole) is added
followed by copper sulfate (500 mg, 2.0 mmole) and sodium ascorbate
(400 mg, 2.0 mmole) and the resultant mixture stirred at 55-60 C
(oil bath) for 6 hours, cooled to room temperature and stirred
overnight. The resulting mixture is concentrated under vacuum to
one half volume and filtered thru a micro-glass filter. The
filtrate is loaded on a resin column (Dowex 50w 50.times.4-100) and
eluted with water (6.times.75 mL) until neutral. The column is then
eluted with 15% ammonium hydroxide (10.times.75 mL) and the
fractions positive to ninhydrin are pooled and concentrated to a
glassy foam.
Example 11
Amine-Functionalized Drug Conjugation with Multivalent Activated
Esters in Organic Solvent (Drug Added First)
[0447] A framework containing N-terminal activated esters is
dissolved at 60 mM in 1.0 ml of anhydrous DMSO followed by the
addition of 400 ul (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. The
amine-bearing drug is then dissolved separately in 7.9 ml of DMSO
at a concentration of 7.4 mM. Once dissolved, the entire drug
solution is added dropwise over the course of 10 minutes to the
framework/DMSO/TEA solution followed by room temperature mixing for
two hours. The remaining activated esters are then reacted with
amine-functionalized ligands in the following manner. A 370 mM
solution of ligand is prepared in an appropriate volume of dry
DMSO. Once dissolved, enough solution is added to provide a number
of reactive equivalents equal to three times the number of initial
activated ester groups, N, minus one. For example, if there are N=3
initial activated ester groups per framework, then
(3.times.(3-1).times.60 mM/370 mM)=0.973 ml of ligand solution are
added. If there are N=4 initial activated ester groups per
framework, then (3.times.(4-1).times.60 mM/370 mM)=1.46 ml of
ligand solution are added, and so on. After the ligand solution is
added, the solution is stirred for one more hour at room
temperature to ensure complete reaction.
[0448] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 10 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC on a Waters C8, 7 um, 19.times.150 mm column.
Buffer A is deionized water containing 0.1% TFA and Buffer B is
acetonitrile containing 0.1% TFA. Before purification, the column
is equilibrated at 15 ml/minutes with a 80% A/20% B mobile phase
using a Waters DeltraPrep 600 system. Approximately 5 ml of the
crude solution is injected onto the column over the course of 2
minutes at a flow rate of 15 ml/minutes after which a linear
gradient is employed from 80% A/20% B to 75% A/25% B over the next
5 minutes followed by a slower linear gradient from 75% A/25% B to
62% A/38% B over the next 22 minutes. The retention time of the
desired peak will vary depending on the drug, framework, and ligand
used. Once collected, the solution is rotovapped to remove
acetonitrile and lyophilized to obtain pure conjugate whose
identity may be verified by LC-MS (HT Laboratories, San Diego,
Calif.).
Example 12
B1-Insulin Conjugates with Multivalent Saccharides--Homogeneous
Ligand
[0449] Using the method described in Example 11 and the
amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-insulin (MW=6,008
g/mol) of Example 8, insulin conjugates were prepared with the
following frameworks and ligands.
Tris-Succinimidyl-1,3,5-benzenetricarboxylate (TSB),
tris-Succinimidyl aminotriacetate (TSAT), tris-Succinimidyl
(6-aminocaproyl)aminotriacetate (TSAT-C6), and
tetrakis-(N-succinimidyl carboxypropyl)pentaerythritol TSPE
activated ester frameworks were purchased from Molecular
Biosciences (Boulder, Colo.) and used without further purification.
The TSB-C4 and TSB-C6 frameworks were synthesized according to
Example 9. The AEM, AEBM, and AETM ligands were synthesized
according to Examples 1-5. The appropriately sized size exclusion
medium is Biogel P2 (Bio-Rad Laboratories, Hercules, Calif.), and
the appropriately sized ultrafiltration membrane molecular weight
cutoff is 3 kD.
[0450] In all cases, the BOC protecting groups were removed by
dissolving the lyophilized powder obtained according to Example 11
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150M NaCl.
The pH was adjusted to between 7.0 and 8.0 using NaOH solution
after which the material is passed through a Biogel P2 column to
remove anisole, BOC and other low MW byproducts of deprotection, as
well as any other contaminating salts. The deprotected, purified
aqueous conjugate solution was then concentrated using Amicon 3K
membranes (Millipore, Billerica, Mass.) to approximately 58 U of
insulin/ml (based on A280 measurements) and stored at 4 C until
needed. Because the starting NH.sub.2--B1-BOC2(A1,B29)-insulin
material only possesses one free amine group at the Phe-B1
terminus, the Phe-B1 is the only site of insulin conjugation to the
framework as verified in each deprotected final product by
N-terminal sequencing.
[0451] In the following table (and others like it in the Examples)
the framework MW values are for the activated esters of the
frameworks. This is so one can immediately calculate the mass of
activated ester framework to add to the reaction mixture. Once
reacted, the framework loses the activated esters and so the MW
contribution in the final product is much lower.
TABLE-US-00003 Frame- AE- MW work sugar Purity (LC- Sugar/
Conjugate Framework MW Ligand MW (HPLC) MS) Insulin TSB-AEM-2 (B1)
TSB 501 AEM 223 97% 6410 2.0 TSB-AEBM-2 TSB 501 AEBM 385 94% 6734
2.0 (B1) TSB-AETM-2 TSB 501 AETM 547 96% 7057 2.0 (B1) TSB-C4-AEM-2
TSB-C4 755 AEM 223 95% 6665 2.0 (B1) TSB-C4-AEBM-2 TSB-C4 755 AEBM
385 97% 6989 2.0 (B1) TSB-C4-AETM-2 TSB-C4 755 AETM 547 95% 7313
2.0 (B1) TSB-C6-AEM-2 TSB-C6 882 AEM 223 99% 6791 2.0 (B1)
TSB-C6-AEBM-2 TSB-C6 882 AEBM 385 99% 7114 2.0 (B1) TSB-C6-AETM-2
TSB-C6 882 AETM 547 95% 7438 2.0 (B1) TSAT-AEM-2 TSAT 482 AEM 223
98% 6390 2.0 (B1) TSAT-AEBM-2 TSAT 482 AEBM 385 95% 6714 2.0 (B1)
TSAT-AETM-2 TSAT 482 AETM 547 94% 7038 2.0 (B1) I-1: TSAT-C6-
TSAT-C6 822 AEM 223 97% 6730 2.0 AEM-2 (B1) I-3: TSAT-C6- TSAT-C6
822 AEBM 385 99% 7054 2.0 AEBM-2 (B1) I-2: TSAT-C6- TSAT-C6 822
AETM 547 97% 7378 2.0 AETM-2 (B1) I-16: TSPE- TSPE 813 AEM 223 98%
6829 3.0 AEM-3 (B1) TSPE-AEBM-3 TSPE 813 AEBM 385 97% 7314 3.0 (B1)
TSPE-AETM-3 TSPE 813 AETM 547 94% 7802 3.0 (B1)
Example 13
B1-Insulin Conjugates with Multivalent Saccharides--Mixed
Ligands
[0452] Using the method described in Example 11 and the
amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-Insulin (MW=6,008
g/mol) of Example 8, insulin conjugates were prepared which
possessed a mixture of saccharide ligands connected to the
framework.
[0453] The TSAT-C6 and TSPE activated ester frameworks were
purchased from Molecular Biosciences (Boulder, Colo.) and used
without further purification. The AEM, AEBM, and AETM were
synthesized according to Examples 1-5. The appropriately sized size
exclusion medium is Biogel P2 (Bio-Rad Laboratories, Hercules,
Calif.), and the appropriately sized ultrafiltration membrane
molecular weight cutoff is 3 kD.
[0454] In all cases, the BOC protecting groups were removed by
dissolving the lyophilized powder obtained according to Example 11
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150M NaCl.
The pH was adjusted to between 7.0 and 8.0 using NaOH solution
after which the material was passed through a Biogel P2 column to
remove anisole, BOC and other low MW byproducts of deprotection, as
well as any other contaminating salts. The deprotected, purified
aqueous conjugate solution was then concentrated using Amicon 3K
membranes (Millipore, Billerica, Mass.) to the desired level and
stored at 4 C until needed. Because the starting
NH.sub.2--B1-BOC2(A1,B29)-insulin material only possesses one free
amine group at the Phe-B1 terminus, the Phe-B1 is the only site of
insulin conjugation to the framework as verified in each
deprotected final product by N-terminal sequencing.
TABLE-US-00004 Frame- AE- MW Conjugate work sugar Purity (LC-
Sugar/ Identity Framework MW Mixed Ligand MW (HPLC) MS) Insulin
TSPE- TSPE 813 AEM/AETM 223/547 94% 7478 1.0 AEM-1- (33/67 mol/mol)
AEM, AETM-2 2.0 (B1) AETM TSPE- TSPE 813 AEM/AETM 223/547 94% 7152
2.0 AEM-2- (67/33 mol/mol) AEM, AETM-1 1.0 (B1) AETM TSAT-C6-
TSAT-C6 822 AEM/AEBM 223/385 96% 6892 1.0 AEM-1- (50/50 mol/mol)
AEM, AEBM-1 1.0 (B1) AEBM I-4: TSAT-C6 822 AEBM/AETM 385/547 95%
7216 1.0 TSAT-C6- (50/50 mol/mol) AEBM, AEBM-1- 1.0 AETM-1 AETM
(B1)
Example 14
B1-Insulin Conjugates with Multivalent Saccharides Using Premade
Multivalent Saccharides
[0455] Using the method described in Example 11 and the
amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-insulin (MW=6,008
g/mol) of Example 8, the following insulin conjugates are prepared
from pre-synthesized multivalent amine-containing ligands. The
disuccinimidyl suberate (DSS) and TSAT-C6 activated ester
frameworks are purchased from Molecular Biosciences (Boulder,
Colo.) and used without further purification. Divalent AEM-2,
AEBM-2, and AETM-2 molecules containing a terminal reactive amine
are prepared by conjugating two of each ligand to a suitable
framework to which a reactive amine is also conjugated. Trivalent
AEM-3, AEBM-3, and AETM-3 molecules containing a terminal reactive
amine are prepared by conjugating three of each ligand to a
suitable framework to which a reactive amine is also conjugated.
The appropriately sized size exclusion medium is Biogel P2 (Bio-Rad
Laboratories, Hercules, Calif.), and the appropriately sized
ultrafiltration membrane molecular weight cutoff is 3 kD.
[0456] In all cases, the BOC protecting groups are removed by
dissolving the lyophilized powder obtained according to Example 11
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150 M NaCl.
The pH is adjusted to between 7.0 and 8.0 using NaOH solution after
which the material is passed through a Biogel P2 column to remove
anisole, BOC and other low MW byproducts of deprotection, as well
as any other contaminating salts. The deprotected, purified aqueous
conjugate solution is then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to the desired level and stored at 4
C until needed. Because the starting
NH.sub.2--B1-BOC2(A1,B29)-Insulin material only possesses one free
amine group at the Phe-B1 terminus, the Phe-B1 is the only site of
insulin conjugation to the framework as verified in each
deprotected final product by N-terminal sequencing.
TABLE-US-00005 Expected Conjugate Synthesis Conditions
Characterization AE- MW Conjugate Framework sugar (LC- Sugar/
Identity Framework MW Ligand MW MS) Insulin DSS-(AEM-2)-1 DSS 368
AEM-2 676 6621 2.0 AEM (B1) DSS-(AEM-2)-1 DSS 368 AEBM-2 1000 6945
2.0 AEBM (B1) DSS-(AEM-2)-1 DSS 368 AETM-2 1324 7269 2.0 AETM (B1)
DSS-(AEM-3)-1 DSS 368 AEM-3 1085 7031 3.0 AEM (B1) DSS-(AEM-3)-1
DSS 368 AEBM-3 1571 7517 3.0 AEBM (B1) DSS-(AEM-3)-1 DSS 368 AETM-3
2057 8003 3.0 AETM (B1) TSAT-C6- TSAT-C6 822 AEM-2 676 7637 4.0 AEM
(AEM-2)-2 (B1) TSAT-C6- TSAT-C6 822 AEBM-2 1000 8285 4.0 AEBM
(AEBM-2)-2 (B1) TSAT-C6- TSAT-C6 822 AETM-2 1324 8933 4.0 AETM
(AEM-2)-2 (B1) TSAT-C6- TSAT-C6 822 AEM-3 1085 8046 6.0 AEM
(AEM-3)-2 (B1) TSAT-C6- TSAT-C6 822 AEBM-3 1571 9018 6.0 AEBM
(AEBM-3)-2 (B1) TSAT-C6- TSAT-C6 822 AETM-3 2057 9990 6.0 AETM
(AEM-3)-2 (B1)
Example 15
B1-Insulin Conjugates with Multivalent Saccharides Using Dendritic
Framework--Homogeneous Ligand
[0457] 0.1 gm (0.098 mmol) dendron containing an amino core and
four terminal alkyne groups prepared in Example 10b is dissolved at
100 mg/ml in anhydrous DMSO. The solution is added dropwise to a
solution containing disuccinimidyl suberate (DSS, Molecular
Biosciences, 0.098 mmol) and triethylamine (400 uL) and allowed to
react for 1 hour at room temperature. This mixture is then added
dropwise to a 50 mg/ml solution containing the
NH.sub.2--B1-BOC2(A1,B29)-insulin (MW=6,008 g/mol) of Example 8
(0.588 g, 0.098 mmol) and allowed to react for 2 hours.
[0458] The resulting conjugate is superdiluted in water, and the pH
adjusted to 8.0. The solution is desalted using BioGel P2, followed
by concentration using Amicon 3k ultrafiltration devices. The
resulting solution is purified by reverse phase chromatography,
rotovapped to remove acetonitrile, and lyophilized. The BOC
protecting groups are removed by dissolving the lyophilized powder
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150 M NaCl.
The pH is adjusted to between 7.0 and 8.0 using NaOH solution after
which the material is passed through a Biogel P2 column to remove
anisole, BOC and other low MW byproducts of deprotection, as well
as any other contaminating salts. The deprotected, purified aqueous
conjugate solution is then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to the desired level and stored at 4
C until needed. Because the starting
NH.sub.2--B1-BOC2(A1,B29)-insulin material only possesses one free
amine group at the Phe-B1 terminus, the Phe-B1 is the only site of
insulin conjugation to the framework and is verified in each
deprotected final product by N-terminal sequencing.
Example 16
Synthesis of NH.sub.2--B29-BOC2(A1,B1)-Insulin
a. Fmoc-1-(B29)-Insulin
[0459] In a typical synthesis, 4 gm of powdered insulin (Sigma
Aldrich, St. Louis, Mo.) is dissolved in 100 ml of anhydrous DMSO
at room temperature followed by the addition of 4 ml of
triethylamine (TEA). The solution is stirred for 30 minutes at room
temperature. Next, 1.2 equivalents of 9-fluorenylmethyl
N-succinimidyl carbonate (Fmoc-NHS) (Sigma Aldrich, St. Louis, Mo.)
is slowly added to the insulin-TEA solution as a 1.0 M solution of
the Fmoc-NHS in THF. The reaction is mixed for approximately one
hour. The reaction is quenched via the addition of 4 ml of a stock
solution containing 250 ul of ethanolamine in 5 ml of DMSO followed
by mixing for five minutes. After quenching, the entire solution is
poured into 1600 ml of acetone and mixed briefly with a spatula.
Next, 8.times.400 .mu.l aliquots of a 18.9% HCl:water solution are
added dropwise over the surface of the mixture to precipitate the
reacted insulin. The precipitated material is then centrifuged and
the supernatant decanted into a second beaker while the precipitate
cake is set aside. To the supernatant solution, another 8.times.400
.mu.l aliquots of a 18.9% HCl:water solution are added dropwise
over the surface of the mixture to obtain a second precipitate of
reacted insulin. This second precipitate is centrifuged and the
supernatant is discarded. The combined centrifuge cakes from the
two precipitation steps are washed once with acetone followed by
drying under vacuum at room temperature to yield the crude powder
which typically contains 20% of the Fmoc1 product, 65% of the Fmoc2
product, and 15% of unreacted insulin.
[0460] A preparative reverse phase HPLC method is used to isolate
the pure desired Fmoc1-insulin from the crude powder. Buffer A is
deionized water containing 0.1% TFA and Buffer B is acetonitrile
containing 0.1% TFA. The crude powder is dissolved at 25 mg/ml in a
70% A/30% B mixture and syringe filtered prior to injection on the
column. Before purification, the column (Waters SymmetryPrep C18, 7
um, 19.times.150 mm) is equilibrated at 15 ml/minutes with a 70%
A/30% B mobile phase using a Waters DeltraPrep 600 system.
Approximately 5 ml of the crude powder solution is injected onto
the column at a flow rate of 15 ml/minutes over the course of 5
minutes after which a linear gradient is employed from 70% A/30% B
to 62% A/38% B over the course of the next 3.5 minutes and held
there for an additional 2.5 minutes. Using this method, the desired
Fmoc1 peak elutes at approximately 3 minutes after the unreacted
RHI peak, followed closely by the Fmoc2-insulin peak. Once
collected, the solution is rotovapped to remove acetonitrile and
lyophilized to obtain pure Fmoc1-insulin powder. Identity is
verified by LC-MS (HT Laboratories, San Diego, Calif.) and site of
conjugation determined by N-terminal sequencing (Western
Analytical, St. Louis, Mo.).
b. BOC2(A1,B1)-Fmoc-(B29)-Insulin
[0461] In a typical synthesis, 1 g of Fmoc1-(B29)-insulin is
dissolved in 25 ml of anhydrous DMSO at room temperature followed
by the addition of 1 ml of triethylamine (TEA). The solution is
stirred for 30 minutes at room temperature. Next, 0.379 ml (2.2
equivalents) of di-tert-butyl-dicarbonate/THF solution (Sigma
Aldrich, St. Louis, Mo.) is slowly added to the insulin-TEA
solution and mixed for approximately one hour. The reaction is
quenched via the addition of 1 ml of a stock solution containing
250 ul of ethanolamine in 5 ml of DMSO followed by mixing for five
minutes. After quenching, the entire solution is poured into 400 ml
of acetone and mixed briefly with a spatula. Next, 8.times.100
.mu.l aliquots of a 18.9% HCl:water solution are added dropwise
over the surface of the mixture to precipitate the reacted insulin.
The precipitated material is then centrifuged and the supernatant
decanted into a second beaker while the precipitate cake is set
aside. To the supernatant solution, another 8.times.100 .mu.l
aliquots of a 18.9% HCl:water solution are added dropwise over the
surface of the mixture to obtain a second precipitate of reacted
insulin. This second precipitate is centrifuged and the supernatant
is discarded. The combined centrifuge cakes from the two
precipitation steps are washed once with acetone followed by drying
under vacuum at room temperature to yield the crude powder which
typically contains greater than 90% of the desired BOC2-Fmoc-1
product.
[0462] A preparative reverse phase HPLC method is used to isolate
the pure BOC2-Fmoc-1-insulin from the crude powder. Buffer A is
deionized water containing 0.1% TFA and Buffer B is acetonitrile
containing 0.1% TFA. The crude powder is dissolved at 25 mg/ml in a
70% A/30% B mixture and syringe filtered prior to injection on the
column. Before purification, the column (Waters SymmetryPrep C18, 7
um, 19.times.150 mm) is equilibrated at 15 ml/minutes with a 70%
A/30% B mobile phase using a Waters DeltraPrep 600 system.
Approximately 5 ml of the crude powder solution is injected onto
the column at a flow rate of 15 ml/minutes over the course of 5
minutes after which a linear gradient is employed from 70% A/30% B
to 62% A/38% B over the course of the next 3.5 minutes and held
there for an additional 2.5 minutes. Using this method, the desired
BOC2-Fmoc-1 peak elutes at approximately 5 minutes after the
Fmoc1-insulin starting material. Once collected, the solution is
rotovapped to remove acetonitrile and lyophilized to obtain pure
BOC2(A1,B1)-Fmoc(B29)-insulin powder. Identity is verified by LC-MS
(HT Laboratories, San Diego, Calif.) and site of conjugation
determined by N-terminal sequencing (Western Analytical, St. Louis,
Mo.).
c. NH.sub.2--(B29)-BOC2(A1,B1)-Insulin
[0463] The Fmoc protecting group of the BOC2(A1,B1)-Fmoc(B29) is
removed by dissolving the lyophilized powder obtained according to
the previous step in 20% piperidine in dimethylformamide (DMF) for
30 minutes at 4 C followed by 10.times. superdilution in 25 mM
HEPES pH 8.2 buffer containing 0.150 M NaCl. The pH is adjusted to
between 7.0 and 8.0 using NaOH solution after which the material is
passed through a Biogel P2 column to remove Fmoc, DMF, and any
other contaminating salts. The NH.sub.2--(B29)-BOC2(A1,B1)-insulin
is lyophilized into a powder if needed or used directly in aqueous
solution if desired.
Example 17
Synthesis of NH.sub.2--B29-BOC2(A1,B1)-Insulin Conjugates
[0464] All of the multivalent-ligand-drug conjugates described in
previous examples using the NH.sub.2--B1-BOC2(A1,B29)-insulin of
Example 8 may be prepared instead using the
NH.sub.2--B29-BOC2(A1,B1)-insulin of Example 16. All of the
resulting conjugates will possess the same MW and degree of
substitution characteristics, but the site of conjugation to the
insulin molecule will be at the epsilon B29 amino group and not the
N-terminal Phe-B1. This can be confirmed by N-terminal
sequencing.
Example 18
Amine-Functionalized Drug Conjugation with Multivalent Activated
Esters in Organic Solvent (Drug Added Last)
[0465] This example describes an alternative to the method
described in Example 11 in which the drug is added to the framework
before the ligand(s). In this example the ligand(s) are added to
the framework before the drug.
[0466] A framework containing N terminal activated esters is
dissolved at 60 mM in 1 ml of anhydrous DMSO followed by the
addition of 400 ul (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. In parallel, a
122 mM solution of ligand is prepared in an appropriate volume of
anhydrous DMSO. Once dissolved, enough ligand solution is added
dropwise over the course of ten minutes to provide a number of
reactive equivalents equal to exactly the number of activated ester
groups on the framework, N, minus one. For example, if there are
N=3 activated ester groups on the framework, then
(1.times.(3-1).times.60 mM/122 mM)=0.98 ml of ligand solution are
added. If there are N=4 activated ester groups on the framework,
then (1.times.(4-1).times.60 mM/122 mM)=1.5 ml of ligand solution
are added, and so on. After the ligand solution is added, the
solution is stirred for two hours at room temperature.
