Note: Descriptions are shown in the official language in which they were submitted.
CA 02864366 2015-07-07
TITLE OF THE INVENTION
Lipid Construct Comprising An Amphipathic Lipid, Cholesterol, Dicetyl
Phosphate, and a
Hepatocyte Receptor Binding Molecule
BACKGROUND OP THE INVENTION
Diabetes is a disorder affecting large numbers of people worldwide.
Management approaches to control Type I and Type II diabetes aim primarily at
normalizing blood glucose levels to prevent short- and long-term
complications.
Many patients require multiple daily injections of an insulin to control their
diabetes.
Several insulin products have been produced that control blood sugar levels
over
differing time intervals. Several products combine various forms of insulin in
an
attempt to provide a preparation which controls glucose levels over a wider
period of
time.
Previous attempts to normalize blood glucose levels in Type I and
Type II diabetic patients have centered on the subcutaneous administration of
insulin
in various time-released formulations, such as ultralente and humulin NPH
insulin
pharmaceutical products. These formulations have attempted to delay and
subsequently control the bio-distribution of insulin by regulating release of
insulin to
peripheral tissues with the expectation that sustained management of insulin
bio-
availability will lead to better glucose control. Glargine insulin is a long-
acting form
of insulin in which insulin is released from the subcutaneous tissue around
the site of
injection into the bloodstream at a relatively constant rate throughout the
day.
Although glargine insulin is released at a constant rate throughout the day,
the
released insulin reaches a wide range of systems within the body rather than
being
delivered to targeted areas of the body. What is needed is a composition of
insulin
where a portion of the dosed insulin is released at a relatively constant rate
throughout
the day and another portion of insulin that is time released from the site of
administration and targeted for delivery to the liver to better control
glucose
production.
There is, therefore, an unmet need in the art for compositions and
methods of managing blood glucose levels in Type I and Type II diabetic
patients.
The present invention meets these needs by providing a long-acting composition
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comprising insulin that is free and insulin that is associated with a lipid
construct
targeted for delivery to hepatocytes. A lipid construct is a
lipid/phospholipid particle
in which individual lipid molecules cooperatively interact to create a bipolar
lipid
membrane which encloses and isolates a portion of the medium in which it was
formed. The lipid construct releases free insulin over time as well as targets
a portion
of the remaining insulin to the hepatocytes in the liver to better control
glucose
storage and production.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention includes a lipid construct
comprising an amphipathic lipid and an extended amphipathic lipid, wherein the
extended amphipathic lipid comprises proximal, medial and distal moieties,
wherein
the proximal moiety connects the extended amphipathic lipid to the construct,
the
distal moiety targets the construct to a receptor displayed by a hepatocyte,
and the
medial moiety connects the proximal and distal moieties.
In another aspect, the lipid construct further comprises at least one
insulin.
In still another aspect, the at least one insulin is selected from the
group consisting of insulin lispro, insulin aspart, regular insulin, insulin
glargine,
insulin zinc, human insulin zinc extended, isophane insulin, human buffered
regular
insulin, insulin glulisine, recombinant human regular insulin, recombinant
human
insulin isophane, premixed combinations of any of the aforementioned insulins,
a
derivative thereof, and a combination of any of the aforementioned insulins.
In another aspect, the lipid construct further comprises an insoluble
form of at least one insulin associated with the lipid construct.
In yet another aspect, the amphipathic lipid comprises at least one
lipids selected from the group consisting of 1,2-diste,aroyl-sn-glycero-3-
phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipalmitoyl-sn-glycerol-[3-
phospho-rac-(1-glycero)], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl), derivatives
thereof; and
mixtures of any of the foregoing compounds.
In one aspect, the proximal moiety of the extended amphipathic lipid
comprises at least one, but not more than two, long acyl hydrocarbon chains
bound to
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a backbone, wherein each hydrocarbon chain is independently selected from the
group
consisting of a saturated hydrocarbon chain and an unsaturated hydrocarbon
chain.
In another aspect, the backbone comprises glycerol.
In still another aspect, the distal moiety of the extended amphipathic
lipid comprises at least one member selected from the group consisting of
biotin, a
biotin derivative, iminobiotin, an iminobiotin derivative, biocytin, a
biocytin
derivative, iminobiocytin, an iminobiocytin derivative and a hepatocyte
specific
molecule that binds to a receptor in a hepatocyte.
In yet another aspect, the extended amphipathic lipid is selected from
the group consisting of N-hydroxysuccinimide (NHS) biotin; sulfo-NHS-biotin; N-
hydroxysuccinimide long chain biotin; sulfo-N-hydroxysuccinimide long chain
biotin;
D-biotin; biocytin; sulfo-N-hydroxysuccinimide-S-S-biotin; biotin-BMCC; biotin-
HPDP; iodoacetyl-LC-biotin; biotin-hydrazide; biotin-LC-hydrazide; biocytin
hydrazide; biotin cadaverine; carboxybiotin; photobiotin; p-aminobenzoyl
biocytin
trifluoroacetate; p-diazobenzoyl biocytin; biotin DHPE; biotin-X-DHPE; 12-
((biotinyl)amino)dodecanoic acid; 12-((biotinyl)amino)dodecanoic acid
succinimidyl
ester; S-biotinyl homocysteine; biocytin-X; biocytin x-hydrazide;
biotinethylenediamine; biotin-XL; biotin-X-ethylenediamine; biotin-XX
hydrazide;
biotin-XX-SE; biotin-XX, SSE; biotin-X-cadaverine; a-(t-B0C)biocytin; N-
(biotiny1)-N'-(iodoacetyl) ethylenediamine; DNP-X-biocytin-X-SE; biotin-X-
hydrazide; norbiotinamine hydrochloride; 3-(N-maleimidylpropionyl)biocytin;
ARP;
biotin-l-sulfoxide; biotin methyl ester; biotin-maleimide; biotin-
poly(ethyleneglycol)amine; (-I-) biotin 4-amidobenzoic acid sodium salt;
Biotin 2-N-
acetylamino-2-deoxy-O-D-gluc,opyranoside; Biotin-a-D-N-acetylneuraminide;
Biotin-
a-L-fucoside; Biotin lacto-N-bioside; Biotin¨Lewis-A trisaccharide;
Biotin¨Lewis-Y
tetrasaccharide; Biotin-n-D-mannopyranoside; biotin 6-0-phospho-a-D-
mannopyranoside; and polychromium-poly(bis)-N-[2,6-(diisopropylphenyl)
carbamoyl methylimino] diacetic acid.
In one aspect, the medial moiety of the extended amphipathic lipid
comprises a thio-acetyl triglycine polymer or a derivative thereof, wherein
the
extended amphipathic lipid molecule extends outward from the surface of the
lipid
construct.
In another aspect, the lipid construct further comprises at least one
insulin associated with a water insoluble target molecule complex, wherein the
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complex comprises a plurality of linked individual units, the individual units
comprise: a bridging component selected from the group consisting of a
transition
element, an inner transition element, a neighbor element of the transition
element and
a mixture of any of the foregoing elements, and a complexing component,
provided
that when the transition element is chromium, a chromium target molecule
complex is
formed.
In yet another aspect, the lipid construct further comprises at least one
insulin that is not associated with the target molecule complex.
In a further aspect, the bridging component is chromium.
In one aspect, the complexing component comprises poly(bis)-[(N-
(2,6-diisopropylphenyl)carbamoyl methyl) iminodiacetic acid].
In another aspect, the distal component of the extended amphipathic
lipid comprises a non-polar derivatized benzene ring or a heterobicyclic ring
structure.
In still another aspect, the construct comprises a positive charge, a
negative charge or combinations thereof.
In one aspect, the extended amphipathic lipid comprises at least one
carbonyl moiety positioned at a distance about 13.5 angstroms or less from the
terminal end of the distal moiety.
In another aspect, the extended amphipathic lipid comprises at least
one carbamoyl moiety comprising a secondary amine.
In yet another aspect, the extended amphipathic lipid comprises
charged chromium in the medial position.
In a further aspect, the lipid construct further comprises cellulose
acetate hydrogen phthalate.
In yet another aspect, the lipid construct further comprises at least one
charged organic molecule bound to the insulin.
In one aspect, the charged organic molecule is selected from the group
consisting of protamines, derivatives of polylysine, highly basic amino acid
polymers,
poly (arg-pro-t1n)n in a mole ratio of 1:1:1, poly (DL-Ala-poly-L-lys)n in a
mole ratio
of 6:1, histones, sugar polymers that contain a positive charge contributed by
a
primary amino group, polynucleotides with primary amino groups, carboxylated
polymers and polymeric amino acids, fragments of proteins that contain large
amounts of amino acid residues with carboxyl (C00¨) or sulfhydral (S¨)
functional
groups, derivative of proteins with negatively charged terminal acidic
carboxyl
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groups, acidic polymers, sugar polymers containing negatively charged carboxyl
groups, derivative thereof and combinations of the aforemention compounds.
In another aspect, a method of manufacturing a lipid construct
comprising an amphipathic lipid and an extended amphipathic lipid, wherein the
extended amphipathic lipid comprises proximal, medial and distal moieties,
wherein
the proximal moiety connects the extended amphipathic lipid to the construct,
the
distal moiety targets the construct to a receptor displayed by a hepatocyte,
and the
medial moiety connects the proximal and distal moieties, comprises: creating a
mixture comprising the amphipathic lipid and an extended amphipathic lipid;
and
forming a suspension of the lipid construct in water.
In still another aspect, the method of manufacturing the lipid construct
comprising an insulin, an amphipathic lipid and an extended amphipathic lipid,
wherein the extended amphipathic lipid comprises proximal, medial and distal
moieties, wherein the proximal moiety connects the extended amphipathic lipid
to the
construct, the distal moiety targets the construct to a receptor displayed by
a
hepatocyte, and the medial moiety connects the proximal and distal moieties,
comprises: creating a mixture comprising the amphipathic lipid and an extended
amphipathic lipid; forming a suspension of the lipid construct in water; and
loading
the insulin into the lipid construct
In another aspect, the step of loading the insulin into the lipid construct
comprises equilibrium loading and non-equilibrium loading.
The still another aspect, the step of loading the insulin into the lipid
construct comprises adding a solution containing free insulin to a mixture of
the lipid
construct in water and allowing the insulin to remain in contact with the
mixture until
equilibrium is reached.
In yet another aspect, the method further comprises the step of
terminally loading the insulin into the lipid construct after the mixture
reaches
equilibrium, wherein the solution containing free insulin is removed from the
construct, further wherein the construct contains insulin associated with the
construct.
In one aspect, the method further comprises the step of removing the
solution containing free insulin from the lipid construct containing insulin
associated
with the construct by a process selected from the group consisting of a rapid
filtration
procedure, centrifugation, filter centrifugation, and chromatography using an
ion-
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exchange resin or streptavidin agarose affinity-resin gel having affinity for
biotin,
iminobiotin or derivates thereof.
In another aspect, the method further comprises the step of adding a
chromium complex comprising a plurality of linked individual units to the
lipid
construct.
In still another aspect, the method further comprises the step of adding
cellulose acetate hydrogen phthalate to the lipid construct.
In yet another aspect, the method further comprises the step of
reclaiming from the process at least one material selected from the group
consisting of
insulin, ion-exchange resin and streptavidin agarose affinity-gel.
In another aspect, the step of loading the insulin into the lipid construct
comprises the step of adding at least one charged organic molecule to the
insulin
before the insulin is loaded into the lipid construct.