[0467] The amine-bearing drug is then dissolved separately in 7.5
ml of anhydrous DMSO at a concentration of 8.1 mM. Once dissolved,
the entire drug solution is added over the course of one minute to
the framework/DMSO/ligand/TEA solution followed by room temperature
mixing for an additional two hours to ensure complete reaction.
[0468] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 10 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC on a Waters SymmetryPrep C18, 7 um column,
19.times.150 mm. Buffer A is deionized water containing 0.1% TFA
and Buffer B is acetonitrile containing 0.1% TFA. Before
purification, the column is equilibrated at 15 ml/minutes with a
80% A/20% B mobile phase using a Waters DeltraPrep 600 system.
Approximately 5 ml of the crude solution is injected onto the
column over the course of 2 minutes at a flow rate of 15 ml/minutes
after which a linear gradient is employed from 80% A/20% B to 75%
A/25% B over the next 5 minutes followed by a slower linear
gradient from 75% A/25% B to 62% A/38% B over the next 22 minutes.
The retention time of the desired peak will vary depending on the
drug, framework, and ligand used. Once collected, the solution is
rotovapped to remove acetonitrile and lyophilized to obtain pure
conjugate whose identity may be verified by LC-MS (HT Laboratories,
San Diego, Calif.).
Example 19
B29-Insulin Conjugates with Multivalent Saccharides Produced in
Organic Solvent from Unprotected Insulin
[0469] This example makes use of the fact that in unprotected
insulin, the Lys-B29 epsilon-amino moiety is the most reactive
amine, followed by the A1 and then the B1. Therefore, when
unprotected insulin is used as the amine-containing drug the
resulting conjugate should be predominantly substituted at the
Lys-B29 position. Using the method described in Example 18 and
recombinant human insulin (MW=5808 Da, Sigma Aldrich, St. Louis,
Mo.) as the amine-containing drug, the following insulin conjugates
were prepared using the TSAT-C6 activated ester framework purchased
from Molecular Biosciences (Boulder, Colo.). The AEM and AETM were
synthesized as described previously. The appropriately sized size
exclusion medium was Biogel P2 (Bio-Rad Laboratories, Hercules,
Calif.), and the appropriately sized ultrafiltration membrane
molecular weight cutoff was 3 kDa.
TABLE-US-00006 AE- MW Framework sugar Purity (LC- Sugar/ Conjugate
Framework MW Ligand MW (HPLC) MS) Insulin I-7: TSAT- TSAT-C6 822
AEM 223 93% 6729 2.0 C6-AEM-2 (B29) I-6: TSAT- TSAT-C6 822 AETM 547
95% 7378 2.0 C6-AETM-2 (B29)
[0470] According to N-terminal sequencing, approximately 85% of the
AEM-containing framework was conjugated to insulin via the Lys-B29
and approximately 87% of the AETM-containing framework was
conjugated to insulin via the Lys-B29.
Example 20
Amine-Functionalized Drug Conjugation with Multivalent Activated
Esters in Aqueous Solvent (Drug Added Last)
[0471] This example describes an alternative to the method
described in Example 18 in which the reaction is performed in
aqueous solvent instead of organic solvent.
[0472] The framework containing N terminal activated esters is
dissolved at 60 mM in 6.25 ml of anhydrous DMSO followed by the
addition of 2 ml (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. In parallel, a
448 mM solution of ligand is prepared in an appropriate volume of
anhydrous DMSO. Once dissolved, enough ligand solution is added
dropwise over the course of ten minutes to provide a number of
reactive equivalents equal to 1.5 times the number of activated
ester groups on the framework, N, minus one. For example, if there
are N=3 activated ester groups on the framework, then
(1.5.times.(3-1).times.60 mM/448 mM).times.6.25 ml=2.5 ml of ligand
solution are added. If there are N=4 activated ester groups on the
framework, then (1.5.times.(4-1).times.60 mM/448 mM).times.6.25
ml=3.8 ml of ligand solution are added, and so on. After the ligand
solution is added, the solution is stirred for one hour at room
temperature.
[0473] The amine-bearing drug is then dissolved separately at 17.2
mM in 2.67 ml of a 0.1M, pH 11 sodium carbonate buffer and the pH
subsequently adjusted to 10.8 with 1.0N sodium hydroxide. Once
dissolved, the entire framework/DMSO/ligand/TEA solution is added
dropwise over the course of 75 minutes to the drug/carbonate buffer
solution. During the addition, the pH of the resulting mixture is
adjusted every 5 minutes to 10.8 if necessary using dilute HCl or
NaOH. The solution is allowed to stir for an additional 15 minutes
after the dropwise addition to ensure complete reaction.
[0474] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 40 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC on a Waters SymmetryPrep C18, 7 um, 19.times.150
mm column. Buffer A is deionized water containing 0.1% TFA and
Buffer B is acetonitrile containing 0.1% TFA. Before purification,
the column is equilibrated at 15 ml/minutes with a 80% A/20% B
mobile phase using a Waters DeltraPrep 600 system. Approximately 5
ml of the crude solution is injected onto the column over the
course of 2 minutes at a flow rate of 15 ml/minutes after which a
linear gradient is employed from 80% A/20% B to 75% A/25% B over
the next 5 minutes followed by a slower linear gradient from 75%
A/25% B to 62% A/38% B over the next 22 minutes. The retention time
of the desired peak will vary depending on the drug, framework, and
ligand used. Once collected, the solution is rotovapped to remove
acetonitrile and lyophilized to obtain pure conjugate whose
identity may be verified by LC-MS (HT Laboratories, San Diego,
Calif.).
Example 21
B29-AEM-2-Insulin Conjugate Synthesized in Aqueous Solvent from
Unprotected Insulin
[0475] This example makes use of the fact that in unprotected
insulin, the Lys-B29 epsilon-amino moiety is the most reactive
amine, followed by the A1 and then the B1. Therefore, when
unprotected insulin is used as the amine-containing drug the
resulting conjugate should be predominantly substituted at the
Lys-B29 position. Using the method described in Example 20 and
recombinant human insulin (MW=5808, Sigma Aldrich, St. Louis, Mo.)
as the amine-containing drug, an AEM-2 insulin conjugate was
prepared using the TSAT-C6 activated ester framework purchased from
Molecular Biosciences (Boulder, Colo.). The AEM used as the insulin
analog was synthesized as described previously. The appropriately
sized size exclusion medium was Biogel P2 (Bio-Rad Laboratories,
Hercules, Calif.), and the appropriately sized ultrafiltration
membrane molecular weight cutoff was 3 kD. The final product (95%
pure by HPLC) was found to have the desired MW of 6729 g/mol
(LC-MS), representing a total of 2.0 AEM molecules conjugated per
insulin, with greater than 85% of the conjugate molecules
conjugated at the Lys-B29 site (N-terminal sequencing).
Example 22
Generalized Amine-Functionalized Drug Conjugation with
Aldehyde-Containing Framework
[0476] a. Framework Functionalized with More than One Ligand and
One Terminal Aldehyde
[0477] First, a framework containing N terminal activated esters is
dissolved at 60 mM in 27.0 ml of anhydrous DMSO followed by the
addition of 800 ul (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. A stock
solution of amine-bearing diethyl acetal is prepared at 580 mM in 5
ml of anhydrous DMSO. Once dissolved, 2.9 ml of the diethyl acetal
solution are added dropwise over the course of 5 minutes to the
framework/DMSO/TEA solution followed by room temperature mixing for
an additional 15 minutes. The remaining activated esters are then
reacted with amine-functionalized ligands in the following manner.
A 370 mM solution of ligand is prepared in an appropriate volume of
dry DMSO. Once dissolved, enough solution is added to provide a
number of reactive equivalents equal to 1.5 times the number of
initial activated ester groups, N, minus one. For example, if there
are N=3 initial activated ester groups per framework, then
(1.5.times.(3-1).times.60 mM.times.27/370 mM)=13 ml of ligand
solution are added. If there are N=4 initial activated ester groups
per framework, then (1.5.times.(4-1).times.60 mM.times.27/370
mM)=20 ml of ligand solution are added, and so on. After the ligand
solution is added, the solution is stirred for an additional hour
and 45 minutes at room temperature to ensure complete reaction.
After reaction, the entire solution is diluted by a factor of ten
with diethyl ether, mixed vigorously, and centrifuged to separate
the dense bottom phase containing the desired material from the
supernatant. After discarding the supernatant, the same volume of
ethanol is added to generate a solid precipitated mass. After
centrifuging and discarding the supernatant, the material is washed
extensively with ethanol and ether and then dried under vacuum to
yield the crude framework containing multiple ligands and a diethyl
acetal group.
[0478] b. Conjugation of Amine-Functionalized Drug with Terminal
Aldehyde
[0479] Once dried, the aldehyde group is generated from the diethyl
acetal by dissolving the collected material in 60 ml of DI water
with the solution pH adjusted to 1.0. The solution is mixed for 30
minutes after which 6 ml of a 200 mM HEPES pH 8.2 buffer containing
1.5 M NaCl is added and the solution pH adjusted to 6.5 using
dilute NaOH solution. 48 mmol of the amine containing drug are
added to the solution and the pH readjusted to 6.5 if necessary.
Separately, a stock solution of reducing agent is prepared by
dissolving 1.5 g of sodium cyanoborohydride (Sigma Aldrich, St.
Louis, Mo.) in 15 ml of a 20 mM HEPES pH 7.0 buffer containing
0.150 M NaCl and the pH carefully adjusted to 6.5 with dilute HCl
solution. 13 ml of the cyanoborohydride stock solution are added to
the drug/framework/aldehyde solution and allowed to react overnight
at room temperature.
[0480] The resulting aqueous solution is first purified by size
exclusion using an appropriate solid phase for the desired
separation of conjugated and unconjugated materials. The solution
passing through the column void volume is then concentrated using
an appropriately sized ultrafiltration membrane to approximately 10
ml. This solution is further purified to obtain the desired product
using preparative reverse phase HPLC on a Waters SymmetryPrep C18,
7 um column, 19.times.150 mm. Buffer A is deionized water
containing 0.1% TFA and Buffer B is acetonitrile containing 0.1%
TFA. Before purification, the column is equilibrated at 15
ml/minutes with a 80% A/20% B mobile phase using a Waters
DeltraPrep 600 system. Approximately 5 ml of the crude solution is
injected onto the column over the course of 2 minutes at a flow
rate of 15 ml/minutes after which a linear gradient is employed
from 80% A/20% B to 75% A/25% B over the next 5 minutes followed by
a slower linear gradient from 75% A/25% B to 62% A/38% B over the
next 22 minutes. The retention time of the desired peak will vary
depending on the drug, framework, and ligand used. Once collected,
the solution is rotovapped to remove acetonitrile and lyophilized
to obtain pure conjugate whose identity may be verified by LC-MS
(HT Laboratories, San Diego, Calif.).
Example 23
AEM-2-Framework Containing a Terminal Reactive Aldehyde Group and
Subsequent Insulin Conjugation at B1
a. TSAT Functionalized with 2 AEM and 1 Aminobutyraldehyde Diethyl
Acetal (ABDA)
[0481] This material was synthesized according to the method
described in Example 22a using TSAT (Molecular Biosciences,
Boulder, Colo.) as the multivalent activated ester framework and
4-aminobutyraldehyde diethyl acetal (Sigma Aldrich, St. Louis, Mo.)
as the amine-bearing diethyl acetal. AEM (MW=223 g/mol),
synthesized as described previously was used as the ligand.
b. Conjugation of TSAT-AEM-2-ABDA with
NH.sub.2--B1-BOC2(A1,B29)-Insulin
[0482] This material was synthesized using the method described in
Example 22b and the TSAT-AEM-2-ABDA produced in (a) above along
with the amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-insulin
(MW=6,008 g/mol), synthesized according to Example 8. The
appropriately sized size exclusion medium was Biogel P2 (Bio-Rad
Laboratories, Hercules, Calif.), and the appropriately sized
ultrafiltration membrane molecular weight cutoff was 3 kD. Because
the starting NH.sub.2--B1-BOC2(A1,B29)-insulin material only
possesses one free amine group at the Phe-B1 terminus, the Phe-B1
is the only site of insulin conjugation to the framework. The BOC
protecting groups were removed by dissolving the lyophilized powder
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150 M NaCl.
The pH was adjusted to between 7.0 and 8.0 using NaOH solution
after which the material was passed through a Biogel P2 column to
remove anisole, BOC and other low MW byproducts of deprotection, as
well as any other contaminating salts. The deprotected, purified
aqueous conjugate solution was then concentrated using Amicon 3K
membranes (Millipore, Billerica, Mass.) to the desired level and
stored at 4 C until needed.
[0483] The final product (95% pure by HPLC) was found to have the
desired MW of 6462 g/mol (LC-MS), representing a total of 2.0 AEM
molecules conjugated per insulin, 99% of which were conjugated at
the Phe-B1 site (N-terminal sequencing).
Example 24
AEM-3-Framework Containing a Terminal Reactive Aldehyde Group and
Subsequent Insulin Conjugation at B1
a. TSPE Functionalized with 3 AEM and 1 Aminobutyraldehyde Diethyl
Acetal (ABDA)
[0484] This material was synthesized according to the method
described in Example 22a using TSPE (Molecular Biosciences,
Boulder, Colo.) as the multivalent activated ester framework and
4-aminobutyraldehyde diethyl acetal (Sigma Aldrich, St. Louis, Mo.)
as the amine-bearing diethyl acetal. AEM (MW=223 g/mol),
synthesized as described previously, was used as the ligand.
b. Conjugation of TSPE-AEM-3-ABDA with
NH.sub.2--B1-BOC2(A1,B29)-Insulin
[0485] This material was synthesized using the method described in
Example 22b and the TSPE-AEM-3-ABDA produced in (a) above along
with the amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-insulin
(MW=6,008 g/mol), synthesized according to Example 8. The
appropriately sized size exclusion medium was Biogel P2 (Bio-Rad
Laboratories, Hercules, Calif.), and the appropriately sized
ultrafiltration membrane molecular weight cutoff was 3 kD. Because
the starting NH.sub.2--B1-BOC2(A1,B29)-insulin material only
possesses one free amine group at the Phe-B1 terminus, the Phe-B1
is the only site of insulin conjugation to the framework. The BOC
protecting groups are removed by dissolving the lyophilized powder
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150 M NaCl.
The pH was adjusted to between 7.0 and 8.0 using NaOH solution
after which the material was passed through a Biogel P2 column to
remove anisole, BOC and other low MW byproducts of deprotection, as
well as any other contaminating salts. The deprotected, purified
aqueous conjugate solution was then concentrated using Amicon 3K
membranes (Millipore, Billerica, Mass.) to the desired level and
stored at 4 C until needed.
[0486] The final product (95% pure by HPLC) was found to have the
desired MW of 6897 g/mol (LC-MS), representing a total of 3.0 AEM
molecules conjugated per insulin, 99% of which were conjugated at
the Phe-B1 site (N-terminal sequencing).
Example 25
AEM-3-Scaffold Containing a Terminal Reactive Aldehyde Group and
Subsequent Insulin Conjugation at B1 Using Unprotected Insulin
a. TSPE Functionalized with 3 AEM and 1 Aminobutyraldehyde Diethyl
Acetal (ABDA)
[0487] This material is synthesized according to the method
described in Example 22a using TSPE (Molecular Biosciences,
Boulder, Colo.) as the multivalent activated ester scaffold and
4-aminobutyraldehyde diethyl acetal (Sigma Aldrich, St. Louis, Mo.)
as the amine-bearing diethyl acetal. AEM (MW=223 g/mol),
synthesized as described previously, was used as the ligand.
b. Conjugation of TSPE-AEM-3-ABDA with
NH.sub.2--B1-BOC2(A1,B29)-Insulin
[0488] This material was synthesized using the method described in
Example 22b and the TSPE-AEM-3-ABDA produced in (a) above along
with the amine-bearing drug, unmodified insulin (MW=5,808 g/mol,
Sigma-Aldrich, St. Louis, Mo.). The appropriately sized size
exclusion medium was Biogel P2 (Bio-Rad Laboratories, Hercules,
Calif.), and the appropriately sized ultrafiltration membrane
molecular weight cutoff was 3 kD. Although the starting unprotected
insulin material possesses three free amine groups, the Phe-B1 is
the predominant site of insulin conjugation to the scaffold due to
the fact that the Phe-B1 (pKa .about.6.8) is the most reactive
amine at pH 6.5. The lyophilized powder was dissolved in 25 mM
HEPES pH 8.2 buffer containing 0.150 M NaCl. The pH was adjusted to
between 7.0 and 8.0 using NaOH solution after which the material
was then concentrated using Amicon 3K membranes (Millipore,
Billerica, Mass.) to the desired level and stored at 4 C until
needed.
[0489] The final product (95% pure by HPLC) was found to have the
desired MW of 6897 g/mol (LC-MS), representing a total of 3.0 AEM
molecules conjugated per insulin, >85% of which were conjugated
at the Phe-B1 site (N-terminal sequencing).
Example 26
Mixed Framework Chemistry and Corresponding Separate Conjugation of
Drug and Ligands
[0490] Succinimidyl-3,5-dimaleimidophenyl benzoate (SDMB) can be
purchased from Molecular Biosciences (Boulder, Colo.) and used in
the following example without further purification. SDMB is
dissolved at 60 mM in 1.0 ml of anhydrous DMSO followed by the
addition of 400 ul (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. The
amine-bearing drug is then dissolved separately in 7.5 ml of
anhydrous DMSO at a concentration of 8.1 mM. Once dissolved, the
entire SDMB solution is added dropwise over the course often
minutes to the DMSO-drug solution followed by room temperature
mixing for an additional two hours to ensure complete reaction.
[0491] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 10 ml.
[0492] Separately, 6.0 mmol of an amine-containing ligand is
dissolved in a 20 mM pH 8.2 HEPES buffered saline solution
containing 0.150 M NaCl at a concentration of 450 mM. To this
solution, 6.6 mmol of iminothiolane (Sigma-Aldrich, St. Louis, Mo.)
is added and allowed to react at pH 8.2 for 30 minutes at room
temperature to convert the amine-terminal groups to terminal
sulfhydryl groups. The resulting material is mixed with the 10 ml
solution of drug-framework-di-maleimide conjugate produced in the
previous step. The maleimide groups are allowed to react with the
indicator-analog sulfhydryl groups at pH 8.2 for 2 hours to ensure
complete reaction. The resulting solution is then purified by size
exclusion using an appropriate solid phase for the desired
separation of conjugated and unconjugated materials. The solution
passing through the column void volume is then concentrated using
an appropriately sized ultrafiltration membrane to approximately 10
ml.
[0493] Finally, this solution is further purified to obtain the
desired product using preparative reverse phase HPLC on a Waters
SymmetryPrep C18, 7 um column, 19.times.150 mm. Buffer A is
deionized water containing 0.1% TFA and Buffer B is acetonitrile
containing 0.1% TFA. Before purification, the column is
equilibrated at 15 ml/minutes with a 80% A/20% B mobile phase using
a Waters DeltraPrep 600 system. Approximately 5 ml of the crude
solution is injected onto the column over the course of 2 minutes
at a flow rate of 15 ml/minutes after which a linear gradient is
employed from 80% A/20% B to 75% A/25% B over the next 5 minutes
followed by a slower linear gradient from 75% A/25% B to 62% A/38%
B over the next 22 minutes. The retention time of the desired peak
will vary depending on the drug, framework, and ligand used. Once
collected, the solution is rotovapped to remove acetonitrile and
lyophilized to obtain pure conjugate whose identity may be verified
by LC-MS (HT Laboratories, San Diego, Calif.).
Example 27
Insulin-Conjugated to Aminoethyl Saccharides Using Mixed Framework
Chemistry
[0494] Using the method described in Example 26 and the
amine-bearing drug, NH.sub.2--B1-BOC2(A1,B29)-insulin (MW=6,008
g/mol), synthesized according to Example 8, the following specific
drug conjugates are obtained. AEM (MW=223 g/mol), AEBM (MW=385
g/mol), and AETM (MW=547 g/mol) are synthesized as previously
described and used as the ligands in the synthesis. The
appropriately sized size exclusion medium is Biogel P2 (Bio-Rad
Laboratories, Hercules, Calif.), and the appropriately sized
ultrafiltration membrane molecular weight cutoff is 3 kD.
[0495] In all cases, the BOC protecting groups are removed by
dissolving the lyophilized powder obtained according to Example 26
in 90% TFA/10% anisole for one hour at 4 C followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150 M NaCl.
The pH is adjusted to between 7.0 and 8.0 using NaOH solution after
which the material is passed through a Biogel P2 column to remove
anisole, BOC and other low MW byproducts of deprotection, as well
as any other contaminating salts. The deprotected, purified aqueous
conjugate solution is then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to approximately 58 U of insulin/ml
(based on A280 measurements) and stored at 4 C until needed.
Because the starting NH.sub.2--B1-BOC2(A1,B29)-insulin material
only possesses one free amine group at the Phe-B1 terminus, the
Phe-B1 will be the only site of insulin conjugation to the
framework. This can be verified in each deprotected final product
by N-terminal sequencing.
TABLE-US-00007 Expected Conjugate Synthesis Conditions
Characterization AE-sugar AE-iminothiolane MW Sugar/ Ligand MW
intermediate MW (LC-MS) Insulin AEM 223 360 6822 2.0 AEM .sup. AEBM
385 522 7146 2.0 AEBM AETM 547 684 7470 2.0 AETM
Example 28
Generalized Click Chemistry for Drug Conjugation with Complementary
Frameworks
[0496] A framework (8.3 mmol) containing at least one amino
functionality and one or more terminal alkyne groups is taken into
THF (40 mL), water (40 mL) and stirred into solution. An azidoethyl
group-bearing drug (10.51 mmole) is added followed by copper
sulfate (500 mg, 2.0 mmole) and sodium ascorbate (400 mg, 2.0
mmole). The resulting mixture is stirred at 55-60 C (oil bath) for
6 hours, cooled to room temperature, stirred overnight and
concentrated under vacuum to one half volume and filtered thru a
micro-glass filter. The filtrate is loaded on a resin column (Dowex
50w 50.times.4-100) and eluted with water (6.times.75 mL) until
neutral. The column is then eluted with 15% ammonium hydroxide
(10.times.75 mL) and the fractions positive to ninhydrin are pooled
and concentrated to a glassy foam.
Example 29
Conjugates Prepared Using Natural Insulins from Other Species Such
as Bovine and Porcine
[0497] Insulins from other species which contain at least one
reactive amine functionality (e.g., bovine and porcine insulin) may
be coupled using any of the methods used to conjugate human
insulin. Those skilled in the art will appreciate that the
molecular weights of the resulting conjugates made from bovine or
porcine insulins will differ from those made from human insulin by
the amounts listed in the following table.
TABLE-US-00008 Molecular Weight Difference in MW human Type of
Insulin (g/mol) insulin (g/mol) Human insulin 5808 -- Porcine
insulin 5778 -30 Bovine insulin 5733 -75
[0498] Those skilled in the art will also appreciate that the
resulting conjugates made from bovine or porcine insulin may have
chromatographic peak retention times that differ slightly from
those conjugates made from human insulin, due to the small
differences in structures between the insulins.
Example 30
Conjugates Prepared with Insulin Analogs Such as Lispro, Aspart,
Glulysine, Glargine, and Detemir
[0499] All known insulin analogs which contain at least one
reactive amine functionality (e.g., lispro, aspart, glulisine,
glargine, and detemir) may be coupled using any of the methods used
to conjugate human insulin. Those skilled in the art will
appreciate that the molecular weights of the resulting conjugates
made from insulin analogs will differ from those made from human
insulin by the amounts listed in the following table.
TABLE-US-00009 Molecular Weight Difference in MW human Type of
Insulin (g/mol) insulin (g/mol) Human insulin 5808 -- Insulin
lispro 5808 -- Insulin aspart 5832 +24 Insulin glulisine 5823 +15
Insulin glargine 6063 +255 Insulin detemir 5913 +105
[0500] Those skilled in the art will also appreciate that the
resulting conjugates made from insulin analogs may have
chromatographic peak retention times that differ slightly from
those conjugates made from human insulin, due to the small
differences in structures between the insulins.
[0501] The use of insulin glulisine (which does not contain a B29
lysine, but rather a B3 lysine) will give predominantly B3
conjugates when using unprotected insulin glulisine. However, if
B1-insulin glulisine conjugates are desired, then
BOC-(A1,B3)-insulin glulisine is first synthesized using the same
protocol as BOC-(A1,B29)-human insulin as described in Example
8.
Example 31
Conjugates Prepared with Peptidic Insulin Secretagogue
Conjugates
[0502] Peptidic insulin secretagogues (e.g., without limitation
GLP-1 or the GLP-1 analog exanitide) which contain an N-terminal
amine functionality may be coupled using any of the methods used to
conjugate insulin.
II. In Vitro Assays of Exemplary Conjugates
[0503] This second set of examples describes various experiments
investigating the in vitro properties of some exemplary
conjugates.
Example 32
Synthesis of Insulin-Glycogen Conjugates
[0504] This comparative example describes the synthesis of an
insulin-glycogen conjugate according to U.S. Patent Application
Publication No. 20070099820. Briefly, 1 gm of commercially
available, unpurified oyster glycogen (Type II, Sigma-Aldrich, St.
Louis, Mo.) 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 of 0.68 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 of 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. The resulting purified
material contained 1.0 wt % of insulin per insulin-glycogen
conjugate as measured using amino acid analysis (UCLA Biopolymers
Laboratory, Los Angeles, Calif.).
Example 33
Liquid Chromatography Analysis
[0505] This example describes the differences between the RP-HPLC
profiles of insulin-glycogen synthesized according to Example 32
and an exemplary conjugate synthesized according to the present
invention. 100 ul of a 5 mg/ml solution of insulin-glycogen
synthesized according to Example 32 and 100 ul of a 1 mg/ml
solution of exemplary conjugate were injected separately onto a
Waters Symmetry C8 5 um column (4.6 mm.times.250 mm), equilibrated
with a 80% Water/20% Acetonitrile (CH3CN) mobile phase (each
containing 0.1% TFA). The exemplary conjugate used in this study
was synthesized using TSAT-C6 as the framework, AEM as the ligand,
and NH.sub.2--B1-BOC2(A1,B29)-insulin as the drug.
[0506] The samples were eluted at 1.0 ml/minutes using the
following gradient method: 0-5 minutes--constant 80% Water/20%
CH3CN, 5-35 minutes--linear gradient to 50% Water/50% CH3CN.
[0507] The elution profiles in FIG. 1 show a single spike for the
exemplary conjugate indicating a single chemically distinct species
as compared to a broad and heterogenous elution profile for the
insulin-glycogen conjugate, indicating a broad distribution of
different chemical and/or molecular weight entities.
Example 34
Molecular Weight Distribution Analysis
[0508] This example describes the difference in MW and MW
distribution between the insulin-glycogen synthesized according to
Example 32 and the same exemplary conjugate. The MW and MW
distribution of the insulin-glycogen conjugate was determined by
injecting 1 ml of a 25 mg/ml solution in pH 7 HEPES buffered saline
onto an Ultrahydrogel Size Exclusion Column (Waters Corporation,
Millford, Mass.) equilibrated with HEPES buffered saline. The
column was eluted over the course of 30 minutes at 0.5 ml per min,
and the elution profile was measured as an absorbance at 280 nm. In
separate experiments using the same protocol, dextran MW standards
of 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, and
410000 g/mol (Sigma-Aldrich, St. Louis, Mo.) were injected to
establish a calibration curve of MW versus retention time. Based on
the calibration curve and the elution profile of the
insulin-glycogen conjugate, the average MW was determined to be
500,000 g/mol with 67% of the distribution eluting over the broad
range of 250,000 to 1,000,000 g/mol (data not shown). In contrast,
the exemplary conjugate was determined to have just a single MW of
exactly 6,730 g/mol as determined by LC/MS (HT Laboratories, San
Diego, Calif.) (data not shown).
Example 35
Chemical and Physical Stability of Conjugates
[0509] This example compares the stability of an exemplary
conjugate with that of unconjugated insulin under accelerated
conditions according to the method described in Hinds et al.
(Bioconj. Chem. 11:195-201, 2000) at 37 C and a mechanical
agitation rate of 150 strokes/min. Pharmaceutical grade recombinant
human insulin (RHI) was selected as the control for the accelerated
stability study. Holcombe et al. (Diabetes Care 27:1241-1242, 2004)
describes that under non-accelerated conditions RHI stability is
maintained for at least 30 days at room temperature (RT) and
considerably longer when refrigerated. FIG. 2 shows the results
from the aggregation stability assay for RHI and two exemplary
conjugates in pH 7.4 phosphate buffered saline (PBS) at 50 U/ml. In
all cases, the % remaining in solution was determined by
centrifuging (4500.times.g, 5 min) the solution at a given time
point, measuring the A280 of the supernatant, and dividing the
supernatant A280 by that of the original starting solution.
Conjugate I-1 (see FIG. 45) was synthesized using TSAT-C6 as the
framework, AEM as the ligand, and NH.sub.2--B1-BOC2(A1,B29)-insulin
as the drug. Conjugate I-16 (see FIG. 45) was synthesized using
TSPE as the framework, AEM as the ligand, and
NH.sub.2--B1-BOC2(A1,B29)-insulin as the drug.
[0510] After 48 hours of continuous agitation at 37 C, less than 6%
of the RHI remained stable in solution, while the majority of the
RHI precipitated out as insoluble aggregates. After the same amount
of time, both conjugates remained substantially more stable, as
96%-99% of the conjugates remained intact and soluble in the PBS
solution. The data conclusively show that the conjugates are
significantly more stable than RHI under these conditions.
[0511] RP-HPLC was used to assess the chemical stability of the
conjugates (see FIG. 3a). After 48 hours of accelerated stability
the conjugate solutions were analyzed using a C8-reverse phase
column using a water-acetonitrile elution gradient. The retention
times of the pre- and post-stability conjugate samples are shown
along with the percentage of unconjugated (free) insulin and
desamido insulin found in the resulting LC traces. No detectable
amounts of free insulin or desamido were observed, indicating that
(i) the covalent linkage between the saccharides and the insulin
molecule is stable, and (ii) no significant chemical degradation of
the conjugate occurs during the accelerated stability test (AST).
Prior to and in parallel with the AST, the conjugate was also
subjected to a 90-day non-accelerated stability test that included
daily thermal cycling between 4.degree. C. and RT. At the
conclusion of the parallel study, RP-HPLC demonstrated that the
conjugate was still chemically and physically stable (data not
shown).
[0512] Further confirmation of the conjugate chemical stability in
HEPES buffer is provided from the LC-MS data obtained before and
after subjecting the conjugate to the AST. Interestingly, the 48
hour AST conjugate samples in PBS showed that substantial
degradation had occurred, while the 48 hour AST conjugate samples
in HEPES buffer were completely intact and stable (see FIG. 3b).
Conjugate I-7 stored in HEPES has a MW of 6730 Da before and after
the AST, demonstrating that both mannose residues, the conjugate
framework, and insulin are all chemically unchanged and quite
stable. To ensure conjugate stability, all buffers used for
storage, in vitro testing, and in vivo testing contain HEPES as the
buffering agent. In certain embodiments, the present disclosure
provides a composition comprising an inventive conjugate in a HEPES
buffer.
[0513] The LC-MS data greatly enhances FDA manufacturing regulatory
compliance, as the LC-MS test can readily act as the chemical
identity assay of the conjugate. Since the drug (e.g., insulin),
conjugate framework, and conjugate all have discrete molecular
weights, the resulting ligand ratio can be readily calculated by
subtracting the conjugate framework MW from the conjugate MW to
give the remaining mass due to the saccharide groups. In the case
of conjugate I-7, the mannose:insulin molar ratio is calculated as
exactly 2.0.
Example 36
Functional Stability of Conjugates
[0514] After demonstrating that the conjugate was chemically and
physically stable, a 72 hour AST conjugate was assessed for its
subcutaneous bioactivity in vivo vs. fresh conjugate using
Sprague-Dawley rats at 5 U/kg (see FIG. 4).
[0515] Analysis of the 72 hour HEPES AST conjugate data showed that
the time to reach the glucose nadir (T.sub.nadir) was 60 minutes,
and the time to return to 70% of the fasting blood glucose values
(T.sub.70% BG) was less than 128.+-.15 min. A comparison of fresh
conjugate vs. 72 hour AST conjugate bioactivity curves at each
timepoint using the student t-test (n=4 for each group) showed no
significant differences (all p-values >0.21). These results were
within specified targets for the formulation, indicating that
preserved conjugate chemical stability translates into preserved in
vivo functional performance.
III. In Vivo Assays of Exemplary Conjugates
[0516] This third set of examples describes various experiments
investigating the in vivo properties of some exemplary
conjugates.
Example 37
Conjugate Bioactivity Versus RHI and Dextran or Glycogen
Conjugates
[0517] (a) Insulin-Dextran Bioactivity
[0518] This comparative example evaluates the in vivo
pharmacodynamic profile of subcutaneously administered
insulin-dextran (Sigma-Aldrich, MW.about.70K). As shown below, the
insulin-dextran conjugates synthesized according to U.S. Patent
Publication No. 20040202719 act relatively slowly after
subcutaneous injection, because the high MW of the conjugate
polymer significantly hinders the absorption rate into systemic
circulation. 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 minutes. 300
mg of recombinant human insulin (RHI) 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. The degree of insulin conjugation was
10% (w/w) as determined by amino acid analysis (UCLA Biopolymers
Laboratory, Los Angeles, Calif.).
[0519] Subcutaneous injections of the insulin-dextran were
administered using 0.25 ml of a sterilized 1.times.PBS solution (20
U of equivalent insulin/ml) behind the neck of fasted normal
non-diabetic rats (Male Sprague-Dawley, 200-250 g, n=4). Blood
samples were 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 were measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). As shown in FIG. 5, the times to reach the glucose
nadir (T.sub.nadir) concentration was found to be about 3 hours
after injection, and the serum glucose levels remain depressed for
at least five hours post injection.
[0520] (b) Insulin-Glycogen Bioactivity
[0521] This example evaluates the in vivo pharmacodynamic profile
of subcutaneously administered insulin-glycogen. The
insulin-glycogen conjugate was synthesized according to Example 32.
The bioactivity of the insulin-glycogen conjugate was evaluated by
injecting a 2.5 equivalent U of insulin/kg dose behind the neck of
fasted normal non-diabetic rats (Male Sprague-Dawley, 200-250 g,
n=4). Blood samples were 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 were measured
using commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). As compared to the
insulin-dextran conjugates above, the high MW insulin-glycogen
conjugates lower glucose levels much more rapidly and to a greater
extent (see FIG. 6). This rapid action and elimination profile is
due to the rapid enzymatic digestion of the high MW glycogen
polymer chain following subcutaneous injection.
[0522] (c) an Exemplary Conjugate and RHI Bioactivity
[0523] This example evaluates and compares the in vivo
pharmacodynamic profile of a subcutaneously administered exemplary
conjugate and recombinant human insulin (RHI). The exemplary
conjugate, I-1 in FIG. 45, was synthesized using TSAT-C6 as the
scaffold, AEM as the indicator analog, and
NH.sub.2--B1-BOC2(A1,B29)-insulin as the drug. In each case, the
conjugate or RHI was injected at 3.5 U/kg behind the neck of fasted
normal non-diabetic rats (Male Sprague-Dawley, 400-500 g, n=6).
Blood samples were collected via tail vein bleeding at 0 minutes
and at 30, 60, 90, 120, 150, 180, 210, 240, and 300 minutes after
injection. Blood glucose values were measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). As shown in FIG. 7, the glucose depression profiles
for RHI and the exemplary conjugate are nearly identical despite
the inability for the exemplary conjugate to be enzymatically
digested in vivo. The rapid action and elimination profiles of the
conjugate are most likely due to the fact that the conjugate is
only 14% larger than RHI making any effect of increased MW almost
negligible in terms of pharmacodynamic properties.
Example 38
PK Comparison with RHI
[0524] This example describes and compares the serum insulin
profiles obtained for a subcutaneously administered exemplary
conjugate and recombinant human insulin (RHI). The exemplary
conjugate, I-1 in FIG. 45, was synthesized using TSAT-C6 as the
framework, AEM as the ligand, and NH.sub.2--B1-BOC2(A1,B29)-insulin
as the drug. In each case, the conjugate or RHI was injected at 3.5
U/kg behind the neck of fasted normal non-diabetic rats (Male
Sprague-Dawley, 400-500 g, n=6). Blood samples were collected via
tail vein bleeding at 0 minutes and at 30, 60, 90, 120, 150, 180,
210, 240, and 300 minutes after injection. Blood from each
timepoint was centrifuged at 4 C to collect the serum. Serum
insulin concentrations were subsequently measured with a
commercially available ELISA kit (Human Insulin ELISA, Mercodia,
Uppsala, Sweden). As can be seen in FIG. 8, the pharmacokinetic
profile for the conjugate is statistically indistinguishable from
that of RHI, demonstrating that this conjugate is rapidly absorbed
into and eliminated from serum following a subcutaneous
injection.
Example 39
PK and Bioactivity of a B29-Substituted Version of the
AEM-2-TSAT-C6-Insulin Conjugate
[0525] This example describes the serum insulin and blood glucose
depression profiles obtained for a subcutaneously administered
exemplary conjugate. The exemplary conjugate, I-7 in FIG. 45, was
synthesized using TSAT-C6 as the framework, AEM as the ligand, and
recombinant human insulin as the drug (to produce a B29-substituted
conjugate instead of a B1-substituted conjugate as in Examples 37
and 38). In this case, the conjugate was injected at 5 U/kg behind
the neck of fasted normal non-diabetic rats (Male Sprague-Dawley,
400-500 g, n=3). Blood samples were collected via tail vein
bleeding at 0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240,
and 300 minutes after injection. Blood glucose values were measured
using commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In addition, blood from each
timepoint was centrifuged at 4 C to collect the serum. Serum
insulin concentrations were subsequently measured with a
commercially available ELISA kit (Human Insulin ELISA, Mercodia,
Uppsala, Sweden). As can be seen in FIG. 9, the pharmacokinetic
profile for the B29-substituted conjugate is statistically
indistinguishable from that of RHI as well as the B1-substituted
conjugate from Example 38, demonstrating that this conjugate is
also rapidly absorbed into and eliminated from serum following a
subcutaneous injection.
Example 40
PK and Bioactivity Comparison with Lispro
[0526] This example compares the serum insulin and blood glucose
profiles obtained for a subcutaneously administered exemplary
conjugate and insulin lispro. Insulin lispro (HUMALOG.RTM.) is a
rapid acting insulin analog in which the penultimate lysine and
proline residues on the C-terminal end of the B-chain have been
reversed. This modification blocks the formation of insulin
multimers. Data from soluble recombinant human insulin (RHI) is
also provided for comparison (see Example 38 and FIG. 8).
[0527] The exemplary conjugate, I-1 in FIG. 45, was synthesized
using TSAT-C6 as the framework, AEM as the ligand, and
NH.sub.2--B1-BOC2(A1,B29)-insulin as the drug. In each case, the
conjugate or insulin lispro was injected at 3.5 U/kg behind the
neck of fasted normal non-diabetic rats (Male Sprague-Dawley,
400-500 gm, n=6). Blood samples were collected via tail vein
bleeding at 0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240,
and 300 minutes after injection. Blood glucose values were measured
using commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In addition, blood from each
timepoint was centrifuged at 4 C to collect the serum. Serum
insulin concentrations were subsequently measured with a
commercially available ELISA kit (Human Insulin ELISA, Mercodia,
Uppsala, Sweden). As can be seen in FIG. 12, the pharmacokinetic
profile for the conjugate is statistically indistinguishable from
that of insulin lispro.
Example 41
Effect of Ligand on Bioactivity
[0528] This example compares the blood glucose profiles obtained
for a series of subcutaneously administered exemplary conjugates.
The exemplary conjugates were synthesized using TSAT-C6 as the
framework, and NH.sub.2--B1-BOC2(A1,B29)-insulin as the drug. The
ligand composition was varied across the conjugates to cover a
range of affinities: AEM-2, AEBM-2, AETM-1-AEBM-1 and AETM-2 (from
lowest to highest affinity). The insulin conjugates are shown as
I-1, 1-2, 1-3, and I-4 in FIG. 45. In each case, the conjugates
were injected at 5 U/kg (3.5 U/kg for AEM-2) behind the neck of
fasted normal non-diabetic rats (Male Sprague-Dawley, 400-500 gm,
n=6). Blood samples were collected via tail vein bleeding at 0
minutes and at 30, 60, 90, 120, 150, 180, 210, 240, and 300 minutes
after injection. Blood glucose values were measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In addition, blood from each
timepoint was centrifuged at 4 C to collect the serum. Serum
insulin concentrations were subsequently measured with a
commercially available ELISA kit (Human Insulin ELISA, Mercodia,
Uppsala, Sweden).
[0529] As can be seen in FIG. 13, the glucose lowering response
decreased as the affinity of the ligand increased. This data
provided the first indication that the nature of the ligand may
affect the bioactivity of the conjugate. FIGS. 14-16 show the blood
glucose levels alongside the serum insulin levels for each of the
four conjugates tested. These results show quite clearly that the
reduced glucose response for conjugates with higher affinity
ligands results from the reduced PK profile of the conjugate
(compare FIG. 14 for AEM-2 with FIG. 17 for AETM-2).
Example 42
Effect of Exogenous Inhibitors on PK and Bioactivity
[0530] In view of the data described in Example 41 we hypothesized
that the reduced PK profile and bioactivity observed with
conjugates having higher ligands might result from stronger binding
to endogenous saccharide binding molecules. For example, without
wishing to be limited to any particular theory, we hypothesized
that binding to endogenous "lectin-like" proteins such as
surfactant proteins A and D or members of the selectin family might
be causing these conjugates to be cleared more rapidly than
conjugates with lower affinity ligands.
[0531] In order to test this hypothesis we ran a set of experiments
to determine whether exogenous ligands could compete for binding to
these proposed endogenous saccharide binding molecules and thereby
increase the PK profile of the conjugates post-administration. The
high affinity AETM-2 conjugate I-2 was used for all experiments. In
each case, the same dose of conjugate was injected behind the neck
of fasted normal non-diabetic rats (Male Sprague-Dawley, 400-500
gm, n=6). After a 15 minute delay a dose of the exogenous ligand
was injected IP. Blood samples were collected via tail vein
bleeding at 0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240,
and 300 minutes after the initial conjugate injection. Blood
glucose values were measured using commercially available test
strips (Precision Xtra, Abbott Laboratories, Abbott Park, Ill.). In
addition, blood from each timepoint was centrifuged at 4 C to
collect the serum. Serum insulin concentrations were subsequently
measured with a commercially available ELISA kit (Human Insulin
ELISA, Mercodia, Uppsala, Sweden). A control was performed by
injecting saline instead of the exogenous ligand after 15
minutes.
[0532] FIG. 18 shows the results obtained when alpha-methyl mannose
was administered. Alpha-methyl mannose is a very high affinity
saccharide which is capable of competing with AETM for binding to
lectins such as Con A. As shown, the change in PK/PD profile that
resulted from injection of alpha-methyl mannose was very
significant (p<0.05).
[0533] FIG. 19 is a control experiment in which soluble recombinant
human insulin (RHI) was used for the initial injection instead of
the AETM-2 conjugate. As shown, there was no change in PK/PD
profile when alpha-methyl mannose was injected (p>>0.05).
[0534] The endogenous mannan-binding lectin (MBL) is known to bind
saccharides with the following relative affinities: D-mannose,
L-fucose>D-glucose, N-acetyl-D-glucosamine>>D-galactose.
We therefore decided to run two experiments comparing the effects
of L-fucose (high affinity ligand), D-glucose (intermediate
affinity ligand) and D-galactose (low affinity ligand) on the PK/PD
profile for the AETM-2 conjugate. The results with L-fucose are
compared with the results obtained with alpha-methyl mannose in
FIG. 20. As shown, alpha-methyl mannose and L-fucose appear to
exhibit the same kind of effect. The results with D-glucose and
D-galactose are compared in FIG. 21. Galactose exhibits no effect
as compared to saline. Glucose appears to exhibit a small effect;
however, this is complicated by the fact that the exogenous insulin
from the conjugate quickly lowers the glucose, so the sustained
effect observed with alpha-methyl mannose and L-fucose does not
occur.
Example 43
Effect of Exogenous Inhibitor at the Local Injection Site
[0535] In view of the data described in Example 42, we set out to
determine whether the alpha-methyl mannose (a-MM) induced increase
in serum conjugate concentration and bioactivity was a result of an
increased rate of absorption from the subcutaneous injection site.
The high affinity TSAT-C6-AETM-2 conjugate I-2 was used for this
experiment. First, the conjugate was diluted to a concentration of
5 U/ml (0.2 mg/ml insulin equivalent) using either a buffered
saline solution or a buffered saline solution containing 1 M a-MM.
Each solution was injected sub-Q at a dose of 5 U/kg into the back
of the neck of each of three non-diabetic, male SD rats at time 0
and blood samples were collected via tail vein bleeding at 0
minutes and at 15, 30, 45, 60, 90, 120, 150, 180, 240, and 300
minutes after injection. Blood glucose values were measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In addition, blood from each
timepoint was centrifuged at 4 C to collect the serum. Serum
insulin concentrations were subsequently measured with a
commercially available ELISA kit (Iso Insulin ELISA, Mercodia,
Uppsala, Sweden). In this experiment, rats that received the a-MM
conjugate solution also received an injection of buffered saline
solution sub-Q at a separate hind quarter injection site at 15
minutes. Rats that received the saline conjugate solution also
received an injection of a-MM solution sub-Q at a separate hind
quarter injection site at 15 minutes. These 15-minute delayed
injections were used to make sure that the amount of a-MM injected
sub-Q with the conjugate solution would not raise the systemic
concentration of a-MM high enough to invoke the kind of a-MM
induced effects exhibited in FIG. 18.