In still another aspect, a method of increasing the bioavailability of at
least one insulin in a patient comprises: combining at least one insulin with
a lipid
construct, wherein the lipid construct comprises a plurality of non-covalent
multi-
dentate binding sites; and administering the construct containing insulin to
the patient.
In another aspect, increasing the bioavailability further comprising the
step of modulating the isoelectric point of at least one active ingredient.
In yet another aspect, the insulin is selected from the group consisting
of insulin lispro, insulin aspart, regular insulin, insulin glargine, insulin
zinc, human
insulin zinc extended, isophane insulin, human buffered regular insulin,
insulin
glulisine, recombinant human regular insulin, recombinant human insulin
isophane,
premixed combinations of any of the aforementioned insulins, a derivative
thereof,
and a combination of any of the aforementioned insulins.
The method of claim 33, wherein the lipid construct comprises insulin,
1,2-distearoyl-sn-glycero-3-phophocholine, cholesterol, dicetyl phosphate, 1,2-
dipalmitoyl-sn-glycero-[3-phospho-rac-(1-glycerol)), 1,2-distearoyl-sn-glycero-
3-
phosphoethanolamine, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl) or derivatives, and a hepatocyte receptor binding molecule.
In one aspect, the method further comprises the step of adding at least
one charged organic molecule to the insulin before the insulin is combined
with the
lipid construct.
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In another aspect, a method of forming a time-release composition that
provides increased bio-distribution of insulin in a host comprises: removing a
lipid
construct from a bulk phase media by binding the construct through lipids
comprising
iminobiotin or an irninobiotin derivative to streptavidin agarose affinity-gel
at pH 9.5
or greater; separating the construct from the bulk phase media; and releasing
the
construct from the affinity-gel by adjusting the pH of an aqueous mixture of
the
affinity gel to pH 4.5, wherein, the released construct contains insoluble
insulin;
wherein upon administration of the construct to a warm-blooded host the
insulin is
resolubilized under the physiological pH conditions in the host.
In still another aspect, a method of treating a patient afflicted with
diabetes comprises administering to the patient an effective amount of a lipid
construct comprising insulin associated with the construct.
In yet another aspect, the insulin is selected from the group consisting
of insulin lispro, insulin aspart, regular insulin, insulin glargine, insulin
zinc, human
insulin zinc extended, isophane insulin, human buffered regular insulin,
insulin
glulisine, recombinant human regular insulin, recombinant human insulin
isophane,
premixed combinations of any of the aforementioned insulins, a derivative
thereof,
and a combination of any of the aforementioned insulins.
In one aspect, the lipid construct further comprises a target molecule
complex, wherein the complex comprises a plurality of linked individual units,
further
wherein the linked individual units comprises: a bridging component selected
from
the group comprising a transition element, an inner transition element, a
neighbor
element of the transition element and a mixture of any of the foregoing
elements; and
a complexhig component; provided that when the transition element is chromium,
a
chromium target molecule complex is formed.
In another aspect, the lipid construct further comprises insulin not
associated with the target molecule complex.
In still another aspect, the administration is oral or subcutaneous.
In yet another aspect, the insulin associated with the construct
comprises at least one charged organic molecule bound to the insulin.
In one aspect, the invention includes a method for increasing the
delivery of insulin to hepatocytes in the liver of a patient afflicted with
diabetes by
administering to the patient a lipid construct comprising insulin, an
amphipathic lipid,
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and an extended lipid, wherein the extended lipid comprises a moiety that
binds to
hepatocyte receptors, wherein the lipid construct is present in a plurality of
sizes.
In another aspect the at least one insulin is selected from the group
consisting of insulin lispro, insulin aspart, regular insulin, insulin
glarginc, insulin
zinc, human insulin zinc extended, isophane insulin, human buffered regular
insulin,
insulin glulisine, recombinant human regular insulin, recombinant human
insulin
isophane, premixed combinations of any of the aforementioned insulins, a
derivative
thereof, and a combination of any of the aforementioned insulins.
In still another aspect, the method further comprises protecting the
insulin within the lipid construct from hydrolytic degradation by providing a
three-
dimensional structural array of lipid molecules so as to prevent access to the
insulin
by hydrolytic enzymes.
In yet another aspect, the method further comprises adding cellulose
acetate hydrogen phthalate to the lipid construct to react with individual
lipid
molecules.
In still another aspect, the method further comprises producing an
insolubilized dosage form of insulin within the lipid construct.
In one aspect, the invention includes a kit for use in treating a mammal
inflicted with diabetes, the kit comprising a lipid construct, a physiological
buffer
solution, an applicator, and an instructional material for the use thereof,
wherein the
lipid construct comprises an amphipathic lipid and an extended amphipathic
lipid,
wherein the extended amphipathic lipid comprises proximal, medial and distal
moieties, wherein the proximal moiety connects the extended amphipathic lipid
to the
construct, the distal moiety targets the construct to a receptor displayed by
a
hepatocyte, and the medial moiety connects the proximal and distal moieties.
In another aspect, the kit further comprises at least one insulin.
In one aspect the invention includes a hepatocyte-targeting
composition comprises : at least one free insulin; at least one insulin
associated with a
water-insoluble target molecule complex and a lipid construct matrix
comprising at
least one lipid component; wherein the target molecule complex is comprised of
a
combination of: multiple linked individual units, the individual units
comprising: at
least one bridging component selected from the group consisting of a
transition
element, an inner transition element, and a neighbor element of the transition
element;
and a complexing component provided that when the transition element is
chromium,
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a chromium target molecule complex is created; further wherein the target
molecule
complex comprises a negative charge.
In another aspect, the at least one insulin is selected from the group
consisting of insulin lispro, insulin aspart, regular insulin, insulin
glargitte, insulin
zinc, human insulin zinc extended, isophane insulin, human buffered regular
insulin,
insulin glulisine, recombinant human regular insulin, recombinant human
insulin
isophane, premixed combinations of any of the aforementioned insulins, a
derivative
thereof, and a combination of any of the aforementioned insulins.