[0536] The results shown in FIG. 22 indicate that within
experimental error, the conjugate PK and bioactivity profiles were
not enhanced at all by purposefully co-injecting a high
concentration of a-MM inhibitor with the conjugate. These results
are consistent with the hypothesis that the exogenous saccharides
do not act on the conjugates by changing the absorption profile
into systemic circulation from the site of injection.
Example 44
Effect of Delaying the Exogenous Inhibitor Injection after the
Initial Conjugate Injection
[0537] Based on the results in FIG. 22, the a-MM-enhanced PK and
bioactivity results cannot be explained by increased injection site
absorption but rather must be the result of a systemic effect that
occurs after the conjugate has been absorbed. The following two
hypotheses could explain such behavior: (a) the conjugate is being
eliminated from the body via a lectin dependent mechanism that can
be disrupted by the competitive saccharide or (b) the conjugate is
binding to lectins within the body and is only released into
circulation in the presence of the competitive saccharide. In the
case of (b) one would expect that introduction of a-MM into the
animal after the conjugate has been fully absorbed from the sub-Q
depot would cause the absorbed conjugate to release from the lectin
sites in the body thereby increasing its serum concentration and
bioactivity. However, if an elimination mechanism is at work as
described in (a) then injecting a-MM after the conjugate has been
fully absorbed from the sub-Q depot will not produce any increase
in serum concentration or bioactivity because the conjugate will
have already been completely eliminated from the body.
[0538] To determine the likely mechanism, the high affinity
TSAT-C6-AETM-2 conjugate I-2 was injected at 5 U/kg behind the neck
of fasted normal non-diabetic rats (Male Sprague-Dawley, 400-500
gm, n=3 per group). After four different delay times (15 minutes,
60 minutes, 120 minutes, and 240 minutes) a 4 g/kg dose of a-MM
solution was injected IP. Blood samples were collected via tail
vein bleeding at 0 minutes and at the following intervals for each
experiment:
TABLE-US-00010 IP a-MM injection delay time (min) Sample time
points (min post injection) 15 15, 30, 45, 60, 90, 120, 150, 180,
240, 300, 360 60 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, 300,
360 120 15, 30, 45, 60, 90, 120, 135, 150, 180, 240, 300, 360 240
15, 30, 45, 60, 120, 180, 240, 255, 270, 300, 360
[0539] Blood glucose values were measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). In addition, blood from each timepoint was centrifuged
at 4 C to collect the serum. Serum insulin concentrations were
subsequently measured with a commercially available ELISA kit (Iso
Insulin ELISA, Mercodia, Uppsala, Sweden).
[0540] As shown in FIG. 23, the increase in serum conjugate
concentration and bioactivity due to IP a-MM injection is less and
less prevalent the longer the delay time between the conjugate
injection and the IP a-MM injection. For example, after 240 minutes
no increase in serum conjugate concentration is observed indicating
that there is no lectin-bound depot of conjugate present in the
body after complete conjugate absorption. These results are
consistent with proposed mechanism (a) and inconsistent with
proposed mechanism (b). It is to be understood that while we
hypothesize that mechanism (a) may be responsible for the PK
properties observed with the conjugates of the present disclosure,
the claims presented herein are in no way limited to a specific
mechanism of action.
Example 45
Effect of a-MM on PK and Bioactivity as a Function of Ligand
Affinity
[0541] In view of the data described in Example 42, we set out to
determine the pharmacokinetic and pharmacodynamic behavior of
conjugates synthesized using different saccharide ligands than the
AETM ligand used in Example 42. In this example, the TSAT-C6
framework was used and the following conjugates were synthesized
according to the methods described in Example 20 (note
glucosamine-HCl or GA-HCl was purchased from Sigma-Aldrich (St.
Louis, Mo.) and used without further purification):
TABLE-US-00011 Frame- AE- MW work sugar Purity (LC- Sugar/
Conjugate Framework MW Ligand MW (HPLC) MS) Insulin I-7: TSAT-C6-
TSAT-C6 822 AEM 223 95% 6729 2.0 AEM-2 (B29) I-5: TSAT-C6- TSAT-C6
822 GA- 216 95% 6641 2.0 GA-2 (B29) HCl
According to N-terminal sequencing, approximately 90% of each
saccharide-containing framework was conjugated to insulin via the
Lys-B29. TSAT-C6-AEM-2 (B29) and TSAT-C6-GA-2 (B29) are shown in
FIG. 45 as conjugates I-7 and I-5, respectively.
[0542] The same type of experiments described in Example 42 were
repeated for the conjugates described in the table above. In each
case, the same dose of conjugate (5 U/kg) was injected behind the
neck of fasted normal non-diabetic rats (Male Sprague-Dawley,
400-500 gm, n=3). After a 15 minute delay a 4 g/kg dose of a-MM was
injected IP. Blood samples were collected via tail vein bleeding at
0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240, and 300
minutes after the initial conjugate injection. Blood glucose values
were measured using commercially available test strips (Precision
Xtra, Abbott Laboratories, Abbott Park, Ill.). In addition, blood
from each timepoint was centrifuged at 4 C to collect the serum.
Serum insulin concentrations were subsequently measured with a
commercially available ELISA kit (ISO Insulin ELISA, Mercodia,
Uppsala, Sweden). A control was performed by injecting saline
instead of a-MM after 15 minutes.
[0543] FIGS. 24 and 25 show the results obtained when a-MM was
administered by IP injection 15 minutes after the sub-Q injection
of I-7 and I-5, respectively. As shown, the increase in PK/PD
profile that resulted from injection of a-MM was very significant
(p<0.05) for conjugate I-7 when compared to the saline injection
control group. However, the extent of the a-MM-induced increase in
serum conjugate concentration was less than that obtained for the
AETM-2 conjugates from Example 42. The I-5 conjugate profile was
unaffected by the a-MM injection, just like the results obtained
for RHI in FIG. 19. Taken together, these data illustrate that both
mannose-derived conjugates (AEM-2 and AETM-2) exhibit the
a-MM-enhanced PK/PD profile while the lower affinity-glucosamine
derived conjugates do not. Furthermore, the relative change in PK
profile for the lower affinity AEM-2 conjugates is less than the
change observed for the higher affinity AETM-2 conjugates.
Example 46
Effect of a-MM on PK and Bioactivity as a Function of Ligand
Valency
[0544] In this example, we set out to determine the pharmacokinetic
and pharmacodynamic behavior of conjugates to which an increasing
number of exemplary saccharide ligands have been covalently
attached. All conjugates were synthesized according to the methods
described in Example 20 using the frameworks and saccharide ligands
specified below:
TABLE-US-00012 Frame- AE- MW work sugar Purity (LC- Sugar/
Conjugate Framework MW Ligand MW (HPLC) MS) Insulin I-8: DSS-AEM-1
DSS 368 AEM 223 >95% 6168 1.0 (B29) I-9: TSPE-AEM- TSPE 813 AEM
223 >95% 6829 3.0 3 (B29) I-10: DSS- DSS 368 AETM 547 >95%
6491 1.0 AETM-1 (B29) I-11: TSPE- TSPE 813 AETM 547 >95% 7800
3.0 AETM-3 (B29)
According to N-terminal sequencing, approximately 90% of each
saccharide-containing framework was conjugated to insulin via the
Lys-B29. The conjugates are shown in FIG. 45 as I-8, I-9, I-10, and
I-11.
[0545] The same type of experiment described in Example 45 was
repeated for the conjugates described in the table above. In each
case, the same dose of conjugate (5 U/kg) was injected behind the
neck of fasted normal non-diabetic rats (Male Sprague-Dawley,
400-500 gm, n=3). After a 15 minute delay a 4 g/kg dose of a-MM was
injected IP. Blood samples were collected via tail vein bleeding at
0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240, and 300
minutes after the initial conjugate injection. Blood glucose values
were measured using commercially available test strips (Precision
Xtra, Abbott Laboratories, Abbott Park, Ill.). In addition, blood
from each timepoint was centrifuged at 4 C to collect the serum.
Serum insulin concentrations were subsequently measured with a
commercially available ELISA kit (ISO Insulin ELISA, Mercodia,
Uppsala, Sweden). A control was performed by injecting saline
instead of a-MM after 15 minutes.
[0546] FIGS. 26 and 27 show the results obtained when a-MM was
administered by IP injection 15 minutes after the sub-Q injection
of I-8 and I-9, respectively. As shown, the increase in PK/PD
profile that resulted from injection of a-MM was very significant
(p<0.05) for I-9 and less so for I-8 when compared to the saline
injection control group. Furthermore, the I-9 conjugate exhibited
approximately the same a-MM-induced PK profile as the I-2 conjugate
from Example 42, both of which were much more pronounced than that
obtained from I-8.
[0547] FIGS. 28 and 29 show the results obtained when a-MM was
administered by IP injection 15 minutes after the sub-Q injection
of I-10 and I-11, respectively. As shown, the increase in PK/PD
profile that resulted from injection of a-MM was very significant
(p<0.05) for I-11 and slightly less so for I-10 when compared to
the saline injection control group. Furthermore, the I-11 conjugate
exhibited approximately the same a-MM-induced PK profile as the I-2
conjugate from Example 42, both of which were slightly more
pronounced than that obtained from I-10.
Example 47
In Vivo Half Life/Elimination Rate Comparison
[0548] The results obtained in Example 44 are consistent with the
exemplary conjugates being eliminated from the body via a lectin
dependent mechanism that can be disrupted by the presence of a
competitive saccharide. In order to explore this mechanism in more
detail, we conducted the following experiments on exemplary
conjugates to determine the rate at which they were cleared from
serum in vivo versus unconjugated insulin. All conjugates used in
this study were synthesized according to the general methods
described in Example 20.
[0549] In each case the soluble conjugate was dosed at 0.4 mg
conjugate/kg body weight into dual jugular vein cannulated male
Sprague-Dawley rats (Taconic, JV/JV, 350-400 g, n=3). A sterile
conjugate solution or control insulin was injected intravenously
via one JV cannula, followed immediately by a chase solution of
heparin-saline to ensure that all of the conjugate dose was
administered into the animal. The second cannula was used to
collect blood samples at t=0 (pre-dose), and at 1, 2, 4, 8, 15, 30,
60, 90, 120, and 180 minutes post-dose.
[0550] Blood glucose values were measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). In addition, blood from each timepoint was centrifuged
at 4 C to collect the serum. Serum insulin or serum conjugate
concentrations were subsequently measured with a commercially
available ELISA kit (Iso-Insulin ELISA, Mercodia, Uppsala,
Sweden).
[0551] FIG. 30 shows the serum concentration of either RHI or
TSAT-C6-AETM-2 conjugate, shown as I-6 in FIG. 45, as a function of
time following the intravenous injection. Clearly, I-6 is
eliminated much more rapidly from serum than is RHI. The data is
best fit using a two-compartment bi-exponential model with the
following general formula: C(t)=A.sub.o EXP(-at)+B.sub.o EXP(-bt)
where t is time, C(t) is the concentration in serum as a function
of time, A.sub.o is the first compartment concentration constant, a
is the first compartment exponential time constant, B.sub.o is the
second compartment concentration constant, and b is the second
compartment exponential time constant. The elimination half-lives
(in minutes) associated with each compartment are t1/2(a)=0.693/a
and t1/2(b)=0.693/b. In FIG. 30, for RHI the t1/2(a)=0.76 and
t1/2(b)=11.46 and for I-6 the t1/2(a)=0.47 and t1/2(b)=2.87. In
other words, the t1/2(b) for I-6 is about four times shorter than
the t1/2(b) for RHI.
[0552] The following table summarizes the t1/2 parameters for a
number of conjugates tested using exactly the same procedure
described above (structures are shown in FIG. 45):
TABLE-US-00013 Ratio to RHI Ratio to RHI Formulation t1/2 (a) t1/2
(b) t1/2 (a) t1/2 (b) RHI 0.76 11.46 1.00 1.00 I-5: TSAT-C6-GA-2
0.81 12.02 1.07 1.05 I-8: DSS-AEM-1 0.90 9.61 1.18 0.84 I-7:
TSAT-C6-AEM-2 0.45 2.77 0.60 0.24 I-9: TSPE-AEM-3 0.66 2.62 0.87
0.23 I-10: DSS-AETM-1 0.82 4.48 1.08 0.39 I-6: TSAT-C6-AETM-2 0.47
2.87 0.62 0.25 I-11: TSPE-AETM-3 0.22 1.33 0.29 0.12
This data is consistent with the hypothesis that the exemplary
conjugates are eliminated from serum more rapidly than unconjugated
insulin, the extent of which is governed by the affinity of the
particular conjugate for the endogenous lectin and the number of
ligands substituted per conjugate. Furthermore, the a-MM induced
increase in PK/PD profiles demonstrated in Examples 42, and 44-46
correlates well with the reduction in Phase b half-life for each of
the conjugates tested.
Example 48
In Vivo Half Life/Elimination Rate Under Glucose Infusion
[0553] In this example, it was further hypothesized that the
clearance rate of exemplary conjugates could be inhibited by the
presence of physiological concentrations of glucose. In order to
determine the rate at which the conjugates were cleared from serum
in vivo under hyperglycemic conditions, the following experiment
was conducted. In each case, I-7 was dosed at 0.4 mg conjugate/kg
body weight into dual jugular vein cannulated male Sprague-Dawley
rats (Taconic, JV/JV, 350-400 g, n=3).
[0554] One hour before the start of the experiment one rat cannula
was connected to a syringe infusion pump containing a sterile 50%
w/v glucose solution. The pump infusion rate was adjusted by the
experimenter to ensure that the blood glucose levels in the animal
remained above 300 mg/dL at all times during the experiment. Blood
glucose was measured using commercially available test strips
(Precision Xtra, Abbott Laboratories, Abbott Park, Ill.). In a
typical experiment, it was found that the infusion pump rate
required to keep the animals above 300 mg/dL was typically greater
than 85 uL/min. A blood sample was taken at t=0 min, after which a
sterile conjugate solution or control insulin was injected
intravenously via the second rat cannula, followed immediately by a
chase solution of heparin-saline to ensure that all of the
conjugate dose was administered into the animal. After an
additional flush of the cannula line with heparin-saline, the
second cannula was used to collect blood samples at t=1, 2, 4, 8,
15, 30, 60, 90, 120, and 180 minutes post-dose.
[0555] Blood from each timepoint was centrifuged at 4 C to collect
the serum, and serum insulin or serum conjugate concentrations were
subsequently measured with a commercially available ELISA kit
(Iso-Insulin ELISA, Mercodia, Uppsala, Sweden). Insulin or
conjugate serum concentration vs. time data was best fit with the
sum of two independent decaying exponentials (C(t)=a
exp(-k.sub.at)+b exp(-k.sub.bt)) according to the two-compartment
model, where t1/2(a)=(ln 2)/k.sub.a and t1/2(b)=(ln 2)/k.sub.b. The
following table summarizes the t1/2 parameters for I-7 with and
without the glucose infusion along with those obtained for RHI from
Example 47:
TABLE-US-00014 Ratio Ratio t1/2 t1/2 to RHI to RHI Infusion
Formulation (a) (b) t1/2 (a) t1/2 (b) None RHI 0.76 11.46 1.00 1.00
Saline TSAT-C6-AEM-2 (I-7) 0.45 2.77 0.60 0.24 Glucose
TSAT-C6-AEM-2 (I-7) 0.64 5.11 0.84 0.45 (400 mg/dl)
We can conclude from these data that glucose is able to inhibit the
accelerated serum elimination for this conjugate thereby doubling
the Phase b elimination half life from 2.77 to 5.11 minutes.
Example 49
In Vivo Half Life/Elimination Rate Under a-MM Infusion
[0556] In this example, it was further hypothesized that the
clearance rate of insulin-saccharide conjugates could be inhibited
by the presence of arbitrarily high concentrations of inhibitory
saccharides other than glucose, such as .alpha.-methyl-mannose
(a-MM). In order to determine the rate at which exemplary
conjugates were cleared from serum in vivo in the presence of a-MM,
the following experiment was conducted. In each case the soluble
conjugate was dosed at 0.4 mg conjugate/kg body weight into dual
jugular vein cannulated male Sprague-Dawley rats (Taconic, JV/JV,
350-400 g, n=3).
[0557] One hour before the start of the experiment one rat cannula
was connected to a syringe infusion pump containing a sterile 25%
w/v a-MM solution. The pump infusion rate was adjusted by the
experimenter, but was typically set at 85 uL/min. A blood sample
was taken at t=0 min, after which a sterile conjugate solution or
control insulin was injected intravenously via the second rat
cannula, followed immediately by a chase solution of heparin-saline
to ensure that all of the conjugate dose was administered into the
animal. After an additional flush of the cannula line with
heparin-saline, the second cannula was used to collect blood
samples at t=1, 2, 4, 8, 15, 30, 60, 90, 120, and 180 minutes
post-dose.
[0558] In addition, blood glucose was measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). Blood from each timepoint was centrifuged at 4 C to
collect the serum, and serum insulin or serum conjugate
concentrations were subsequently measured with a commercially
available ELISA kit (Iso-Insulin ELISA, Mercodia, Uppsala, Sweden).
Insulin or conjugate serum concentration vs. time data was best fit
with the sum of two independent decaying exponentials (C(t)=a
exp(-k.sub.at)+b exp(-k.sub.bt)) according to the two-compartment
model, where t1/2(a)=(ln 2)/k.sub.a and t1/2(b)=(ln 2)/k.sub.b. The
following table summarizes the t1/2 parameters for the
TSAT-C6-AEM-2 conjugate with and without the a-MM infusion along
with those obtained with glucose infusion from Example 48 and those
obtained for RHI with no saccharide infusion from Example 47:
TABLE-US-00015 Ratio Ratio t1/2 t1/2 to RHI to RHI Infusion
Formulation (a) (b) t1/2 (a) t1/2 (b) None RHI 0.76 11.46 1.00 1.00
Saline TSAT-C6-AEM-2 (I-7) 0.45 2.77 0.60 0.24 a-MM TSAT-C6-AEM-2
(I-7) 0.92 10.09 1.21 0.88 Glucose TSAT-C6-AEM-2 (I-7) 0.64 5.11
0.84 0.45 (400 mg/dl)
We can conclude from these data that not only does a-MM inhibit the
accelerated serum elimination for this conjugate, it does so to an
even greater extent than does glucose. In this case, the Phase b
elimination half life nearly quadruples from 2.77 to 10.09
minutes.
IV. Other Examples
[0559] This fourth set of examples describes various experiments
investigating the synthesis, formulation, and properties of some
exemplary conjugates.
Example 50
Long Acting Insulin Conjugates Using Protamine, Zinc, and Other
Excipients
[0560] Given that the data from previous examples is consistent
with a saccharide-dependent serum elimination mechanism, we set out
to develop formulations of conjugates that would provide a steady,
sustained rate of absorption from a subcutaneous injection site. At
a steady absorption rate, the serum conjugate concentration at any
point in time will be governed primarily by the
saccharide-dependent elimination rate. In such a way, we can
formulate a long acting, sustained-release insulin exhibiting a
saccharide-responsive PK profile.
[0561] In order to generate long acting conjugates, we prepared PZI
(protamine zinc insulin) formulations from the conjugate solutions.
Conjugates substituted with insulin at the B1-terminus do not form
amorphous or crystalline PZI formulations as readily, so we used
B29-substituted conjugates prepared based on the methods of Example
20. The excipients used in these formulations comprise protamine,
zinc, m-cresol, and salt all of which were obtained commercially
from Sigma-Aldrich (St. Louis, Mo.). The concentrations of these
components may be varied in order to obtain an optimally flat,
sustained absorption rate. In addition, in some cases it was found
that the addition of a small amount of unmodified insulin helped
stabilize the formulation. In these cases, the concentration of
unmodified insulin contained in the sample was varied to obtain an
optimally flat, sustained absorption rate. In all formulations
tested, the following recipe was used:
TABLE-US-00016 Component Variable Volume (ml) Conjugate solution at
% unmodified insulin content 1.000 2.7 mg/ml (M/M) 250 mM HEPES
NaCl concentration (M) 0.111 buffered saline Zinc acetate solution
Zinc concentration (mg/ml) 0.124 Cresol solution in water v/v %
0.159 pH 7.2 Protamine Protamine concentration (mg/ml) 4 .times.
0.194 solution in 25 mM aliquots HEPES buffered saline
Unless otherwise specified, once the formulations were prepared
after addition of the components in the order described in the
table above, they were gently mixed for 30 minutes prior to in vivo
testing.
[0562] To test the sustained release profile for a given
formulation as well as the glucose-responsive PK profile, the
following experiment was conducted. The formulation was injected at
a predetermined dose (.about.15 U/kg in most cases unless otherwise
specified) behind the neck of fasted normal non-diabetic rats (Male
Sprague-Dawley, 400-500 gm, n=3). After a 240 minute delay, a
glucose dose (4 g/kg) was injected IP. Blood samples were collected
via tail vein bleeding at 0 minutes and at 30, 60, 90, 120, 150,
180, 210, 240, and 300 minutes after the initial conjugate
injection. Blood glucose values were measured using commercially
available test strips (Precision Xtra, Abbott Laboratories, Abbott
Park, Ill.). In addition, blood from each timepoint was centrifuged
at 4 C to collect the serum. Serum insulin concentrations were
subsequently measured with a commercially available ELISA kit
(Iso-Insulin ELISA, Mercodia, Uppsala, Sweden). According to the
manufacturer's assay specifications, the Iso-Insulin ELISA is 71%
cross-reactive with rat insulin. The serum samples were diluted by
10.times. in order to minimize the amount of endogenous rat insulin
detected in each sample but the possibility of rat insulin
detection could not be completely ruled out. Therefore, the results
are generally reported as "measured insulin," which can consist of
some amount of endogenous rat insulin in addition to the conjugate
or RHI, depending on the experiment. Nevertheless, all samples
collected in each of the following examples were treated
identically and can be directly compared for differences in
performance.