In still another aspect, the insulin comprises insulin-like moieties,
including fragments of insulin molecules, that have the biological activity of
insulins.
In yet another aspect, the lipid component comprises at least one lipid
selected from the group consisting of 1,2-distearoyl-sn-glycero-3-
phosphocholine,
1,2-dipahnitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-
phosphocholine, cholesterol, cholesterol oleate, dicetylphosphate, 1,2-
distearoyl-sn-
glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-phosphate, and 1,2-
dimyristoyl-
=
sn-glycero-3-phosphate.
In one aspect, the lipid component comprises at least one lipid selected
from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine,
cholesterol,
and dieetyl phosphate.
In another aspect, the lipid component comprises a mixture of 1,2-
distearoyl-sn-glycero-3-phosphocholine, cholesterol and dicetyl phosphate.
In still another aspect, the bridging component is chromium.
In yet another aspect, the complexing component comprises at least
one member selected from the group consisting of:
N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,6-diethylphenylearbamoylmethyl) iminodiacetic acid;
N-(2,6-dimethylphenylcarbamoylmetlayl) iminodiacetic acid;
N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;
N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,3-dimethylphenylcarbarnoylmethyl) iminodiacetic acid;
N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,5-dimethylphenylcarbarnoylmethyl) iminodiacetic acid;
N-(3,4-dimethylphenylcarbamoylinethyl) iminodiacetic acid;
N-(3,5-dimethylphenylcarbamoyhnethyl) iminodiacetic acid;
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N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2-butylphenylcarbamoylmethyl) irninodiacetic acid;
N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;
N-(2-hexy1oxypheny1carbamoylmethyl) iminodiacetic acid;
N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;
aminopyrrol iminodiacetic acid;
N-(3-bromo-2,4,6-trimethylphenylcarbamoylmethyl) iminodiacetic
acid;
benzimidazole methyl iminodiacetic acid;
N-(3-cyano-4,5-dimethy1-2-pyrrylcarbamoylmethyl) iminodiacetic
acid;
N-(3-cyano-4-methyl-5-benzy1-2-pyrrylcarbamoyhnethyl)
iminodiacetic acid; and
N-(3-cyano-4-methyl-2-pyrrylcarbamoylmethyl) iminodiacetic acid.
In still another aspect, the complexing component comprises
poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid].
In one aspect, the present invention includes a method of
manufacturing a hepatocyte-targeting composition comprises: creating a target
molecule complex, wherein the complex comprises multiple linked individual
units
and a lipid construct matrix; forming a suspension of the target molecule
complex in
buffer; and combining the insulin and the target molecule complex.
In another aspect, a method of manufacturing a hepatocyte-targeting
composition comprises: creating a target molecule complex, wherein the complex
comprises multiple linked individual units and a lipid construct matrix;
forming a
suspension of the target molecule complex in water; adjusting the pH of the
water
suspension to approximately pH 5.3; adjusting the pH of the glargine insulin
to
approximately 4.8; and combining the glargine insulin and the target molecule
complex, wherein the insulin is glargine insulin.
In still another aspect, a method of manufacturing a hepatocyte-
targeting composition comprises: creating a target molecule complex, wherein
the
complex comprises multiple linked individual units and a lipid construct
matrix;
forming a suspension of the target molecule complex in water; adjusting the pH
of the
water suspension to approximately pH 5.3; adjusting the pH of the glargine
insulin to
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approximately 4.8; and combining the glargine insulin, the non-glargine
insulin and
the target molecule complex, wherein the insulin comprises glargine insulin
and at
least one non-glargine insulin.
In one aspect the present invention includes a method of treating a
patient for Type I or Type II diabetes comprising administering to the patient
an
effective amount of a hepatocyte-targeting composition.
In another aspect, the route of administration is selected from the group
consisting of oral, parenteral, subcutaneous, pulmonary and buccal.
In still another aspect, the route of administration is oral or
subcutaneous.
In one aspect the present invention includes a method of treating a
patient for Type I or Type II diabetes comprising administering to the patient
an
effective amount of a hepatocyte targeted composition, wherein insulin
comprises
glargine insulin and at least one non-glargine insulin, further wherein the
non-glargine
insulin is selected from the group consisting of insulin lispro, insulin
aspart, regular
insulin, insulin glargine, insulin zinc, human insulin zinc extended, isophane
insulin,
human buffered regular insulin, insulin glulisine, recombinant human regular
insulin,
recombinant human insulin isophane, premixed combinations of any of the
aforementioned insulins, a derivative thereof, and a combination of any of the
aforementioned insulins.
In another aspect, the non-glargine insulin comprises insulin-like
moieties, including fragments of insulin molecules, that have biological
activity of
insulins.
In still another aspect, the present invention includes a method of
treating a patient for Type I or Type II diabetes comprising administering to
the
patient an effective amount of a hepatocyte-targeting composition.
In another aspect, the route of administration is selected from the group
consisting of oral, parenteral, subcutaneous, pulmonary and buccal.
In still another aspect, the route of administration is oral or
subcutaneous.
In yet another aspect, the present invention includes a method of
treating a patient for Type I or Type II diabetes comprising administering to
the
patient an effective amount of a hepatocyte targeted composition, wherein
insulin
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comprises recombinant human insulin isophane and at least one insulin that is
not
recombinant human insulin isophane.
In another aspect, the at least one insulin that is not recombinant
human insulin isophane comprises insulin-like moieties, including fragments of
insulin molecules, that have biological activity of insulins.
In one aspect, the present invention includes a kit for use in treating
Type I or Type II diabetes in a mammal, the kit comprising a physiological
buffered
solution, an applicator, instructional material for the use thereof, and a
water insoluble
target molecule complex, wherein the complex comprises multiple linked
individual
units and a lipid construct matrix containing a negative charge, the multiple
linked
individual units comprising: a bridging component selected from the group
consisting
of a transition element, an inner transition element, a neighbor element of
the
transition element and a mixture of any of the foregoing elements, and a
complexing
component, provided that when the transition element is chromium, a chromium
target molecule complex is created, wherein the multiple linked individual
units are
combined with the lipid construct matrix.