Example 51
Effect of Protamine Concentration on Long Acting Insulin Conjugate
Performance
[0563] The purpose of this example was to demonstrate the effect of
protamine concentration on the time action and glucose-responsive
PK profile of an exemplary conjugate. In this example I-6,
synthesized according to the methods described in Example 20, was
tested using the generalized formulation and in vivo protocol
described in Example 50:
TABLE-US-00017 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 16.7% 1.000 6) solution at 2.7 mg/ml 250 mM
HEPES NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration (mg/ml) = 0.124 see below Cresol
solution in water 3% v/v 0.159 pH 7.2 Protamine Protamine
concentration 4 .times. 0.194 solution in 25 mM (mg/ml) = see below
aliquots HEPES buffered saline Zinc concentration Protamine
concentration Formulation (mg/ml) (mg/ml) 1xP-1xZ 1.15 1.25 4xP-4xZ
4.60 5.00 10xP-4xZ 4.60 12.50
The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of each of the three formulations described
above. The results shown in FIG. 31a-c demonstrate that as the
protamine concentration in the formulation increases, the more
protracted the resulting formulation and the more pronounced the
measured increase in serum insulin profile after the four hour
glucose injection. The 1.times.P-1.times.Z formulation released a
significant portion of the insulin conjugate payload over a short
period of time immediately following the injection such that very
little signal was detected after the IP glucose challenge. On the
other hand, the 10.times.P-4.times.Z formulation released a low
basal amount of insulin over the first four hours with no
hypoglycemia and subsequently attained >4.times. increase in
measured insulin concentration immediately following the IP glucose
injection.
Example 52
Effect of Zinc Concentration on Long Acting Insulin Conjugate
Performance
[0564] The purpose of this example was to demonstrate the effect of
zinc concentration on the formulation stability, time action and
glucose-responsive PK profile of an exemplary conjugate. In this
example I-6, synthesized according to the methods described in
Example 20, was tested using the generalized formulation and in
vivo protocol described in Example 50:
TABLE-US-00018 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 0% 1.000 6) solution at 2.7 mg/ml 250 mM HEPES
NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration (mg/ml) = 0.124 see below Cresol
solution in water 3% v/v 0.159 pH 7.2 Protamine Protamine
concentration 4 .times. 0.194 solution in 25 mM (mg/ml) = see below
aliquots HEPES buffered saline Zinc concentration Protamine
concentration Formulation (mg/ml) (mg/ml) 4xP-1xZ 1.15 5.00 4xP-2xZ
2.30 5.00 10xP-1xZ 1.15 12.5 10xP-2xZ 2.30 12.5
The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of each of the four formulations described
above. The results shown in FIGS. 32a-b and 33a-b demonstrate that
within experimental error, the concentration of zinc did not have a
significant effect on the overall sustained release nature of the
formulation or the glucose-responsive profile. In all cases, a
statistically significant increase in measured insulin
concentration was observed following the IP glucose injection. As
demonstrated in Example 51, the higher protamine (10.times.P)
formulations released less conjugate over time than the lower
protamine (4.times.P) formulations regardless of the zinc
concentration. However, when the formulations were left at room
temperature for greater than 24 hours, both 10.times.P formulations
transformed from an easily dispersible particulate solution into a
sticky, agglomerated, two-phase solution. This did not happen with
the corresponding 10.times.P-4.times.Z formulation. Similarly, the
4.times.P-1.times.Z formulation was found to transform the same way
as the 10.times.P-1.times.Z formulation whereas the
4.times.P-2.times.Z was relatively stable at room temperature for
weeks. Therefore, the zinc concentration for a given protamine
concentration can be adjusted to prepare easily dispersible
formulations that are stable over long periods of time.
Example 53
Effect of m-Cresol Concentration on Long Acting Insulin Conjugate
Performance
[0565] The purpose of this example was to demonstrate the effect of
m-cresol concentration on the time action and glucose-responsive PK
profile of an exemplary conjugate. In this example I-6, synthesized
according to the methods described in Example 20, was tested using
the generalized formulation and in vivo protocol described in
Example 50:
TABLE-US-00019 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 0% 1.000 6) solution at 2.7 mg/ml 250 mM HEPES
NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in
water v/v % = see below 0.159 pH 7.2 Protamine Protamine
concentration = 4 .times. 0.194 solution in 25 mM 12.5 mg/ml
aliquots HEPES buffered saline Formulation Cresol concentration No
cresol 0 4x cresol 12% v/v
The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of the two formulations described above. The
results shown in FIG. 34a-b demonstrate that the presence of
m-cresol maintains a more protracted formulation. The no cresol
formulation releases a significant portion of the insulin conjugate
payload over a short period of time immediately following the
injection such that very little increase in measured insulin
concentration was observed when challenged with IP glucose. On the
other hand, the 4.times. cresol formulation releases a low basal
amount of insulin over the first four hours with no hypoglycemia
and subsequently attains a 3-4.times. increase in measured insulin
concentration immediately following the IP glucose injection.
Example 54
Effect of Salt/Isotonic Agent Concentration on Long Acting Insulin
Conjugate Performance
[0566] The purpose of this example was to demonstrate the effect of
salt concentration and choice of isotonic agent on the time action
and glucose-responsive PK profile of an exemplary conjugate. In
this example I-6, synthesized according to the methods described in
Example 20, was tested using the generalized formulation and in
vivo protocol described in Example 50:
TABLE-US-00020 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 16.7% 1.000 6) solution at 2.7 mg/ml 250 mM
HEPES NaCl or glycerol concentration 0.111 buffered saline (M) =
see below Zinc acetate solution Zinc concentration = 4.6 mg/ml
0.124 Cresol solution in water 3% v/v 0.159 pH 7.2 Protamine
Protamine concentration = 4 .times. 0.194 solution in 25 mM 12.5
mg/ml aliquots HEPES buffered saline Formulation NaCl concentration
No salt 0.0M 3.3x salt 5.0M Glycerol Neat glycerol solution instead
of buffered saline
The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of each of the three formulations described
above. The results shown in FIG. 35a-c demonstrate that the
presence of salt in the formulation maintains a more protracted
formulation. The no salt formulation released a significant portion
of the insulin conjugate payload over the first four hours of the
experiment such that very little increase in measured insulin
concentration was observed when challenged with IP glucose. On the
other hand, the 3.3.times. salt formulation released a low basal
amount of conjugate over the first four hours with no hypoglycemia
and subsequently attained a .about.4.times. increase in measured
insulin concentration immediately following the IP glucose
injection. This performance was similar to that obtained with the
10.times.P-4.times.Z formulation from Example 51, which was exactly
the same as the 3.3.times. salt formulation but contained
approximately 1/3 the salt concentration (1.5 M as compared to 5.0
M). Finally, substituting glycerol for NaCl as the isotonic agent
does not appear to adversely affect the protracted nature of the
formulation.
Example 55
Effect of Unmodified Insulin Concentration on Long Acting Insulin
Conjugate Performance
[0567] The purpose of this example was to demonstrate the effect of
including different concentrations of unmodified insulin on the
time action and glucose-responsive PK profile of an exemplary
conjugate. In this example I-6, synthesized according to the
methods described in Example 20, was tested using the generalized
formulation and in vivo protocol described in Example 50:
TABLE-US-00021 Component Variable Volume (ml) TSAT-C6-AETM-2 (I- %
unmodified insulin = see 1.000 6) solution at 2.7 mg/ml below 250
mM HEPES NaCl concentration = 1.5M 0.111 buffered saline Zinc
acetate solution Zinc concentration = 4.6 mg/ml 0.124 Cresol
solution in water 3% v/v 0.159 pH 7.2 Protamine Protamine
concentration = 4 .times. 0.194 solution in 25 mM 12.5 mg/ml
aliquots HEPES buffered saline Formulation % unmodified insulin
1/24 4.17 1/12 8.33 1/6 16.7
The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of each of the three formulations described
above. The results shown in FIG. 36a-c demonstrate that the
presence of unmodified insulin in the formulation beneficially
produces a more protracted formulation with substantial increase in
measured insulin concentration following an IP glucose injection.
Furthermore, the presence of unmodified insulin helps preserve the
formulation performance even after several weeks of room
temperature incubation (see Example 57 below).
Example 56
Long Acting Insulin Conjugates--Dose Response Effect
[0568] In this example, we evaluated the dose-response effect of a
particular formulation of a long-acting exemplary conjugate.
Conjugate I-6, synthesized according to the methods described in
Example 20, was tested using the generalized formulation and in
vivo protocol described in Example 50:
TABLE-US-00022 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 16.7% 1.000 6) solution at 2.7 mg/ml 250 mM
HEPES NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in
water 3% v/v 0.159 pH 7.2 Protamine Protamine concentration = 4
.times. 0.194 solution in 25 mM 12.5 mg/ml aliquots HEPES buffered
saline
The four hour IP glucose injection (4 g/kg) experiment was
performed by dosing 5 or 15 U/kg (body weight in
grams/1.87=microliters of injection volume) of the formulation
described above.
[0569] As shown in FIG. 37, the conjugate exhibited a flat PK
profile until the glucose was injected. The increase in measured
insulin concentration following the IP glucose challenge was
dramatic and dose-dependent (compare data obtained with a 5 U/kg
(left) and 15 U/kg (right) dose of conjugate). No hypoglycemia was
observed at early or late time points.
Example 57
Stability of Exemplary Long-Acting, Glucose-Responsive
Conjugates
[0570] In this example, we synthesized the same exact long acting
formulation from Example 56 at a 2.times. scale. Half of the
material was stored at 2-8 C and the other half stored at room
temperature for one week or two weeks. After the specified storage
time, the material was redispersed and tested using the same four
hour IP glucose injection protocol described in Example 56 at a 15
U/kg dose (body weight in grams/1.87=microliters of injection
volume). As shown in FIGS. 38-39, this formulation demonstrates
similar performance even after being stored refrigerated (FIG. 38)
or at room temperature (FIG. 39) for at least two weeks.
Example 58
Performance of Long Acting Conjugates Prepared from Conjugates with
Varying Ligand Affinity and Multivalency
[0571] In this example, we set out to determine the time action and
glucose-responsive PK profile of long-acting formulations of
conjugates constructed from different types and numbers of ligands.
All conjugates for this example were synthesized according to the
methods described in Example 20 using the frameworks and saccharide
ligands specified below:
TABLE-US-00023 Frame- AE- MW work sugar Purity (LC- Sugar/
Conjugate Framework MW Ligand MW (HPLC) MS) Insulin I-8: DSS-AEM-1
DSS 368 AEM 223 >95% 6168 1.0 (B29) I-7: TSAT-C6- TSAT-C6 822
AEM 223 95% 6729 2.0 AEM-2 (B29) I-9: TSPE-AEM- TSPE 813 AEM 223
>95% 6829 3.0 3 (B29) I-10: DSS- DSS 368 AETM 547 >95% 6491
1.0 AETM-1 (B29) I-11: TSPE- TSPE 813 AETM 547 >95% 7800 3.0
AETM-3 (B29) I-17: C6-amide- C6-amide 838 AEM 223 95% 6745 2.0
AEM-2 (B29)
The following long-acting formulation was used for each
conjugate:
TABLE-US-00024 Component Variable Volume (ml) Conjugate solution at
unmodified insulin = 16.7% 1.000 2.7 mg/ml 250 mM HEPES NaCl
concentration = 1.5M 0.111 buffered saline Zinc acetate solution
Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in water 3%
v/v 0.159 pH 7.2 Protamine Protamine concentration = 4 .times.
0.194 solution in 25 mM 12.5 mg/ml aliquots HEPES buffered
saline
[0572] The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of each of the conjugates described above. As
shown in FIG. 40a-e, all conjugates exhibited a protracted
absorption profile with some element of increase in measured serum
insulin concentration following the 4 hour glucose injection. It
appears that there was some significant conjugate absorption in the
first four hours after injection of the long acting TSPE-AETM-3
conjugate I-11. However, all other conjugates exhibited flat
absorption profiles like the ones observed for TSAT-C6-AETM-2
conjugates. These results correlate well with the fact that the
half-lives of these conjugates are all less than unmodified insulin
as described in Examples 47-48 and that each of them demonstrates
an a-MM-induced increase in PK/PD profile as described in Examples
45-46.
Example 59
Performance of Long Acting Conjugates Under IP a-MM Testing
Conditions
[0573] In this example, we tested the a-MM-responsive profile of
long-acting formulations of conjugates constructed from
TSAT-C6-AETM-2 (I-6) and TSAT-C6-GA-2 (I-5) conjugates. Both
conjugates were prepared according to the general methods described
in Example 20. In addition, the following long-acting formulation
was used for each conjugate:
TABLE-US-00025 Component Variable Volume (ml) Conjugate solution at
unmodified insulin = 16.7% 1.000 2.7 mg/ml 250 mM HEPES NaCl
concentration = 1.5M 0.111 buffered saline Zinc acetate solution
Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in water 3%
v/v 0.159 pH 7.2 Protamine Protamine concentration = 4 .times.
0.194 solution in 25 mM 12.5 mg/ml aliquots HEPES buffered
saline
[0574] To test the sustained release nature of the formulations as
well as the a-MM-responsive PK profile, the following experiment
was conducted. The formulations were injected at 15 U/kg (body
weight in grams/1.87=microliters of injection volume) behind the
neck of fasted normal non-diabetic rats (Male Sprague-Dawley,
400-500 gm, n=3). After a 240 minute delay, an a-MM dose (4 g/kg)
was injected IP. Blood samples were collected via tail vein
bleeding at 0 minutes and at 30, 60, 90, 120, 150, 180, 210, 240,
and 300 minutes after the initial conjugate injection. Blood
glucose values were measured using commercially available test
strips (Precision Xtra, Abbott Laboratories, Abbott Park, Ill.). In
addition, blood from each timepoint was centrifuged at 4 C to
collect the serum. Serum insulin concentrations were subsequently
measured with a commercially available ELISA kit (Iso-Insulin
ELISA, Mercodia, Uppsala, Sweden).
[0575] As shown in FIG. 41a, the peak:baseline serum concentration
ratio following the IP a-MM injection for the long-acting
TSAT-C6-AETM-2 (I-6) formulation was even higher than that observed
for the same formulation when glucose is substituted for a-MM.
Furthermore, the TSAT-C6-GA-2 (I-5) formulation (FIG. 41b) did not
demonstrate any increase in serum conjugate concentration following
the a-MM challenge. These results correlate well with the fact that
that the elimination half life of the TSAT-C6-GA-2 (I-5) conjugate
was nearly identical to unmodified insulin (Example 47), and that
it exhibited no a-MM-induced change in PK (Example 45).
Example 60
Long Acting Conjugates in Diabetics and Non-Diabetics
[0576] In order to confirm the in vivo utility of the long acting
TSAT-C6-AETM-2 (I-6) formulation, we administered it (5, 10 and 20
U/kg) to normal and STZ-induced diabetic rats (Male Sprague-Dawley,
400-500 gm, n=6). The formulation was prepared using the following
procedure:
TABLE-US-00026 Component Variable Volume (ml) TSAT-C6-AETM-2 (I-
unmodified insulin = 0% 1.000 6) solution at 2.7 mg/ml 250 mM HEPES
NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in
water 3% v/v 0.159 pH 7.2 Protamine Protamine concentration = 4
.times. 0.194 solution in 25 mM 12.5 mg/ml aliquots HEPES buffered
saline
[0577] No external IP injections of glucose were used to trigger
the bioactivity of the conjugates. Instead we relied on the
endogenous levels of glucose in the rats to control the PK and PD
profile of the conjugate formulation. Blood samples were collected
via tail vein bleeding at various time points after the initial
conjugate injection. Blood glucose values were measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). As shown in FIG. 42, no
hypoglycemia was observed at early or late time points for the
normal or diabetic rats. The glucose profiles observed with the
diabetic rats are dramatic and demonstrate that the conjugates were
activated by the higher glucose concentrations and exerted their
glucose-lowering effect in a dose proportional manner over a long
time period (over 8 hours at the highest dose).
[0578] The experiment was repeated using different doses (7, 14 and
28 U/kg) and a longer time period (24 hours). Results from that
experiment are shown in FIG. 43.
Example 61
Conjugation with Fatty Acid Esters to Generate Long Acting
Formulations
[0579] It will be appreciated that we could have used an
alternative approach to protamine zinc insulin for making a long
acting form of an exemplary conjugate. In this example, we
demonstrate how to convert a B1-substituted insulin conjugate into
a long-acting formulation by covalently modifying the
B29-epsilon-amine group with a long chain fatty acid. As described
in U.S. Pat. No. 6,869,930, insulin acylation with C14-myristic
acid, for example, leads to a soluble material with a flat,
protracted time action profile. B1-substituted TSAT-C6-AETM-2 (I-2)
is synthesized according to the methods in Example 12. The material
may then be lyophilized into a dry powder and used as the starting
material in the following procedure.
[0580] Myristic acid-NHS ester is dissolved at 60 mM in 1 ml of
anhydrous DMSO followed by the addition of 400 ul (excess) of
triethylamine (TEA). The solution is stirred rapidly for 10 minutes
at room temperature. Conjugate I-2 is then dissolved separately in
7.5 ml of anhydrous DMSO at a concentration of 8.1 mM. Once
dissolved, the entire solution is added over the course of one
minute to the myristic acid-NHS ester solution followed by room
temperature mixing for an additional two hours to ensure complete
reaction.
[0581] The resulting solution is then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution is first purified by size exclusion using
an appropriate solid phase for the desired separation of conjugated
and unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 10 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC on a Waters SymmetryPrep C18, 7 um column,
19.times.150 mm. Buffer A is deionized water containing 0.1% TFA
and Buffer B is acetonitrile containing 0.1% TFA. Before
purification, the column is equilibrated at 15 ml/minutes with a
80% A/20% B mobile phase using a Waters DeltraPrep 600 system.
Approximately 5 ml of the crude solution is injected onto the
column over the course of 2 minutes at a flow rate of 15 ml/minutes
after which a linear gradient is employed from 80% A/20% B to 75%
A/25% B over the next 5 minutes followed by a slower linear
gradient from 75% A/25% B to 62% A/38% B over the next 22 minutes.
The retention time of the desired peak will vary depending on the
drug, framework, and ligand used. Once collected, the solution is
rotovapped to remove acetonitrile and lyophilized to obtain pure
conjugate whose identity may be verified by LC-MS (HT Laboratories,
San Diego, Calif.). The protracted and saccharide-responsive PK/PD
profile of the resulting conjugate may then be evaluated using the
4 hour IP glucose or a-MM conditions described in Examples 50 and
59.
Example 62
Long-Acting Conjugates with Insulin Possessing a Neutral
Isoelectric Point
[0582] It will be appreciated that we could have used yet other
alternative approaches to protamine zinc insulin for making a long
acting form of the conjugate. In this example, we demonstrate how
to synthesize long-acting conjugates by substituting insulin
glargine (LANTUS.RTM.), for example, instead of wild-type human
insulin in preparing the conjugate. Any and all synthesis methods
described in the preceding examples may be used to form insulin
glargine-conjugates. Insulin glargine is an exemplary long acting
insulin analog in which Asp-A21 has been replaced by glycine, and
two arginines have been added to the C-terminus of the B-chain. The
effect of these changes is to shift the isoelectric point,
producing a solution that is completely soluble at pH 4 but
insoluble at physiological pH. Once synthesized and purified, the
protracted and saccharide-responsive PK/PD profile of the resulting
conjugate may then be evaluated using the 4 hour IP glucose or a-MM
conditions described in Examples 50 and 59.
Example 63
Use of Soluble Conjugates in a Pump Delivery System
[0583] Instead of formulating the saccharide-responsive conjugates
into long acting formulations, each may be continuously infused
intravenously, subcutaneously, or intraperitoneally using a pump
delivery system. The sterile solution of conjugate at a known
concentration (typically 25-100 U/ml) is loaded into the pump
reservoir and continuously delivered into the compartment of choice
at a steady rate. This rate is adjusted to the maximal level at
which no hypoglycemia is observed in the subject. Then, using the 4
hour IP glucose or a-MM conditions described in Examples 50 and 59,
the glucose-responsive PK/PD profile may be evaluated. The pump
method is advantageous in that no excipients are required to
provide a steady input and absorption rate for the conjugates. A
variety of insulin pumps have been described in the art and may be
used for this purpose. For example, see any one of the pumps
described in U.S. Pat. Nos. 4,435,173, 4,498,843, 4,923,375,
5,062,841, 6,650,951, 6,744,350, 6,852,104, 7,377,907, and
7,515,060, and U.S. Patent Publication Nos. 20080172028,
20090005726, 20090112165, 20090137957, 20090177142, 20090177154,
and 20100004598, each of which is incorporated herein by
reference.
Example 64
Synthesis of C6-Amide-AEM-2 intermediate
[0584] This example describes a synthetic process for making a
ZC-AEM-2 intermediate having the following chemical structure:
##STR00042##
[0585] a. Intermediate Z2A
[0586] The first phase of the process involved combining reagents
Z1A and Z1B to produce intermediate Z2A as shown in the following
reaction scheme:
##STR00043##
[0587] This was achieved as follows. To a 250 mL two neck flask was
added compound Z1B (Sigma-Aldrich, St. Louis, Mo., 13.9 g),
1-hydroxybenzotriazole hydrate (HOBT, Sigma-Aldrich, St. Louis,
Mo., 11.0 g) and dimethylformamide (DMF, Sigma-Aldrich, St. Louis,
Mo.) at room temperature under nitrogen. The mixture was cooled to
0 C, after which time Compound Z1A (Sigma-Aldrich, St. Louis, Mo.,
9.5 mL) and di-isopropylcarbodiimide (DIC, Sigma-Aldrich, St.
Louis, Mo., 9.8 ml) were added to the mixture under nitrogen. The
reaction solution was stirred at room temperature overnight. After
36 hours of reaction, (dimethylamino)pyridine (DMAP, Sigma-Aldrich,
St. Louis, Mo., 7.71 g) was added, and after 48 hours the reaction
mixture was filtered with a Buchner funnel. The filtrate was kept
at 4 C overnight and worked up the following day.
[0588] The filtrate was evaporated, and the resulting residue was
taken up in 120 mL of ethyl acetate and washed successively with
10% aqueous HCl solution (3.times.30 mL), saturated aqueous
NaHCO.sub.3 solution (3.times.30 mL), brine (3.times.20 mL), and
dried over anhydrous sodium sulfate. The solution was concentrated
to dryness in vacuo to yield the product which could be used
without further purification.
[0589] b. Intermediate Z3A
[0590] The second phase of the process involved converting
intermediate Z2A to intermediate Z3A as shown in the following
reaction scheme:
##STR00044##
[0591] Compound Z2A (16.0 g) was dissolved in methanol
(Sigma-Aldrich, St. Louis, Mo., 85 mL), and the solution was cooled
to 0 C. A 10% aqueous solution of sodium hydroxide (19 mL) was
added dropwise and the reaction was stirred at 0 C for 3 hours.
Afterwards, the suspension was dissolved with a minimum amount of
water (15 mL). To this solution was added Amberlite IR-120
(Sigma-Aldrich, St. Louis, Mo., H+ form), in 15.times.2 g aliquots
at 0 C. The bead suspension was stirred for 0.5 hr, resulting in a
solution pH of .about.5. The resulting mixture was filtered to
remove the Amberlite beads, and the filtrate concentrated and dried
in vacuo to yield a white-red solid, 13.0 g yield.
[0592] c. Intermediate Z4A
[0593] The third phase of the process involved combining
intermediate Z3A with reagent Z3B to produce intermediate Z4A as
shown in the following reaction scheme:
##STR00045##
[0594] Compound Z3B (Sigma-Aldrich, St. Louis, Mo., 19.0 g) was
dissolved in DMF (50 mL). The suspension was cooled to 0 C, after
which time triethylamine (15.0 mL) was added to the solution. The
temperature of the solution was maintained at 0.degree. C. for 0.75
hours. Next, the solution was charged with a solution of Compound
Z3A (13.0 g) dissolved in DMF (24 mL) at 0 C, followed by HOBT
(Sigma-Aldrich, St. Louis, Mo., 15.9 g), DMF (50 mL), and DIC
(Sigma-Aldrich, St. Louis, Mo., 15.0 mL). The resulting solution
was stirred for an additional hour at 0 C and then allowed to warm
to room temperature and react overnight for an additional 18
hours.
[0595] Next the reaction was recooled to 0 C, and
N,N-diisopropylethylamine (DIPEA, Sigma-Aldrich, St. Louis, Mo., 15
mL) was added and the resulting solution stirred for 0.5 hr until
the pH of the solution was approximately 9.0. Anhydrous methylene
chloride (25 mL) and DIC (3.5 mL) were added and stirring was
continued for another 10 hours. More compound Z3B was added (free
base, 2.5 g) and the reaction was allowed to proceed for an
additional 50 hours at room temperature under a nitrogen
atmosphere.
[0596] Finally, the reaction mixture was filtered, and the filtrate
concentrated via rotary evaporation. The residue was dissolved in
methylene chloride (CH.sub.2Cl.sub.2, 400 mL), and the organic
phase washed with a 5% HCl solution (3.times.200 mL). The organic
layer was cooled to 0.degree. C. and neutralized with a saturated
sodium bicarbonate solution (3.times.100 mL) and brine (2.times.200
mL). The organic solution was dried over magnesium sulfate, which
was then separated by filtration and concentrated using rotary
evaporation. The residue was purified by column chromatography on
silica gel (eluent phase: methylene chloride/methanol 50:1 to 5:1).
Column fractions were concentrated and dried in vacuo overnight. A
white solid was obtained as Z4A (18.9 g, yield 81%).
[0597] d. Intermediate Z5A
[0598] The fourth phase of the process involved converting
intermediate Z4A to intermediate Z5A as shown in the following
reaction scheme:
##STR00046##
[0599] Ester Z4A (2.27 g) was dissolved in methanol (10 mL), and to
this solution 5.0 mL of 1.0M sodium hydroxide solution was added at
room temperature. The mixture was stirred at room temperature for
38 hours. At the end of this time, an additional 1.5 mL of 2.0M
aqueous sodium hydroxide solution was added, and the mixture was
stirred at room temperature for another 14 hours.
[0600] Next, the reaction mixture was acidified with a 10% aqueous
solution of HCl at 0.degree. C. The product was extracted with
ethyl acetate (3.times.25 mL). The combined organic layers were
dried over magnesium sulfate, and then concentrated to dryness in
vacuo overnight to yield Z5A as a white solid (2.2 g).
[0601] e. Intermediate Z6A
[0602] The fifth phase of the process involved combining
intermediate Z5A with aminoethylmannose (AEM) to produce
intermediate Z6A as shown in the following reaction scheme:
##STR00047##
[0603] Diacid compound Z5A (2.0 g), aminoethylmannose (1.46 g),
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium
hexafluorophosphate (HATU, Sigma-Aldrich, St. Louis, Mo., 2.7 g)
were dissolved in dry DMF (80 mL) under nitrogen at 0.degree. C.
DIPEA (Sigma-Aldrich, St. Louis, Mo., 3.0 mL) was added dropwise to
the mixture at 0.degree. C. under a nitrogen atmosphere. The
mixture was stirred for 1 hr at 0.degree. C. and then an additional
4 hr at room temperature. At this point, another aliquot of
aminoethylmannose (0.5 g) and HATU (0.52 g) was added to the
reaction solution at room temperature, and the solution was stirred
for another 3 hours. The mixture was concentrated via rotary
evaporation and the residue was purified by column chromatography
on silica gel (methylene chloride/methanol 20:1, then 6:1 eluting
to 1:1). Fractions were collected and evaporated in vacuo to yield
purified product (TLC: upper spot, Rf 0.6, methylene
chloride:methanol 1:3).
[0604] It will be appreciated that this entire process can be
repeated to obtain conjugates with different ligands by replacing
AEM with another amine containing reagent (e.g., without limitation
AEG, AEBM, AETM, etc.) when converting intermediate Z5A to
intermediate Z6A.
[0605] f. Intermediate Z7A
[0606] The sixth phase of the process involved converting
intermediate Z6A to intermediate Z7A as shown in the following
reaction scheme:
##STR00048##
[0607] Compound Z6A (1.43 g) was dissolved in anhydrous methanol
(100 mL). To this solution was added (1.00 g palladium on carbon
(Pd/C)), and hydrogen gas was bubbled into the solution to reduce
the protecting group to give the corresponding amine. It was found
that the reaction had reached complete conversion after
approximately 9 hours of reaction.
[0608] The reaction mixture was filtered through a pad of Celite,
and the filtrate was concentrated and dried in vacuo. Compound Z7A
was obtained as a brown solid (1.16 g, yield 94%).
[0609] g. Intermediate Z8A
[0610] The following scheme shows how the coupling agent Z8A was
prepared:
##STR00049##
[0611] To a cooled solution of N-hydroxysuccinimide (Sigma-Aldrich,
St. Louis, Mo., NHS, 7.0 g) in dry CH.sub.2Cl.sub.2(65 mL) at 0 C
was added triethylamine (9.5 mL) and adipoyl chloride (3.97 mL, 5.0
g). The resulting mixture was stirred for 2 hr at 0 C. The mixture
was washed with a saturated aqueous solution of NaCl (3.times.30
mL) and dried over magnesium sulfate. The solution was filtered and
concentrated in vacuo, and then the residue purified via silica gel
chromatography to obtain a white solid (6.6 g) as product Z8A.
[0612] h. Intermediate Z9A
[0613] The following scheme shows how the coupling agent Z8A was
combined with intermediate Z7A to produce intermediate Z9A:
##STR00050##
[0614] Compound Z7A (0.256 g) was dissolved in dry DMF, and the
solution was cooled to 0 C. To this solution was added a solution
of Z8A (0.710 g) in anhydrous DMF (15 mL) under a nitrogen
atmosphere. The mixture was stirred at 0 C for 2 hours.
[0615] The solution volume was concentrated to one third and
filtered off excess unreacted DSS. The filtrate was further
concentrated to approximately 3 mL of column and purified by silica
gel chromatography (eluent: methylene chloride/methanol 20:1 to
4:1, then 3:1 to 1:1). Collected fractions were concentrated and
dried in vacuo to give 151 mg of purified product. .sup.1H NMR (300
MHz, DMSO) .delta. 1.28-1.63 (band, 20H), 2.07-2.22 (band, 6H),
2.85 (s, 4H), 3.08-3.64 (band, 22H), 3.91 (s, 4H), 4.08 (s, 2H),
4.49 (s, 2H), 4.57 (d, 2H, J=5.70 Hz), 4.64 (s, 2H), 4.73 (t, 4H,
J=5.10 Hz), 7.85 (s, 2H, amide NH), 8.68, 8.19, 7.98 (s, 3H, amide
NH). LC-MS (Found 1074.67 [M+Na+], M=1051.57 Da; Calculated 1052.08
Da).
Example 65
Synthesis of C6-Amide-AEM-2 (B1) Conjugate
[0616] This example describes a method for preparing a
B1-conjugated insulin from intermediate Z9A of Example 64. Compound
Z9A was conjugated to insulin as follows.
NH.sub.2--B1-BOC2(A1,B29)-insulin synthesized according to Example
8 (0.167 g) was dissolved in dry DMSO (3 mL) at room temperature
under nitrogen and stirred for 0.5 hours at room temperature. To
this solution was added anhydrous triethylamine (0.013 mL) at room
temperature under nitrogen. Compound Z9A (0.177 g) in dry DMSO (1.5
mL) was added via syringe pump at a rate of (4.2 uL/min) to the
insulin-triethylamine mixture at room temperature at a stir rate of
80 rpm. The reaction conversion was monitored by analytical HPLC.
After 4 hours, another 3 equivalents of TEA (0.010 mL) were added
to the reaction mixture. After a total of 10.5 hours at room
temperature, the reaction was stopped and placed in a -20 C freezer
overnight.
[0617] The next day, the reaction mixture was thawed and placed
onto a small packed column of ion exchange beads (SP Sephadex
beads, Sigma-Aldrich, St. Louis, Mo., isocratic conditions). The
column was centrifuged briefly for 4 min at 1000.times.g to purify
the insulin conjugate (assessed by analytical HPLC). The collected
fractions from the ion exchange column (6 mL) were added dropwise
to dry acetone (30 mL) with stirring at 140 rpm for 10 min. The
resulting suspension was poured into a 50 mL centrifuge tube which
was spun for 10 min at 3500.times.g. The clear supernatant was
removed from the tubes, and the cake kept and set aside. The
supernatant was added to another 30 mL of acetone to obtain a
second crop of precipitate (after adding a few drops of 5N HCl),
which was then centrifuged for 10 min at 3500.times.g to obtain a
second centrifuge cake.
[0618] The combined cakes were dried in vacuo for 1 hour, to obtain
a white solid (197 mg, 92% yield) at >98% purity by analytical
HPLC. The insulin conjugate BOC groups were removed by the
procedure set forth in Example 12 to obtain the biologically active
insulin conjugate.
[0619] The process described in this example has the advantage of
producing a high yield, high purity insulin conjugate without
requiring reverse phase HPLC.
Example 66
Synthesis of I-17: C6-Amide-AEM-2 (B29) Conjugate
[0620] This example describes one method for preparing a
B29-conjugated insulin from intermediate Z9A of Example 64.
Compound Z9A is conjugated to insulin as follows.
NH.sub.2--B29-BOC2(A1,B1)-insulin synthesized according to Example
16 (0.167 g) is dissolved in dry DMSO (3 mL) at room temperature
under nitrogen and stirred for 0.5 hours at room temperature. To
this solution is added anhydrous triethylamine (0.013 mL) at room
temperature under nitrogen. Compound Z9A (0.177 g) in dry DMSO (1.5
mL) is added via syringe pump at a rate of (4.2 uL/min) to the
insulin-triethylamine mixture at room temperature at a stir rate of
80 rpm. The reaction conversion is monitored by analytical HPLC.
After 4 hours, another 3 equivalents of TEA (0.010 mL) are added to
the reaction mixture. After a total of 10.5 hours at room
temperature, the reaction is stopped and placed in a -20 C freezer
overnight.
[0621] The next day, the reaction mixture is thawed and placed onto
a small packed column of ion exchange beads (SP Sephadex beads,
Sigma-Aldrich, St. Louis, Mo., isocratic conditions). The column is
centrifuged briefly for 4 min at 1000.times.g to purify the insulin
conjugate (assessed by analytical HPLC). The collected fractions
from the ion exchange column (6 mL) are added dropwise to dry
acetone (30 mL) with stirring at 140 rpm for 10 min. The resulting
suspension is poured into a 50 mL centrifuge tube which is spun for
10 min at 3500.times.g. The clear supernatant is removed from the
tubes, and the cake kept and set aside. The supernatant is added to
another 30 mL of acetone to obtain a second crop of precipitate
(after adding a few drops of 5N HCl), which is then centrifuged for
10 min at 3500.times.g to obtain a second centrifuge cake.
[0622] The combined cakes are dried in vacuo for 1 hour, to obtain
a white solid. The insulin conjugate BOC groups are removed by the
procedure set forth in Example 12 to obtain the biologically active
insulin conjugate.
Example 67
Alternative Synthesis of I-17: C6-Amide-AEM-2 (B29) Conjugate
[0623] This example describes another method for preparing a
B29-conjugated insulin from intermediate Z9A of Example 64.
Specifically, this alternative method involves directly coupling
compound Z9A to unprotected insulin at the B29 epsilon amino group.
Compound Z9A was dissolved at 53 mM in 1.0 ml of anhydrous DMSO
followed by the addition of 0.4 ml (excess) of triethylamine (TEA).
The solution was stirred rapidly for 5 minutes at room temperature.
Recombinant human insulin (RHI) powder was then dissolved
separately at 17.2 mM in 1 ml of a 0.1 M, pH 11 sodium carbonate
buffer and the pH was subsequently adjusted to 10.8 with 1.0N
sodium hydroxide. Once dissolved, the entire solution of Compound
Z9A was added dropwise over the course of 10 minutes to the
insulin/carbonate buffer solution. The solution was allowed to stir
for an additional 15 minutes after the dropwise addition to ensure
complete reaction.
[0624] The resulting solution was then superdiluted by 10.times.
into a 20 mM pH 5.0 HEPES buffered saline solution containing 0.150
M NaCl followed by pH adjustment with dilute HCl to a final pH of
8.0. The aqueous solution was first purified by size exclusion
using Biogel P2 (Bio-Rad Laboratories, Hercules, Calif.) for the
desired separation of conjugated and unconjugated materials. The
solution passing through the column void volume was then
concentrated using a 3 kDa ultrafiltration membrane to
approximately 15 ml. This solution was further purified to obtain
the desired product using preparative reverse phase HPLC on a
Waters SymmetryPrep C18, 7 um, 19.times.150 mm column. Buffer A was
deionized water containing 0.1% TFA and Buffer B was acetonitrile
containing 0.1% TFA. Before purification, the column was
equilibrated at 15 ml/minutes with a 80% A/20% B mobile phase using
a Waters DeltraPrep 600 system. Approximately 5 ml of the crude
solution was injected onto the column over the course of 2 minutes
at a flow rate of 15 ml/minutes after which a linear gradient is
employed from 80% A/20% B to 75% A/25% B over the next 5 minutes
followed by a slower linear gradient from 75% A/25% B to 62% A/38%
B over the next 22 minutes. Once the desired fraction was
collected, the solution was rotovapped to remove acetonitrile and
lyophilized to obtain pure conjugate whose identity was verified by
LC-MS (HT Laboratories, San Diego, Calif.). The final product (95%
pure by HPLC) was found to have the desired MW of 6744 g/mol
(LC-MS), representing a total of 2.0 AEM molecules conjugated per
insulin, with greater than 85% of the conjugate molecules
conjugated at the Lys-B29 site (as determined by N-terminal
sequencing).
Example 68
Long Acting I-17 Conjugates
[0625] In this example, we set out to determine the time action and
glucose-responsive PK profile of long-acting formulations of
conjugates constructed from Compound Z9A of Example 64. The
conjugate for this example was synthesized according to the methods
described in Example 67. The following long-acting formulation was
used for this conjugate:
TABLE-US-00027 Component Variable Volume (ml) I-17 conjugate
solution unmodified insulin = 16.7% 1.000 at 2.7 mg/ml 250 mM HEPES
NaCl concentration = 1.5M 0.111 buffered saline Zinc acetate
solution Zinc concentration = 4.6 mg/ml 0.124 Cresol solution in
water 3% v/v 0.159 pH 7.2 Protamine Protamine concentration = 4
.times. 0.194 solution in 25 mM 12.5 mg/ml aliquots HEPES buffered
saline
[0626] The four hour IP glucose injection (4 g/kg) experiments were
performed by dosing 15 U/kg (body weight in grams/1.87=microliters
of injection volume) of the formulation described above. As shown
in FIG. 44, the conjugate exhibits a protracted absorption profile
with a significant increase in measured serum insulin concentration
following the 4 hour glucose injection.
Example 69
Conjugates Prepared from 2-Aminoethyl Alpha-L-Fucopyranoside and
2-Aminoethyl N-Acetyl-Beta-D-Glucosamine and Evaluation
[0627] The data from Example 42 demonstrates the ability of
L-fucose to inhibit the presumed lectin pathway that leads to the
increased PK profile of exemplary conjugates. It is also known that
beta-linked N-acetyl glucosamine may also inhibit the pathways for
which mannose and fucose are inhibitors (e.g. see Haurum et al. in
Biochem. J. 293:873-878, 1993). This example describes how insulin
conjugates such as the ones described above may be synthesized
using 2-aminoethyl alpha-L-fucopyranoside or 2-aminoethyl
N-acetyl-beta-D-glucosamine ligands. 2-aminoethyl
alpha-L-fucopyranoside (MW=207 g/mol) is prepared according to the
method of Ni et al. in Bioconjugate Chem. 14:232-238, 2003.
2-aminoethyl N-acetyl-beta-D-glucosamine (MW=264 g/mol) is
synthesized according to the method of Cai et al. in Organic
Letters 7:4021-4024, 2005. Either one of these ligands may be
readily substituted for the amino-functionalized sugar ligands in
any of the conjugate synthesis methods described above in Examples
11-13, 15, 17-27, and 29-31.
[0628] The PK of the resulting conjugates are tested in vivo for
a-MM, L-fucose, or glucose-induced increases in PK/PD profiles
following subcutaneous injection in rats according to the methods
described in Example 42. The conjugates can also be formulated as
sustained release formulations according to the methods in Examples
50-55 and subsequently evaluated for their protracted and
glucose-responsive pharmacokinetics according to the 4 hour IP
glucose injection protocol outlined in those same examples.
Alternative methods of sustaining the release of these conjugates
may also be employed such as those described in Examples 61-63.
Example 70
Exemplary Conjugate of Formula (VIb-3)
[0629] In certain embodiments the present disclosure provides a
conjugate of formula (VIb-3):
##STR00051##
[0630] wherein:
[0631] W is an insulin molecule; and
[0632] each occurrence of --X is
##STR00052##
[0633] In certain embodiments, the insulin molecule is selected
from the group consisting of human insulin, porcine insulin, and
bovine insulin. In certain embodiments, the insulin molecule is
insulin glargine or insulin detemir. In certain embodiments, the
insulin molecule includes three disulfide bridges.
Example 71
Exemplary Conjugate I-6
[0634] In certain embodiments the present disclosure provides
conjugate I-6:
##STR00053##
Example 72
Exemplary Conjugate of Formula (VIc-2)
[0635] In certain embodiments the present disclosure provides a
conjugate of formula (VIc-2):
##STR00054##
[0636] wherein:
[0637] W is an insulin molecule; and
[0638] each occurrence of --X is
##STR00055##
[0639] In certain embodiments, the insulin molecule is selected
from the group consisting of human insulin, porcine insulin, and
bovine insulin. In certain embodiments, the insulin molecule is
insulin glargine or insulin detemir. In certain embodiments, the
insulin molecule includes three disulfide bridges.
Example 73
Exemplary Conjugate of Formula (VId-1)
[0640] In certain embodiments the present disclosure provides a
conjugate of formula (VId-1):
##STR00056##
[0641] wherein:
[0642] W is an insulin molecule; and
[0643] --X is
##STR00057##
[0644] In certain embodiments, the insulin molecule is selected
from the group consisting of human insulin, porcine insulin, and
bovine insulin. In certain embodiments, the insulin molecule is
insulin glargine or insulin detemir. In certain embodiments, the
insulin molecule includes three disulfide bridges.
Example 74
Exemplary Formulations
[0645] In certain embodiments the present disclosure provides
sustained release formulations that comprise a conjugate, wherein
the formulation comprises protamine and zinc. It is to be
understood that the present disclosure encompasses sustained
release formulations with any one of the conjugates described
herein (e.g., without limitation, any one of the conjugates of FIG.
45, 49, 55, 60, 61, or 62).
[0646] In certain embodiments, the formulation includes from about
1 to about 5 mg protamine/mg conjugate; and from about 0.1 to about
0.25 mg zinc/mg conjugate.
[0647] In certain embodiments, the formulation includes protamine
and zinc in a ratio (w/w) in the range of about 40:1 to about
10:1.
[0648] In certain embodiments, the formulation further comprises an
amount of unconjugated insulin molecule. In certain embodiments,
the formulation comprises a molar ratio of conjugated insulin
molecule to unconjugated insulin molecule in the range of about
25:1 to about 2:1.
[0649] In certain embodiments, the formulation further comprises an
antimicrobial preservative. In certain embodiments, the
antimicrobial preservative is m-cresol. In certain embodiments, the
formulation comprises from about 0.15 to about 0.35% v/v
m-cresol.
[0650] In certain embodiments, the formulation further comprises an
isotonic agent. In certain embodiments, the isotonic agent is
glycerol. In certain embodiments, the isotonic agent is NaCl. In
certain embodiments, the formulation comprises from about 0.1 to
about 0.2 M NaCl.
[0651] In certain embodiments, the formulation comprises:
[0652] protamine and zinc in a ratio (w/w) in the range of about
40:1 to about 10:1;
[0653] a molar ratio of conjugated insulin molecule to unconjugated
insulin molecule in the range of about 25:1 to about 2:1;
[0654] about 0.15 to about 0.35% v/v m-cresol; and
[0655] glycerol or from about 0.1 to about 0.2 M NaCl.
[0656] In certain embodiments, the formulation comprises:
[0657] about 3.6 mg protamine/mg conjugate; and
[0658] about 0.2 mg zinc/mg conjugate.
[0659] In certain embodiments, the formulation comprises:
[0660] about 3.6 mg protamine/mg conjugate;
[0661] about 0.2 mg zinc/mg conjugate; and
[0662] a molar ratio of conjugated insulin molecule to unconjugated
insulin molecule of about 5:1.
[0663] In certain embodiments, the formulation comprises:
[0664] about 3.6 mg protamine/mg conjugate;
[0665] about 0.2 mg zinc/mg conjugate;
[0666] a molar ratio of conjugated insulin molecule to unconjugated
insulin molecule of about 5:1; and
[0667] about 0.2% v/v m-cresol.
[0668] In certain embodiments, the formulation comprises:
[0669] about 3.6 mg protamine/mg conjugate;
[0670] about 0.2 mg zinc/mg conjugate;
[0671] a molar ratio of conjugated insulin molecule to unconjugated
insulin molecule of about 5:1;
[0672] about 0.2% v/v m-cresol; and
[0673] glycerol or about 0.15 M NaCl.