In another aspect, the kit further comprising at least one insulin,
wherein the insulin is associated with the target molecule complex-, wherein
the
complex comprises a charge.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purposes of illustrating the invention, there are depicted in the
drawings certain embodiments of the invention. However, the invention is not
limited
to the precise arrangements and instrumentalities of the embodiments depicted
in the
drawings.
Figure 1 is a depiction of an insulin binding lipid construct comprising
insulin, amphipathic lipid molecules and an extended amphipathic lipid.
Figure 2 is depiction of a route for manufacturing biocytin.
Figure 3 is a depiction of a route for manufacturing iminobiocytin.
Figure 4 is a depiction of a route for manufacturing benzoyl thioacetyl
triglycine iminobiocytin (BTA-3gly-iminobiocytin).
Figure 5 is a depiction of a route for manufacturing benzoyl thioacetyl
triglycine.
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Figure 6 is a depiction of a route for manufacturing benzoyl thioacetyl
triglycine sulfo-N-hydroxysiccinimide (BTA-3-gly-sulfo-NHS).
Figure 7 is a depiction of a route for manufacturing benzoyl thioacetyl
triglycine iminobiocytin (BTA-3-gly-iminobiocytin).
Figure 8 is a depiction of a route for manufacturing a lipid anchoring
and hepatocyte receptor binding molecule (LA-HRBM).
Figure 9 is a depiction of potential sites for binding between cellulose
acetate hydrogen phthalate and insulin.
Figure 10 is a depiction of the change in structure of iminobiotin under
acidic versus basic conditions.
Figure 11 is a depiction of the chemical structure of glargine insulin.
Figure 12 is a depiction of the chemical structure of recombinant
human insulin isophane and a protamine protein.
Figure 13 is a depiction of a pharmaceutical composition that combines
free insulin and insulin associated with a water insoluble target molecule
complex.
Figure 14 is an outline of a method of manufacturing an insulin
binding lipid construct comprising amphipathic lipid molecules and an extended
amphipathic lipid.
Figure 15 is an outline of the method of manufacturing a hepatocyte
targeted pharmaceutical composition that combines free glargine insulin and
glargine
insulin associated with a water insoluble target molecule complex.
Figure 16 is an outline of the method of manufacturing a hepatocyte
targeted pharmaceutical composition that combines free recombinant human
insulin
isophane and recombinant human insulin isophane associated with a water
insoluble
target molecule complex that contains a portion of recombinant human regular
insulin
that is both free and associated with a lipid construct.
Figure 17 indicates the concentration of glycogen present in the liver
of rats treated with various hepatocyte targeted compositions.
Figure 18 is a graph of the concentrations of glucose in blood of
individual patients treated once before breakfast with HDV-glargine insulin.
Figure 19 is a graph of the effect of a single dose of HDV-glargine
insulin on average blood glucose concentrations in patients consuming three
meals
during the day.
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Figure 20 is a graph of the effect of HDV-glargine insulin on blood
glucose concentrations over time relative to blood glucose concentrations
during
fasting.
Figure 21 is a graph of the concentrations of glucose in blood of
individual patients treated once before breakfast with HDV-Humulin NPH
insulin.
Figure 22 is a graph of the effect of a single dose of HDV-Humulin
NPH insulin on average blood glucose concentrations in patients consuming
three
meals during the day.
Figure 23 is a graph of the effect of HDV-Humulin NPH insulin on
blood glucose concentrations over time relative to blood glucose
concentrations
during fasting.
DETAILED DESCRIPTION OF THE INVENTION
The invention includes a hepatocyte targeted pharmaceutical
composition where insulin is associated with a water insoluble target molecule
complex within the construct and the composition is targeted to hepatocytes in
the
liver of a patient to provide an effective means of managing diabetes.
The invention includes a lipid construct comprising insulin, an
amphipathic lipid and an extended amphipathic lipid (a receptor binding
molecule).
The extended amphipathic lipid comprises proximal, medial and distal moieties.
The
proximal moiety connects the extended lipid molecule to the construct, the
distal
moiety targets the construct to a receptor displayed by a hepatocyte, and the
medial
moiety connects the proximal and distal moieties.
A lipid construct is a spherical lipid and phospholipid particle in which
individual lipid molecules cooperatively interact to create a bipolar lipid
membrane
which encloses and isolates a portion of the medium in which it was formed.
The
lipid construct can target the delivery of insulin to the hepatocytes in the
liver and
provide for a sustained release of insulin to better control diabetes.
The invention also includes a hepatocyte targeted pharmaceutical
composition that combines free insulin and insulin associated with a water
insoluble
target molecule complex targeted to hepatocytes in the liver of a patient to
provide an
effective means of managing blood glucose levels. When a mixture of different
forms
of insulin are associated with a target molecule complex to create a unique
mixture of
insulin molecules, an added therapeutic benefit is achieved once these
insulins are
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CA 02864366 2014-09-18
combined in a hepatocyte targeted lipid construct. The composition of the
invention
can be administered by various routes, including subcutaneously or orally, for
the
purpose of treating mammals afflicted with diabetes.
The invention further provides a method of manufacturing a lipid
construct comprising insulin, an amphipathic lipid and an extended amphipathic
lipid.
The extended amphipathic lipid molecule comprises proximal, medial and distal
moieties. The proximal moiety connects the extended lipid to the construct.
The
distal moiety targets the construct to a receptor displayed by a hepatocyte,
and the
medial moiety connects the proximal and distal moieties.
The invention also provides a method of manufacturing a composition
comprising free insulin and insulin associated with a water insoluble target
molecule
complex within the lipid construct that targets delivery of the complex to
hepatocytes.
The target molecule complex comprises a lipid construct matrix containing
multiple
linked individual units of a structure formed by a metal complex.
Additionally, the invention provides methods of treating individuals
afflicted with diabetes by administering an effective dose of a lipid
construct
comprising insulin, an amphipathic lipid and an extended amphipathic lipid,
targeted
for delivery to hepatocytes.
The invention also provides methods of treating individuals afflicted
with diabetes by administering an effective dose of a lipid construct
comprising
insulin, an amphipathic lipid, an extended amphipathic lipid and a water
insoluble
target molecule complex, targeted for delivery to hepatocytes.
The invention also provides methods of treating a patient with insulin
to which a polar organic compound, or mixture of compounds, is bound, thereby
changing the isoelectric point of insulin. This change in the isolelectrie
point will
change the release of insulin into the body of patient treated with the
composition.
Additionally, the invention provides methods of managing blood
glucose levels in individuals with Type I and Type II diabetes by
administering an
effective dose of a hepatocyte targeted pharmaceutical composition that
combines free
insulin and insulin associated with a water insoluble target molecule complex
targeted
for delivery to hepatocytes. The combination of free insulin and insulin
associated
with a water insoluble target molecule complex creates a dynamic equilibrium
process
between the two forms of insulin that occurs in vivo to help control the
movement of
free insulin to the receptor sites of hormonal action, such as the muscle and
adipose
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tissue of a diabetic patient over a designated time period. Hepatocyte
targeted insulin
is also delivered to the liver of a diabetic patient over a different
designated time
period than free insulin thereby introducing new pharmacodynamic profiles of
insulin
when free insulin is released from the lipid construct. In addition, a portion
of insulin
that is associated with the lipid construct is targeted to the liver. This new
phammcodynamic profile of the product provides not only long-acting basal
insulin
for peripheral tissues, but also meal-time hepatic insulin stimulation for the
management of hepatic glucose storage during a meal. Free insulin is released
from
the site of administration and is distributed throughout the body. Insulin
associated
with a water insoluble target molecule complex is delivered to the liver,
where it is
released over time from the complex. The rate of release of insulin associated
with
the target molecule complex is different than the rate of release of free
insulin from
the site of administration. These different release rates of insulin delivery,
combined
with the targeted delivery of insulin associated with a lipid construct to the
liver,
provide for the normalization of glucose concentrations in patients with Type
I and
Type II diabetes. The hepatocyte targeted composition can also comprise other
types
of insulin, or a combination of other types of insulin.
Definitions
Unless defined otherwise, all technical and scientific terms used herein
generally have the same meaning as commonly understood by one of ordinary
skill in
the art to which the invention belongs. Generally, the nomenclature used
herein and
the laboratory procedures in organic chemistry and protein chemistry are those
well
known and commonly employed in the art.
The articles "a" and "an" are used herein to refer to one or to more
than one (i.e., to at least one) of the grammatical object of the article. By
way of
example, "an element" means one element or more than one element.
The term "active ingredient" refers to recombinant human insulin
isophane, recombinant human regular insulin and other insulins.
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As used herein, amino acids are represented by the full name thereof,
by the three-letter code as well as the one-letter code corresponding thereto,
as
indicated in the following table:
3-Letter 1-Letter 3-Letter 1-
Letter
Full Name Code Code Full Name Code Code
Alanine Ala A Leucine Leu
Arginine Arg R Lysine Lys
Asparagine Asn N Methionine Met
Aspartic Acid Asp D Phenylalanine Phe
Cysteine Cys C Proline Pro
Cystine Cys-Cys C-C Serine Ser
Glutamic Acid Glu E Threonine Thr
Glutamine Gin Q Tryptophan Trp
Glycine Gly U Tyrosine Tyr
Histidine His H Valine Val V
Isoleucine Ile
The term "lower" means the group it is describing contains from 1 to 6
carbon atoms.
The term "alkyl", by itself or as part of another substituent means,
unless otherwise stated, a straight, branched or cyclic chain hydrocarbon
having the
number of carbon atoms designated (i.e. C1-C6 means one to six carbons) and
includes
straight, branched chain or cyclic groups. Examples include: methyl, ethyl,
propyl,
isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cycIohexyl
and
cyclopropylmethyl. Most preferred is (C1-C3) alkyl, particularly ethyl, methyl
and
isopropyl.
The term "allcylene", by itself or as part of another substituent means,
unless otherwise stated, a straight, branched or cyclic chain hydrocarbon
having two
substitution sites, e. g., methylene (-CH2-), ethylene (-CH2CH2-),
isopropylene
(-CH(CH3)=CH2), etc.
The term "aryl", employed alone or in combination with other terms,
means, unless otherwise stated, a cyclic carbon ring structure, with or
without
saturation, containing one or more rings (typically one, two or three rings)
wherein
such rings may be attached together in a pendant manner, such as a biphenyl,
or may
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CA 02864366 2014-09-18
be fused, such as naphthalene. Examples include phenyl; anthmcyl; and
naphthyl.
The structure can have one or more substitution sites where functional groups,
such as
alcohol, alkoxy, amides, amino, cyanides, halogen, and nitro, are bound.
The term "arylloweralkyl" means a functional group wherein an aryl
group is attached to a lower allcylene group, e.g., -CH2CH2-phenyl.
The tenn "alkoxy" employed alone or in combination with other terms
means, unless otherwise stated, an alkyl group or an alkyl group containing a
substituent such as a hydroxyl group, having the designated number of carbon
atoms
connected to the rest of the molecule via an oxygen atom, such as, for
example, -
OCHOH-, -OCH2OH, methoxy (-0C113), ethoxY (-0C112CH3), 1-propoxY (-
0CH2CH2CH3), 2-propoxy (isopropoxy), butoxy (-0CH20-12CH2CH3), pentoxY (-
0CH2CH2CH2CH2CH3), and the higher homologs and isomers.