Example 75
Insulin Conjugation with Multivalent Activated Esters in Organic
Solvent (Drug Added First) to Give A1-Substituted Insulin
Conjugates--General Procedure
[0674] Step 1
[0675] Insulin is dissolved in a 66:37 vol:vol mixture of 100 mM
sodium carbonate buffer (pH 11) and acetonitrile at a concentration
of 14.7 mM. Separately, a monofunctional protecting group-activated
ester (e.g., BOC--NHS) is dissolved at 467 mM in acetonitrile. Once
the insulin is dissolved, small aliquots of the monofunctional
protecting group-activated ester (e.g., BOC--NHS) are added to the
insulin solution. The pH is monitored throughout the process and is
maintained between 10.2-11.0 through the addition of 0.1M sodium
hydroxide. The reaction is monitored by reverse-phase HPLC.
Aliquots of the monofunctional protecting group-activated ester are
added until the HPLC chromatogram shows that all of the unmodified
insulin has been reacted and that a substantial portion of the
reaction mixture has been converted to B29-protected insulin.
Typically the protecting group will be more hydrophobic in nature
and, once reacted onto the insulin, will elute at an HPLC retention
time that is longer than the unmodified insulin.
[0676] Step 2
[0677] Separately, a framework containing N-terminal activated
esters is dissolved at 174 mM in 1.267 ml of anhydrous DMSO
followed by the addition of 100 .mu.l (excess) of triethylamine
(TEA). The solution is stirred rapidly for 10 minutes at room
temperature. In another vial, a 370 mM solution of ligand is
prepared in an appropriate volume of dry DMSO. Once dissolved,
enough solution is added to provide a number of reactive
equivalents equal to three times the number of initial activated
ester groups, N, minus one. For example, if there are N=3 initial
activated ester groups per framework, then (3.times.(3-1).times.60
mM/370 mM)=0.973 ml of ligand solution are added. If there are N=4
initial activated ester groups per framework, then
(3.times.(4-1).times.60 mM/370 mM)=1.46 ml of ligand solution are
added, and so on. After the ligand solution is added, the solution
is stirred for one more hour at room temperature to ensure complete
reaction.
[0678] Step 3
[0679] Once the insulin has been sufficiently reacted with
protecting group as described in Step 1, and after sufficient
reaction has occurred between the framework and ligand in Step 2,
the framework-ligand solution from Step 2 is added dropwise to the
insulin solution in aliquots. The resulting reaction is monitored
by HPLC. Aliquots are added until the B29-protected insulin is
fully reacted to give the desired B29-protected,
A1-framework/ligand, insulin-conjugate. Since the ligand-framework
is often more hydrophilic than the insulin, the appearance of the
desired product is signaled by a distinct shift to shorter HPLC
retention times as compared to the B29-insulin (from Step 1). Once
the desired level of reaction has been achieved, the reaction
solution is superdiluted by 10.times. into a 20 mM pH 5.0 HEPES
buffered saline solution containing 0.150 M NaCl followed by pH
adjustment with dilute HCl to a final pH of 8.0. The aqueous
solution is first purified by size exclusion using an appropriate
solid phase for the desired separation of conjugated and
unconjugated materials. The solution passing through the column
void volume is then concentrated using an appropriately sized
ultrafiltration membrane to approximately 10 ml. This solution is
further purified to obtain the desired product using preparative
reverse phase HPLC on a Waters C8, 7 um, 19.times.150 mm column.
Buffer A is deionized water containing 0.1% TFA and Buffer B is
acetonitrile containing 0.1% TFA. Before purification, the column
is equilibrated at 15 ml/minutes with a 80% A/20% B mobile phase
using a Waters DeltraPrep 600 system. Approximately 5 ml of the
crude solution is injected onto the column over the course of 2
minutes at a flow rate of 15 ml/minutes after which a linear
gradient is employed from 80% A/20% B to 75% A/25% B over the next
5 minutes followed by a slower linear gradient from 75% A/25% B to
62% A/38% B over the next 22 minutes. The retention time of the
desired peak will vary depending on the insulin, framework, and
ligand used. Once collected, the solution is rotovapped to remove
acetonitrile and lyophilized to obtain pure mono-protected
insulin-conjugate.
[0680] Step 4
[0681] In all cases the protecting group is then removed from the
insulin-conjugate. In cases where a BOC protecting group is used in
Step 1, the BOC groups are removed by dissolving the lyophilized
powder obtained according to Step 3 in 90% TFA/10% anisole for one
hour at 4 C followed by 10.times. superdilution in 25 mM HEPES pH
8.2 buffer containing 0.150M NaCl. (If a protecting group other
than BOC is present on the amine-bearing drug, then the appropriate
deprotection conditions are employed instead of TFA/anisole. A
listing of protection agents and deprotection conditions may be
found in Protecting Groups in Organic Synthesis, T. W. Greene and
P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, as
described in the Definitions section.) The pH was adjusted to
between 7.0 and 8.0 using NaOH solution after which the material is
passed through a Biogel P2 column to remove anisole, BOC and other
low MW byproducts of deprotection, as well as any other
contaminating salts. The deprotected, purified aqueous conjugate
solution was then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to approximately 66 U of insulin/ml
(based on A280 measurements) and stored at 4 C until needed. The
identity of the final conjugate is verified by LC-MS (HT
Laboratories, San Diego, Calif.). The A1 site of conjugation is
confirmed by N-terminal sequencing (Western Analytical, St. Louis,
Mo.), which reveals >95% of the Phe-B1-chain terminus present
and <5% of the Gly.sup.A1-chain terminus present due to the
substitution of Gly.sup.A1 with the ligand-containing
framework.
[0682] One of ordinary skill in the art will appreciate that other
amine-functionalized drugs can be conjugated to ligand-containing
frameworks using analogous procedures to that described in Example
75. One of ordinary skill in the art will also appreciate that
Example 75 is relevant not only to wild-type insulin, but also to
insulin mutants as described herein.
[0683] The following insulin-conjugates were prepared according to
the procedure in Example 75 using BOC--NHS as the protecting
reagent.
TABLE-US-00028 Frame- AE- MW Frame- work sugar Purity (LC- Sugar/
Conjugate work MW Ligand MW (HPLC) MS) Insulin I-13: TSAT-C6-AEM-2
TSAT- 822 AEM 223 97% 6730 2.0 (A1) C6 I-12: TSAT-C6-AETM- TSAT-
822 AETM 547 97% 7378 2.0 2 (A1) C6 I-15: TSPE-AEM-3 TSPE 813 AEM
223 98% 6829 3.0 (A1) I-14: TSPE-AETM-3 TSPE 813 AETM 547 94% 7802
3.0 (A1)
[0684] The following insulin-conjugates can be prepared according
to the procedure in Example 75. In some embodiments, the
insulin-conjugates are prepared using BOC--NHS as the protecting
reagent.
TABLE-US-00029 Frame- AE- MW Sugar/ Frame- work sugar (LC-MS)
Insulin Conjugate work MW Ligand MW (expected) (expected) DSS-AEM-1
(A1) DSS 368 AEM 223 6169 1.0 DSS-AEBM-1 (A1) DSS 368 AEBM 385 6331
1.0 DSS-AETM-1 (A1) DSS 368 AETM 547 6493 1.0 TSAT-C6-AEBM-2 (A1)
TSAT- 822 AEBM 385 7054 2.0 C6 TSPE-AEBM-3 (A1) TSPE 813 AEBM 385
7314 3.0
Example 76
Insulin Conjugation with Multivalent Activated Esters in Organic
Solvent (Drug Added First) to Give A1,B29-Substituted Insulin
Conjugates--General Procedure
[0685] Step 1
[0686] A framework containing N-terminal activated esters is
dissolved at 147 mM in 2.5 ml of anhydrous DMSO followed by the
addition of 1.0 mL (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. In another
vial, a 272 mM solution of ligand is prepared in an appropriate
volume of dry DMSO. Once dissolved, enough solution is added to
provide a number of reactive equivalents equal to three times the
number of initial activated ester groups, N, minus one. For
example, if there are N=3 initial activated ester groups per
framework, then (3.times.(3-1).times.60 mM/370 mM)=0.973 ml of
ligand solution are added. If there are N=4 initial activated ester
groups per framework, then (3.times.(4-1).times.60 mM/370 mM)=1.46
ml of ligand solution are added, and so on. After the ligand
solution is added, the solution is stirred for one more hour at
room temperature to ensure complete reaction.
[0687] Step 2
[0688] Insulin is dissolved in 1.5 mL of 100 mM sodium carbonate
buffer (pH 11) at a concentration of 17.2 mM. Solution pH is
maintained at .about.11 by addition of 0.1 M NaOH as needed. Once
the insulin is dissolved, small aliquots of the framework-ligand
solution are added to the insulin solution. The pH is monitored
throughout the process and is maintained between 10.2-11.0 through
the addition of 0.1M sodium hydroxide. The reaction is monitored by
reverse-phase HPLC. Aliquots of framework-ligand solution are added
until the HPLC chromatogram shows that substantially all of the
unmodified insulin has been reacted and that a substantial portion
of the reaction mixture has been converted to a small portion of
mono-reacted insulin/framework/ligand conjugate and the majority
product is di-reacted insulin/framework/ligand conjugate. Typically
the framework-ligand construct will be more hydrophilic than the
unmodified insulin, causing the mono and di-reacted amine-bearing
drug products to elute at HPLC retention times shorter than that of
the unmodified insulin. Likewise, the HPLC peak of the desired
product, the disubstituted insulin-conjugate, will appear at a
retention time that is shorter than that of the mono-substituted
insulin-conjugate.
[0689] Step 3
[0690] Once the insulin has been sufficiently reacted with
framework ligand as described in Step 2, the solution is then
superdiluted by 10.times. into a 20 mM pH 5.0 HEPES buffered saline
solution containing 0.150 M NaCl followed by pH adjustment with
dilute HCl to a final pH of 8.0. The aqueous solution is first
purified by size exclusion using an appropriate solid phase for the
desired separation of conjugated and unconjugated materials. The
solution passing through the column void volume is then
concentrated using an appropriately sized ultrafiltration membrane
to approximately 10 ml. This solution is further purified to obtain
the desired product using preparative reverse phase HPLC on a
Waters C8, 7 um, 19.times.150 mm column. Buffer A is deionized
water containing 0.1% TFA and Buffer B is acetonitrile containing
0.1% TFA. Before purification, the column is equilibrated at 15
ml/minutes with a 80% A/20% B mobile phase using a Waters
DeltraPrep 600 system. Approximately 5 ml of the crude solution is
injected onto the column over the course of 2 minutes at a flow
rate of 15 ml/minutes after which a linear gradient is employed
from 80% A/20% B to 75% A/25% B over the next 5 minutes followed by
a slower linear gradient from 75% A/25% B to 62% A/38% B over the
next 22 minutes. The retention time of the desired peak will vary
depending on the insulin, framework, and ligand used. Once
collected, the solution is rotovapped to remove acetonitrile and
lyophilized to obtain pure conjugate. The identity of the final
conjugate is verified by LC-MS (HT Laboratories, San Diego,
Calif.). The A1, B29 sites of conjugation are confirmed by
N-terminal sequencing (Western Analytical, St. Louis, Mo.), which
reveals >95% of the Phe-B1-chain terminus present and <5% of
the Gly.sup.A1-chain terminus present due to the substitution of
Gly.sup.A1 with the ligand-containing framework.
[0691] One of ordinary skill in the art will appreciate that other
amine-functionalized drugs can be conjugated to ligand-containing
frameworks using analogous procedures to that described in Example
76. One of ordinary skill in the art will also appreciate that
Example 76 is relevant not only to wild-type insulin, but also to
insulin mutants as described herein.
[0692] The following insulin-conjugates were prepared according to
the procedure in Example 76.
TABLE-US-00030 Frame- AE- MW Frame- work sugar Purity (LC- Sugar/
Conjugate work MW Ligand MW (HPLC) MS) Insulin II-4:
DSS-Di-sub-AEM-1 DSS 368 AEM 223 92% 6531 2.0 (A1, B29) II-3:
DSS-Di-sub-AETM- DSS 368 AETM 547 94% 7179 2.0 1 (A1, B29) II-1:
TSAT-C6-Di-sub- TSAT- 822 AEM 223 97% 7653 4.0 AEM-2 (A1, B29) C6
II-2: TSAT-C6-Di-sub- TSAT- 822 AETM 547 97% 8949 4.0 AETM-2 (A1,
B29) C6
[0693] The following insulin-conjugates can be prepared according
to the procedure in Example 76.
TABLE-US-00031 Frame- Framework AE-sugar MW Sugar/ Conjugate work
MW Ligand MW (LC-MS) Insulin DSS-Di-sub- DSS 368 AEBM 385 6855 2.0
AEBM-1 (A1, B29) TSAT-C6-Di-sub- TSAT-C6 822 AEBM 385 8301 4.0
AEBM-2 (A1, B29) TSPE-Di-sub- TSPE 813 AEM 223 7852 6.0 AEM-3 (A1,
B29) TSPE-Di-sub- TSPE 813 AEBM 385 8824 6.0 AEBM-3 (A1, B29)
TSPE-Di-sub- TSPE 813 AETM 547 9796 6.0 AETM-3 (A1, B29)
Example 77
Insulin Conjugation with Multivalent Activated Esters in Organic
Solvent (Drug Added First) to Give A1,B1-Substituted Insulin
Conjugates
[0694] Step 1
[0695] A framework containing N-terminal activated esters is
dissolved at 147 mM in 2.5 ml of anhydrous DMSO followed by the
addition of 1.0 mL (excess) of triethylamine (TEA). The solution is
stirred rapidly for 10 minutes at room temperature. In another
vial, a 272 mM solution of ligand is prepared in an appropriate
volume of dry DMSO. Once dissolved, enough solution is added to
provide a number of reactive equivalents equal to three times the
number of initial activated ester groups, N, minus one. For
example, if there are N=3 initial activated ester groups per
framework, then (3.times.(3-1).times.60 mM/370 mM)=0.973 ml of
ligand solution are added. If there are N=4 initial activated ester
groups per framework, then (3.times.(4-1).times.60 mM/370 mM)=1.46
ml of ligand solution are added, and so on. After the ligand
solution is added, the solution is stirred for one more hour at
room temperature to ensure complete reaction.
[0696] Step 2
[0697] An insulin containing three reactive amine groups each with
a distinguishable pKa (e.g., in the case of wild-type insulin, pKa
Gly.sup.A1=8.0, Phe.sup.B1=6.7, Lys.sup..epsilon.B29=11.2; see Mei
et al., Pharm. Res. 16:1680-1686, 1999) that has been previously
mono-protected at the highest pKa amine group (e.g., Lys.sup.B29)
with a monofunctional protecting group-activated ester (e.g.,
BOC--NHS) is dissolved in 1.5 mL of DMSO at a concentration of 17.2
mM. Once the B29-BOC-insulin is dissolved, small aliquots of the
framework-ligand solution are added to the B29-BOC-insulin
solution. The reaction is monitored by reverse-phase HPLC. Aliquots
of framework-ligand solution are added until the HPLC chromatogram
shows that substantially all of the B29-BOC-insulin has been
reacted and that a substantial portion of the reaction mixture has
been converted to a small portion of mono-reacted
B29-BOC-insulin/framework/ligand conjugate and the majority product
is di-reacted B29-BOC-insulin/framework/ligand conjugate. Typically
the framework-ligand construct will be more hydrophilic than the
B29-BOC-insulin, causing the mono-conjugated and di-conjugated
B29-BOC-insulin-conjugates to elute at HPLC retention times shorter
than that of the unmodified B29-BOC-insulin. Likewise, the HPLC
peak for the desired product, the disubstituted
B29-BOC-insulin-conjugate, will appear at a retention time that is
shorter than that of the mono-substituted
B29-BOC-insulin-conjugate.
[0698] Step 3
[0699] Once the B29-BOC-insulin has been sufficiently reacted with
ligand-containing framework as described in Step 2, the solution is
then superdiluted by 10.times. into a 20 mM pH 5.0 HEPES buffered
saline solution containing 0.150 M NaCl followed by pH adjustment
with dilute HCl to a final pH of 8.0. The aqueous solution is first
purified by size exclusion using an appropriate solid phase for the
desired separation of conjugated and unconjugated materials. The
solution passing through the column void volume is then
concentrated using an appropriately sized ultrafiltration membrane
to approximately 10 ml. This solution is further purified to obtain
the desired product using preparative reverse phase HPLC on a
Waters C8, 7 um, 19.times.150 mm column. Buffer A is deionized
water containing 0.1% TFA and Buffer B is acetonitrile containing
0.1% TFA. Before purification, the column is equilibrated at 15
ml/minutes with a 80% A/20% B mobile phase using a Waters
DeltraPrep 600 system. Approximately 5 ml of the crude solution is
injected onto the column over the course of 2 minutes at a flow
rate of 15 ml/minutes after which a linear gradient is employed
from 80% A/20% B to 75% A/25% B over the next 5 minutes followed by
a slower linear gradient from 75% A/25% B to 62% A/38% B over the
next 22 minutes. The retention time of the desired peak will vary
depending on the insulin, framework, and ligand used. Once
collected, the solution is rotovapped to remove acetonitrile and
lyophilized to obtain pure conjugate.
[0700] Step 4
[0701] In all cases the protecting group is then removed from the
conjugate. In cases where a BOC protecting group is used in Step 1,
the BOC groups are removed by dissolving the lyophilized powder
obtained according to Step 3 in 90% TFA/10% anisole for one hour at
4 C followed by 10.times. superdilution in 25 mM HEPES pH 8.2
buffer containing 0.150M NaCl. (If a protecting group other than
BOC is present on the amine-bearing drug, then the appropriate
deprotection conditions are employed instead of TFA/anisole. A
listing of protection agents and deprotection conditions may be
found in Protecting Groups in Organic Synthesis, T. W. Greene and
P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, as
described in the Definitions section.) The pH was adjusted to
between 7.0 and 8.0 using NaOH solution after which the material is
passed through a Biogel P2 column to remove anisole, BOC and other
low MW byproducts of deprotection, as well as any other
contaminating salts. The deprotected, purified aqueous conjugate
solution was then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to approximately 66 U of insulin/ml
(based on A280 measurements) and stored at 4 C until needed. The
identity of the final conjugate is verified by LC-MS (HT
Laboratories, San Diego, Calif.). The A1, B1 sites of conjugation
are confirmed by N-terminal sequencing (Western Analytical, St.
Louis, Mo.), which reveals essentially no Phe-B1-chain terminus
present and no Gly-A1-chain terminus present due to the
substitution at both termini with the ligand-containing
framework.
[0702] One of ordinary skill in the art will appreciate that other
amine-functionalized drugs can be conjugated to ligand-containing
frameworks using analogous procedures to that described in Example
77. One of ordinary skill in the art will also appreciate that
Example 77 is relevant not only to wild-type insulin, but also to
insulin mutants as described herein.
[0703] Conjugate II-5 was prepared according to the procedure in
Example 77 using BOC--NHS as the protecting reagent.
TABLE-US-00032 Frame- AE- MW Frame- work sugar Purity (LC- Sugar/
Conjugate work MW Ligand MW (HPLC) MS) Insulin II-5: TSAT- 822 AETM
547 97% 8949 4.0 TSAT-C6- C6 Di-sub- AETM-2 (A1, B1)
[0704] The following insulin-conjugates can be prepared according
to the procedure in Example 77.
TABLE-US-00033 MW (LC- Sugar/ Frame- Framework AE-sugar MS) Insulin
Identity work MW Ligand MW (expected) (expected) DSS-Di-sub-AEM-
DSS 368 AEM 223 6531 2.0 1 (A1, B1) DSS-Di-sub- DSS 368 AEBM 385
6855 2.0 AEBM-1 (A1, B1) DSS-Di-sub- DSS 368 AETM 547 7179 2.0
AETM-1 (A1, B1) TSAT-C6-Di-sub- TSAT-C6 822 AEM 223 7653 4.0 AEM-2
(A1, B1) TSAT-C6-Di-sub- TSAT-C6 822 AEBM 385 8301 4.0 AEBM-2 (A1,
B1) TSAT-C6-Di-sub- TSAT-C6 822 AETM 547 8949 4.0 AETM-2 (A1, B1)
TSPE-Di-sub- TSPE 813 AEM 223 7852 6.0 AEM-3 (A1, B1) TSPE-Di-sub-
TSPE 813 AEBM 385 8824 6.0 AEBM-3 (A1, B1) TSPE-Di-sub- TSPE 813
AETM 547 9796 6.0 AETM-3 (A1, B1)
Example 78
Insulin Conjugation with Multivalent Activated Esters in Organic
Solvent (Drug Added First) to Give B1,B29-Substituted Insulin
Conjugates
[0705] Step 1
[0706] A framework containing N-terminal activated esters is
dissolved at 147 mM in 2.5 ml of anhydrous DMSO. No base is added,
in contrast with previous examples. The solution is stirred rapidly
for 10 minutes at room temperature. In another vial, a 272 mM
solution of ligand is prepared in an appropriate volume of dry
DMSO. Once dissolved, enough solution is added to provide a number
of reactive equivalents equal to three times the number of initial
activated ester groups, N, minus one. For example, if there are N=3
initial activated ester groups per framework, then
(3.times.(3-1).times.60 mM/370 mM)=0.973 ml of ligand solution are
added. If there are N=4 initial activated ester groups per
framework, then (3.times.(4-1).times.60 mM/370 mM)=1.46 ml of
ligand solution are added, and so on. After the ligand solution is
added, the solution is stirred for one more hour at room
temperature to ensure complete reaction.
[0707] Step 2
[0708] An insulin containing three reactive amine groups each with
a distinguishable pKa (e.g., in the case of wild-type insulin, pKa
Gly.sup.A1=8.0, Phe.sup.B1=6.7, Lys.sup..epsilon.B29=11.2; see Mei
et al., Pharm. Res. 16:1680-1686, 1999) that has been previously
mono-protected at the amine group with the intermediate pKa (e.g.,
Gly.sup.A1 for wild-type insulin) with a monofunctional protecting
group-activated ester (e.g., BOC--NHS) is dissolved in 1.5 mL of
DMSO at a concentration of 17.2 mM. (A1-BOC-insulin can be prepared
using the procedure in Example 8 but reacting with fewer
equivalents of the BOC reagent in order to yield a distribution of
A1,B29-diBOC-insulin, A1-BOC-insulin, and B29-BOC-insulin products.
A1-BOC-insulin can be isolated by RP-HPLC and confirmed by
N-terminal sequencing.) Once the A1-BOC-insulin is dissolved, small
aliquots of the framework-ligand solution are added to the
A1-BOC-insulin solution. The reaction is monitored by reverse-phase
HPLC. Aliquots of framework-ligand solution are added until the
HPLC chromatogram shows that substantially all of the unmodified
A1-BOC-insulin has been reacted and that a substantial portion of
the reaction mixture has been converted to a small portion of
mono-conjugated A1-BOC-insulin/framework/ligand conjugate and the
majority product is di-conjugated A1-BOC-insulin/framework/ligand
conjugate. Typically the framework-ligand construct will be more
hydrophilic than the A1-BOC-insulin, causing the mono and
di-substituted A1-BOC-insulin-conjugates to elute at HPLC retention
times shorter than that of the unmodified A1-BOC-insulin. Likewise,
the HPLC peak of the desired product, the di-substituted
A1-BOC-insulin-conjugate, will appear at a retention time that is
shorter than that of the mono-substituted
A1-BOC-insulin-conjugate.