The term "acyl" means a functional group of the general formula
wherein ¨R is hydrogen, hydrocarbyl, amino or alkoxy. Examples include
acetyl (-C(-0)CH3), propionyl (-C(--=0)CH2CH3), benzoyl (-C(----0)C6H5),
phenylacetyl (-C(1)CH2C6H5), carboethoxy (-CO2 CH2CH3), and
dimethylcarbamoyl (-C(=0)N(CH3)2).
The terms "halo" or "halogen" by themselves or as part of another
substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or
iodine
atom.
The term "heterocycle" or "heterocycly1" or "heterocyclic" by itself or
as part of another substituent means, unless otherwise stated, an
unsubstituted or
substituted, stable, mono- or multicyclic heterocyclic ring system comprising
carbon
atoms and at least one heteroatom selected from the group comprising N, 0, and
S,
and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized,
and the
nitrogen atom may be optionally quaternized. The heterocyclic system may be
attached, unless otherwise stated, at any heteroatom or carbon atom which
affords a
stable structure. Examples include pyrrole, imidazole, benzimidazole,
phthalein,
pyridenyl, pyranyl, furanyl, thiazole, thiophene, oxazole, pyrazole, 3-
pyrroline,
pyrrolidene, pyrimidine, purine, quinoline, isoquinoline, carbazole, etc.
The term "chromium target molecule complex" refers to a complex
comprising a number of individual units, where each unit comprises chromium
(Cr)
atoms capable of accepting up to six ligands contributed by multivalent
molecules,
such as ligands from numerous molecules of N-(2,6-diisopropylphenylcarbamoyl
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CA 02864366 2014-09-18
methyl) iminodiacetic acid. The individual units are linked to each other
forming a
complicated polymeric structure linked in a three-dimensional array. The
polymeric
complex is insoluble in water but soluble in organic solvents.
The term "lipid construct" refers to a lipid and/or phospholipid particle
in which individual lipid molecules cooperatively interact to create a bipolar
lipid
membrane which encloses and isolates a portion of the medium in which the
construct
resides.
The term "amphipathic lipid" means a lipid molecule having a polar
and non-polar end.
The term "extended amphipathic lipid" means an amphipathic
molecule with a structure that, when part of a lipid construct, extends from
the lipid
construct into media around the construct, and can bind or interact with a
receptor.
A "complexing agent" is a compound that will form a polymeric
complex with a selected metal bridging agent, e. g. a salt of chromium,
zirconium,
etc., that exhibits polymeric properties where the polymeric complex is
substantially
insoluble in water and soluble in organic solvents.
By "aqueous media" is meant water or water containing buffer or salt.
By "substantially soluble" is meant that the material, such as the
resultant polymeric chromium target molecule complex or other metal targeting
complexes which may be crystalline or amorphous in composition that are formed
from complexing agents, exhibit the property of being insoluble in water at
room
temperature. Such a polymeric complex or a dissociated form thereof when
associated with a lipid construct matrix forms a transport agent which
functions to
cam, and deliver insulin to hepatocytes in the liver of a warm-blooded host.
By "substantially insoluble" is meant that a polymeric complex, such
as a polymeric chromium target molecule complex or other metal targeting
complexes, exhibits the property of being insoluble in water at room
temperature.
Such a polymeric complex, which may be crystalline, amorphous in composition,
or a
dissociated form thereof, when associated with a lipid construct forms a
transport
agent that carries and delivers insulin to hepatocytes in the liver.
By use of the term "associated with" is meant that the referenced
material is incorporated into or on the surface of, or within, the lipid
construct matrix.
The term "insulin" refers to natural or recombinant forms of insulin,
and derivatives of the aforementioned insulins. Examples of insulin include,
but are
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CA 02864366 2014-09-18
not limited to insulin lispro, insulin aspart, regular insulin, insulin
glargine, insulin
zinc, human insulin zinc extended, isophane insulin, human buffered regular
insulin,
insulin glulisine, recombinant human regular insulin, and recombinant human
insulin
isophane. Also included are animal insulins, such as bovine or porcine
insulin.
The term "free insulin" refers to an insulin that is not associated with a
target molecule complex.
The terms "glargine" and "glargine insulin" both refer to a
recombinant human insulin analog which differs from human insulin in that the
amino
acid asparagine at position A21 is replaced by glycine and two arginines are
added to
the C-terminus of the B-chain. Chemically, it is 21A- Gly-30Ba-L-Arg-30Bb-L-
Arg-
human insulin and has the empirical formula C267H4o4N72078S6 and a molecular
weight of 6063. The structural formula of glargine insulin is provided in
Figure 11.
The term "non-glargine insulin" refers at all insulins, either natural or
recombinant that are not glargine insulin. The term includes insulin-like
moieties,
including fragments of insulin molecules, that have biological activity of
insulins.
The term "recombinant human insulin isophane" refers to a human
insulin that has been treated with protamine. The structural formulas for
recombinant
human insulin isophane and protamine are provided in Figure 12.
The term "at least one insulin that is not recombinant human insulin
isophane insulin" refers at all insulins, either natural or recombinant, that
are not
recombinant human insulin isophane. The term includes insulin-like moieties,
including fragments of insulin molecules that have biological activity of
insulins.
"HDV", or "Hepatocyte Delivery Vehicle", is a water insoluble target
molecule complex comprising a lipid construct matrix containing multiple
linked
individual units of a structure formed by the combination of a metal bridging
agent
and a complexing agent. "HDY" is described in WO 99/59545, Targeted Liposomal
Drug Delivery System.
"HDV-glargine" is a designation for a hepatocyte targeted composition
comprising a mixture of free glargine insulin and glargine insulin associated
with a
water insoluble target molecule complex, wherein the complex comprises
multiple
linked individual units of chromium and N-(2,6-
diisopropylphenylcarbamoylmethyl)
iminodiacetic acid, formed by the combination of a metal bridging agent and a
complexing agent, and a lipid construct matrix.