[0709] Step 3
[0710] Once the A1-BOC-insulin has been sufficiently reacted with
framework ligand as described in Step 2, the solution is then
superdiluted by 10.times. into a 20 mM pH 5.0 HEPES buffered saline
solution containing 0.150 M NaCl followed by pH adjustment with
dilute HCl to a final pH of 8.0. The aqueous solution is first
purified by size exclusion using an appropriate solid phase for the
desired separation of conjugated and unconjugated materials. The
solution passing through the column void volume is then
concentrated using an appropriately sized ultrafiltration membrane
to approximately 10 ml. This solution is further purified to obtain
the desired product using preparative reverse phase HPLC on a
Waters C8, 7 um, 19.times.150 mm column. Buffer A is deionized
water containing 0.1% TFA and Buffer B is acetonitrile containing
0.1% TFA. Before purification, the column is equilibrated at 15
ml/minutes with a 80% A/20% B mobile phase using a Waters
DeltraPrep 600 system. Approximately 5 ml of the crude solution is
injected onto the column over the course of 2 minutes at a flow
rate of 15 ml/minutes after which a linear gradient is employed
from 80% A/20% B to 75% A/25% B over the next 5 minutes followed by
a slower linear gradient from 75% A/25% B to 62% A/38% B over the
next 22 minutes. The retention time of the desired peak will vary
depending on the drug, framework, and ligand used. Once collected,
the solution is rotovapped to remove acetonitrile and lyophilized
to obtain pure conjugate.
[0711] Step 4
[0712] In all cases the protecting group is then removed from the
conjugate. In cases where a BOC protecting group is used in Step 1,
the BOC groups are removed by dissolving the lyophilized powder
obtained according to Step 3 in 90% TFA/10% anisole for one hour at
4 C. If a protecting group other than BOC is present on the
amine-bearing drug, then the appropriate deprotection conditions
are employed instead of TFA/anisole. A listing of protection agents
and deprotection conditions may be found in Protecting Groups in
Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd
edition, John Wiley & Sons, 1999, as described in the
Definitions section. The deprotection step is followed by 10.times.
superdilution in 25 mM HEPES pH 8.2 buffer containing 0.150M NaCl.
The pH is adjusted to between 7.0 and 8.0 using NaOH solution after
which the material is passed through a Biogel P2 column to remove
anisole, BOC and other low MW byproducts of deprotection, as well
as any other contaminating salts. The deprotected, purified aqueous
conjugate solution is then concentrated using Amicon 3K membranes
(Millipore, Billerica, Mass.) to approximately 66 U of insulin/ml
(based on A280 measurements) and stored at 4 C until needed. The
identity of the final conjugate is verified by LC-MS (HT
Laboratories, San Diego, Calif.). The B1 site of conjugation is
confirmed by N-terminal sequencing (Western Analytical, St. Louis,
Mo.), which reveals >95% of the Gly-A1-chain terminus present
and <5% of the Phe-B1-chain terminus present due to the
substitution of Phe-B1 with the ligand-containing framework.
[0713] One of ordinary skill in the art will appreciate that other
amine-functionalized drugs can be conjugated to ligand-containing
frameworks using analogous procedures to that described in Example
78. One of ordinary skill in the art will also appreciate that
Example 78 is relevant not only to wild-type insulin, but also to
insulin mutants as described herein.
[0714] The following insulin-conjugates can be prepared according
to the procedure in Example 78.
TABLE-US-00034 Frame- AE- MW Sugar/ Frame- work sugar (LC-MS)
Insulin Conjugate work MW Ligand MW (expected) (expected) II-7:
DSS-Di-sub-AETM-1 DSS 368 AETM 547 7179 2.0 (B1, B29) II-6:
TSAT-C6-Di-sub- TSAT- 822 AETM 547 8949 4.0 AETM-2 (B1, B29) C6
DSS-Di-sub-AEM-1 (B1, DSS 368 AEM 223 6531 2.0 B29)
DSS-Di-sub-AEBM-1 (B1, DSS 368 AEBM 385 6855 2.0 B29)
TSAT-C6-Di-sub-AEM-2 TSAT- 822 AEM 223 7653 4.0 (B1, B29) C6
TSAT-C6-Di-sub-AEBM-2 TSAT- 822 AEBM 385 8301 4.0 (B1, B29) C6
TSPE-Di-sub-AEM-3 (B1, TSPE 813 AEM 223 7852 6.0 B29)
TSPE-Di-sub-AEBM-3 (B1, TSPE 813 AEBM 385 8824 6.0 B29)
TSPE-Di-sub-AETM-3 (B1, TSPE 813 AETM 547 9796 6.0 B29)
Example 79
In Vivo Half Life/Elimination Rate Comparison
[0715] In order to determine the rate at which the I-6 conjugate
was cleared from serum in vivo in the presence or absence of
inhibitory sugars such as glucose or a-MM, the following experiment
was conducted. In each case the soluble conjugate (or RHI as a
control) was dosed at 0.4 mg conjugate/kg body weight into dual
jugular vein cannulated male Sprague-Dawley rats (Taconic, JV/JV,
350-400 g, n=3).
[0716] To determine the elimination rate in the presence of
elevated glucose levels, one hour before the start of the
experiment one rat cannula was connected to a syringe infusion pump
containing a sterile 50% w/v glucose solution. The pump infusion
rate was adjusted by the experimenter to ensure that the blood
glucose levels in the animal remained above 300 mg/dL at all times
during the experiment. Blood glucose was measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In a typical experiment, it was
found that the infusion pump rate required to keep the animals
above 300 mg/dL was typically greater than 85 uL/min. A blood
sample was taken at t=0 min, after which a sterile conjugate
solution or control insulin was injected intravenously via the
second rat cannula, followed immediately by a chase solution of
heparin-saline to ensure that all of the conjugate dose was
administered into the animal. After an additional flush of the
cannula line with heparin-saline, the second cannula was used to
collect blood samples at t=1, 2, 4, 8, 15, 30, 60, 90, 120, and 180
minutes post-dose.
[0717] To determine the elimination rate in the presence of a-MM,
one hour before the start of the experiment one rat cannula was
connected to a syringe infusion pump containing a sterile 25% w/v
a-MM solution. The pump infusion rate was adjusted by the
experimenter, but was typically set at 85 uL/min. A blood sample
was taken at t=0 min, after which a sterile conjugate solution or
control insulin was injected intravenously via the second rat
cannula, followed immediately by a chase solution of heparin-saline
to ensure that all of the conjugate dose was administered into the
animal. After an additional flush of the cannula line with
heparin-saline, the second cannula was used to collect blood
samples at t=1, 2, 4, 8, 15, 30, 60, 90, 120, and 180 minutes
post-dose.
[0718] Throughout the experiment, blood glucose was measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). Blood from each timepoint was
centrifuged at 4 C to collect the serum, and serum insulin or serum
conjugate concentrations were subsequently measured with a
commercially available ELISA kit (Iso-Insulin ELISA, Mercodia,
Uppsala, Sweden). Insulin or conjugate serum concentration vs. time
data was best fit with the sum of two independent decaying
exponentials (C(t)=a exp(-k.sub.at)+b exp(-k.sub.bt)) according to
the two-compartment model, where t1/2(a)=(ln 2)/k.sub.a and
t1/2(b)=(ln 2)/k.sub.b. Results are shown in FIG. 46. The left
panel demonstrates the significantly higher (>5.times.)
elimination rate for the I-6 conjugate versus RHI in the absence of
a-MM or glucose. The right panel shows that the elimination rate
decreases somewhat (.about.50%) in the presence of glucose (G400
infusion) and quite substantially (.about.400%) in the presence of
a-MM (a-MM infusion).
Example 80
Glucose-Responsive PK for I-6 i.v. Infusion
[0719] In this example, the i.v. elimination rate experiment
described in Example 79 was modified from a single i.v. bolus of
0.4 mg conjugate/kg body weight to a continuous i.v. infusion. The
goal of the experiment was to maintain a constant input rate of
conjugate (or RHI as a control) for six hours with an i.p.
injection of glucose administered at the four hour time point to
determine the resulting effect on serum conjugate (or RHI)
concentration. Dual jugular vein cannulated male Sprague-Dawley
rats (Taconic, JV/JV, 350-400 g, n=3) were used in each experiment
such that one jugular vein line was used for conjugate or RHI
infusion and the other for blood collection.
[0720] For RHI, a 50 mU/ml solution was sterile filtered through a
0.2 um filtration membrane and infused at 0.07 ml/min to provide a
constant input rate of 3.5 mU/min for the entire six hour
experiment. A blood sample was taken at t=0 min, after which the
constant i.v. infusion was initiated. The second cannula was used
to collect blood samples at t=30, 60, 120, 180 and 240 min. At
t=240 min, a 4 g/kg dose of glucose was administered via i.p.
injection followed by blood collection at t=255, 270, 300, 330 and
360 min.
[0721] For the I-6 conjugate, a 150 mU/ml solution was sterile
filtered through a 0.2 m filtration membrane and infused at 0.10
ml/min to provide a constant input rate of 15 mU/min for the entire
six hour experiment. A blood sample was taken at t=0 min, after
which the constant i.v. infusion was initiated. The second cannula
was used to collect blood samples at t=30, 60, 120, 180 and 240
min. At t=240 min, a 1, 2, or 4 g/kg dose of glucose was
administered via i.p. injection followed by blood collection at
t=255, 270, 300, 330 and 360 min.
[0722] Throughout the experiments, blood glucose was measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). Blood from each timepoint was
centrifuged at 4 C to collect the serum, and serum insulin or serum
conjugate concentrations were subsequently measured with a
commercially available ELISA kit (Iso-Insulin ELISA, Mercodia,
Uppsala, Sweden).
[0723] The first two panels of FIG. 47 compare the blood glucose
and serum insulin/conjugate concentration profiles for a 3.5 mU/min
infusion of RHI and 15 mU/min infusion of I-6 before and after a 4
g/kg i.p. glucose injection. RHI infusion causes significant
hypoglycemia prior to glucose injection compared to the I-6
infusion. Following the i.p. glucose injection, the measured serum
insulin concentration of I-6 immediately increases by over 300% as
the blood glucose concentration increases followed by a rapid
return to baseline levels as the glucose concentration decreases.
On the other hand, there is no significant change in measured serum
insulin concentration for RHI after i.p. glucose injection under
the same experimental conditions. The next three panels of FIG. 47
show that the extent to which the measured insulin concentration
increases during i.p. glucose injection is directly related to the
dose of glucose administered and the resulting blood glucose
levels. For example, only a 50% peak to baseline change in serum
insulin concentration is observed for the 1 g/kg glucose injection
versus the 300% peak to baseline change observed for the 4 g/kg
dose.
Example 81
In Vivo Elimination Rate for Insulin-Conjugates with and without
Sugar
[0724] In order to determine the rate at which the I-9 conjugate
was cleared from serum in vivo in the presence or absence of
inhibitory sugars such as glucose or a-MM, the following experiment
was conducted. In each case the soluble conjugate (or RHI as a
control) was dosed at 0.4 mg conjugate/kg body weight into dual
jugular vein cannulated male Sprague-Dawley rats (Taconic, JV/JV,
350-400 g, n=3).
[0725] To determine the elimination rate in the presence of
elevated glucose levels, one hour before the start of the
experiment one rat cannula was connected to a syringe infusion pump
containing a sterile 50% w/v glucose solution. The pump infusion
rate was adjusted by the experimenter to ensure that the blood
glucose levels in the animal remained above 300 mg/dL at all times
during the experiment. Blood glucose was measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). In a typical experiment, it was
found that the infusion pump rate required to keep the animals
above 300 mg/dL was typically greater than 85 .mu.L/min. A blood
sample was taken at t=0 min, after which a sterile conjugate
solution or control insulin was injected intravenously via the
second rat cannula, followed immediately by a chase solution of
heparin-saline to ensure that all of the conjugate dose was
administered into the animal. After an additional flush of the
cannula line with heparin-saline, the second cannula was used to
collect blood samples at t=1, 2, 4, 8, 15, 30, 60, 90, 120, and 180
minutes post-dose.
[0726] To determine the elimination rate in the presence of a-MM,
one hour before the start of the experiment one rat cannula was
connected to a syringe infusion pump containing a sterile 25% w/v
a-MM solution. The pump infusion rate was adjusted by the
experimenter, but was typically set at 85 uL/min. A blood sample
was taken at t=0 min, after which a sterile conjugate solution or
control insulin was injected intravenously via the second rat
cannula, followed immediately by a chase solution of heparin-saline
to ensure that all of the conjugate dose was administered into the
animal. After an additional flush of the cannula line with
heparin-saline, the second cannula was used to collect blood
samples at t=1, 2, 4, 8, 15, 30, 60, 90, 120, and 180 minutes
post-dose.
[0727] Throughout the experiment, blood glucose was measured using
commercially available test strips (Precision Xtra, Abbott
Laboratories, Abbott Park, Ill.). Blood from each timepoint was
centrifuged at 4 C to collect the serum, and serum insulin or serum
conjugate concentrations were subsequently measured with a
commercially available ELISA kit (Iso-Insulin ELISA, Mercodia,
Uppsala, Sweden). Insulin or conjugate serum concentration vs. time
data was best fit with the sum of two independent decaying
exponentials (C(t)=a exp(-k.sub.at)+b exp(-k.sub.bt)) according to
the two-compartment model, where t1/2(a)=(ln 2)/k.sub.a and
t1/2(b)=(ln 2)/k.sub.b. The first panel of FIG. 48 shows that the
elimination rate of unmodified insulin is not affected in the
presence of sugars (glucose G400 or a-MM). For the sake of
comparison, the last panel of FIG. 48 showing conjugate I-6 with
sugar infusion is replotted from the Example 79 results. The middle
panel of FIG. 48 showing conjugate I-9 with sugar infusion shows a
much more pronounced decrease in elimination rate (.about.350% vs.
.about.50%) in the presence of glucose (G400 infusion) versus the
I-6 conjugate. Conjugate I-9 also demonstrates a more significant
decrease in elimination rate (.about.700% vs. .about.400%) in the
presence of a-MM (a-MM infusion) versus the I-6 conjugate.
Example 82
Mechanism Verification and Glucose-Responsive Performance in
Miniature Swine
[0728] In order to determine whether the glucose-responsive
insulin-conjugate results that are described above could be
extended to other species beyond rats, we focused on exploring the
sugar-dependent in vivo elimination rate in human-representative,
non-diabetic, male miniature swine (Yucatan strain), also called
"minipigs" herein. A subset of insulin-conjugates summarized in
FIG. 49 were tested to initially determine the effects of sugar
affinity and multivalency on sugar-dependent elimination rates. The
conjugates are shown in FIG. 45 as I-7, 1-6, I-11, and II-2. All
conjugates used in this study were synthesized according to the
general methods described in Example 20. To produce the
A1,B29-disubstituted AETM-2 insulin-conjugate II-2, approximately
ten times the amount of multivalent active ester framework and AETM
ligand per insulin molecule was used compared to the
B29-monosubstituted AETM-2 insulin-conjugate (I-6) synthesis.
[0729] In each experiment, the insulin-conjugate was dosed i.v. at
0.1 U/kg into non-diabetic, dual-vascular access ported minipigs
and blood was collected at frequent time intervals post-injection.
To determine the serum elimination rate in the presence of glucose,
a sterile 50% w/v glucose solution was infused i.v. into one port
using a syringe pump one hour prior to administering the
insulin-conjugate, and the rate was adjusted throughout the entire
experiment to ensure that the blood glucose levels in the animal
remained at or near 400 mg/dl (typically 80-150 ml/h). To determine
the serum elimination rate in the presence of a-MM, the glucose
solution was replaced with a sterile 25% w/v a-MM solution and the
pump infusion rate held constant throughout the experiment at 80
ml/h. In each case, the resulting insulin-conjugate concentration
vs. time data was fit with the sum of two independent decaying
exponentials (C(t)=.alpha. exp(-k.sub..alpha.t)+.beta.
exp(-k.sub..beta.t)) according to the two-compartment model.
[0730] At 400 mg/dl the high levels of endogenous glucose-induced
porcine insulin crossreacted with our insulin-conjugate
immunoassay. As such, the PK results from the glucose infusion
experiments required subtraction of values obtained from a porcine
insulin-only assay leading to a particularly "noisy" set of data.
Since a-MM does not induce endogenous porcine insulin secretion,
data from the a-MM infusion studies were used as our primary
indicator of sugar-responsive changes in insulin-conjugate
half-life. Interestingly, in the pigs, the AETM-2 insulin-conjugate
(I-6) showed only a modest 1.7.times. increase in t.sub.1/2 in the
presence of a-MM compared to a 4.0.times. increase in the rats
(FIG. 56). However, in the pigs, the A1,B29-di-substituted AETM-2
insulin-conjugate (II-2) demonstrated an almost 10-fold increase in
t.sub.1/2 in the presence of a-MM (FIGS. 50 and 51). Tabular
results for other conjugates are shown in FIG. 57.
[0731] The area over the glucose lowering curve for the i.v. dose
of di-substituted AETM-2 insulin-conjugate (II-2) in the presence
of a-MM was approximately 2.6.times. higher than in the absence of
sugar (FIG. 52). FIG. 59 compares the differences in bioactivity
between RHI, I-6, and II-2 (di-substituted AETM-2
insulin-conjugate), and II-3. All three of the insulin-conjugates
contain the high affinity AETM sugar ligands. In this selected set
of insulin-conjugates, there is no correlation between
sugar-dependent half-life and bioactivity.
[0732] Conjugate II-2 was injected sub-Q as a soluble solution at
doses of 0.25, 0.50, and 1.00 U/kg in both non-diabetic,
normoglycemic and alloxan-diabetic, hyperglycemic minipigs to
determine its ability to lower glucose in diabetics without causing
hypoglycemia in non-diabetic animals. The insulin-conjugate
demonstrated a significant dose-dependent reduction in blood
glucose levels in the diabetics with absolutely no hypoglycemia or
signs of glucose-lowering in the non-diabetics (FIG. 53). In
comparison, RHI injected at 0.063 and 0.125 U/kg caused significant
glucose-lowering in the diabetic animals with noticeable
hypoglycemia and significant glucose-lowering and hypoglycemia in
the non-diabetic animals (FIG. 54). Based on these preliminary
results, a single injection of approximately 0.5 U/kg of soluble
insulin-conjugate II-2 provided hypoglycemia-free glucose control
for 6-8 hours in diabetic minipigs. Serum elimination rates of
sub-Q injected II-2 were determined in diabetic and normal minipigs
(FIG. 58). Similar PK profiles were observed between diabetics and
normals for all doses.
[0733] Taken together, these early results demonstrate that an
endogenous lectin-based mechanism exists in the minipigs that can
be exploited through selection of sugar affinity and multivalency.
It appears that insulin-conjugates with higher affinities and
multivalencies provide improved hypoglycemia-free glycemic control
in minipigs as compared to rats.
Example 83
Optimization Studies in Miniature Swine
[0734] Based on the stark difference in performance of II-2 versus
the other conjugates in FIG. 49, it is desirable to separate and
quantify the effect of insulin conjugation site (A1 vs. B29) from
the effects of sugar affinity and valency. Using similar
conjugation techniques as were used to produce the
insulin-conjugates in FIG. 49, we therefore have synthesized the
array of insulin-conjugates listed in FIG. 55 (shown in FIG. 45 as
I-12, I-13, I-14, I-15, II-1, II-3, and II-4), as well as the
control compounds shown in FIG. 60. All the di-substituted
conjugates to be used in this study were synthesized according to
the general methods described in Example 20. To produce the A1,
B29-disubstituted insulin-conjugates, approximately ten times the
amount of multivalent active ester framework and sugar affinity
ligand per insulin molecule was used compared to the
B29-monosubstituted insulin-conjugate synthesis. The A1-only
substituted materials were also prepared according to the general
methods described Example 20. However, in this case B29-mono-BOC
protected insulin isolated from a BOC protection synthesis
described in Example 8 using half the number of equivalents of
di-tert-butyl-dicarbonate was used. Once purified, the BOC groups
were removed from the conjugate using a TFA/anisole method
described in Example 12.
[0735] In general, the sugar-responsive half-lives and
glucose-lowering effects of each of these insulin-conjugates were
determined as follows. As described above, each insulin-conjugate
was dosed i.v. at 0.1 U/kg into non-diabetic, dual-vascular access
ported minipigs and blood was collected at frequent time intervals
post-injection. To determine the serum elimination rate in the
presence of a-MM, a sterile 25% w/v a-MM solution was infused i.v.
into one port using a syringe pump (80 ml/h) one hour prior to
administering the insulin-conjugate, and the rate was held constant
throughout the entire experiment. In each case, the resulting
insulin-conjugate concentration vs. time data was fit with the sum
of two independent decaying exponentials (C(t)=.alpha.
exp(-k.sub..alpha.t)+.beta. exp(-k.sub..beta.t)) according to the
two-compartment model. Comparison of the 3-phase elimination rates
with and without a-MM infusion was used to identify suitable
conjugates.
[0736] FIG. 61 shows a comparison of glucose levels when various
insulin-conjugates were injected i.v. into minipigs. Conjugate I-12
(A1 substitution with AETM-2) was slightly more effective in
lowering glucose than conjugate I-6 (B29 substitution with AETM-2).
Substitution of conjugate I-6 at the A1 position with polyethylene
oxide to give conjugate C3 reduced overall bioactivity but did not
increase the a-MM induced bioactivity. Substitution of conjugate
I-6 at the A1 position with another TSAT-C6-AETM-2 scaffold to give
conjugate II-2 reduced overall bioactivity but increased the a-MM
induced bioactivity.
[0737] FIG. 62 shows a comparison of glucose levels when various
A1,B29 di-substituted insulin-conjugates were injected i.v. into
minipigs. A1,B29 di-substitution of insulin with acetyl (conjugate
C7) or PEO (conjugate C4) groups did not substantially reduce
overall bioactivity.
[0738] Furthermore, the C7 and C4 conjugates did not display
distinguishable a-MM-induced bioactivity effects. Conjugate II-3
had substantially reduced bioactivity versus conjugates C7 and C4,
but its bioactivity was virtually restored in the presence of
a-MM.
Other Embodiments
[0739] 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.
Sequence CWU 1
1
2121PRTHomo sapiens 1Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys
Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 230PRTHomo
sapiens 2Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala
Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro
Lys Thr 20 25 30
* * * * *