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"HDV-NPH" is a designation for a hepatocyte targeted composition
comprising a mixture of free recombinant human insulin isophane, free non-
humulin
insulin, and recombinant human insulin isophane and non-humulin insulin that
are
associated with a water insoluble target molecule complex, wherein the complex
comprises multiple linked individual units of chromium and N-(2,6-
dlisopropylphenylcarbamoylmethyl) iminodiacetic acid, formed by the
combination
of a metal bridging agent and a complexing agent, and a lipid construct
matrix.
The term "bioavailability" refers to a measurement of the rate and
extent that insulin reaches the systemic circulation and is available at the
sites of
action.
The term "isoelectric point" refers to the pH at which the
concentrations of positive and negative charges on the protein are equal and,
as a
result, the protein will express a net zero charge. At the isoelectric point,
a protein
will exist almost entirely in the form of a zwitterion, or hybrid between
forms of the
protein. Proteins are least stable at their isoelectric points, and are more
easily
coagulated or precipitated at this pH. However, proteins are not denatured
upon
isoelectric precipitation since this process is essentially reversible.
As the term is used herein, "to modulate" or "modulation of' a
biological or chemical process or state refers to the alteration of the normal
course of
the biological or chemical process, or changing the state of the biological or
chemical
process to a new state that is different than the present state. For example,
modulation
of the isoelectric point of a polypeptide may involve a change that increases
the
isolelectric point of the polypeptide. Alternatively, modulation of the
isoelectric point
of a polypeptide may involvea change that decreases the isolelectric point of
a
polypeptide.
"Statistical structure" denotes a structure formed from molecules that
can migrate from one lipid construct to another and the structure is present
in a
plurality of particle sizes that can be represented by a Gaussian
distribution.
"Multi-dentate binding" is a chemical binding process that utilizes
multiple binding sites within the lipid construct, such as cellulose acetate
hydrogen
phthalate, phospholipids and insulin. These binding sites promote hydrogen
bonding,
ion-dipole and dipole-dipole interactions where the individual molecules work
in
tandem to form non-covalent associations that serve to bind or connect two or
more
molecules.
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As used herein, to "treat" means reducing the frequency with which
symptoms of a disease, disorder, or adverse condition, and the like, are
experienced
by a patient.
As used herein, the term "pharmaceutically acceptable carrier" means a
chemical composition with which the active ingredient may be combined and
which,
following the combination, can be used to administer the active ingredient to
a
subject.
As used herein, the term "physiologically acceptable" means that the
ingredient is not deleterious to the subject to which the composition is to be
administered.
Description of the Invention - Composition
Lipid Construct
A depiction of an insulin binding lipid construct comprising insulin, an
amphipathic lipid and an extended amphipathic lipid is shown in Figure 1. The
extended amphipathic lipid, also known as a receptor binding molecule,
comprises
proximal, medial and distal moieties, wherein the proximal moiety connects the
extended lipid molecule to the construct, the distal moiety targets the
construct to a
receptor displayed by a hepatocyte, and the medial moiety connects the
proximal and
distal moieties. Suitable amphipathic lipids generally comprise a polar head
group
and non-polar tail group that are attached to each other through a glycerol-
backbone.
Suitable amphipathic lipids include 1,2-diste,aroyl-sn-glycero-3-
phosphocholine, 1,2-dipalmitoyl-sn-g,lycero-3-phosphocholine, 1,2-dimyristoyl-
sn-
glycero-3-phosphocholine, cholesterol, cholesterol oleate, dicetyl phosphate,
1,2-
distearoyl-sn-glycero-3-phosphate, 1,2-dipahnitoyl-sn-glycero-3-phosphate, 1,2-
dimyristoyl-sn-glycero-3-phosphate, 1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine-N-(Cap Biotinyl), 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl), 1,2-dipalinitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium
salt),
triethylarnmonium 2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-y1) pentanamido)ethyl phosphate and a mixture of any
of the
foregoing lipids or appropriate derivative of these lipids.
In an embodiment, amphipathic lipid molecules include 1,2-distearoyl-
sn-glycero-3-phosphocholine, cholesterol, die,etyl phosphate, 1,2-dipalmitoyl-
sn-
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CA 02864366 2014-09-18
glycero-3-phosphoethanolamine-N-(Cap Biotinyl); 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl), 1,2-dipalmitoyl-sn-glycero-34phospho-mc-(1-glycerol)] (sodium
salt),
triethylammonium 2,3-diacetoxypropy1 2-(5-((3 aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-dlimidazol-4-y1) pentanamido)ethyl phosphate and a mixture of any
of the
foregoing lipids.
The extended amphipathic lipid molecule, also know as a receptor
binding molecule, comprises proximal, medial and distal moieties. The proximal
moiety connects the extended lipid molecule to the construct, and the distal
moiety
targets the construct to a receptor displayed by a hepatocyte. The proximal
and distal
moieties are connected through a medial moiety. The composition of various
receptor
binding molecules is described below. Within a lipid construct, hepatocyte
receptor
binding molecules from one or more of the groups listed below can be present
to bind
the construct to receptors in the hepatocytes.
One group of hepatocyte receptor binding molecules comprises a
terminal biotin or iminobiotin moiety, as well as derivatives thereof. The
structural
formulas of biotin, iminobiotin, carboxybiotin and biocytin are shown in Table
1.
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Table 1
1 1,2-distearoyl-sn-glycero-3-
phosphocholine
2,3-bis(stearoyloxy)propyl o=r-o=
2-(trimethylammonio) ethyl
phosphate r--
.õ
2 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine
2,3-bis(palmitoyloxy)propyl I
2-(trimethylammonio) ethyl
phosphate
3 1,2-dimyristoyl-sn-glycero-
3-phosphocholine
2,3-bis (tetradecanoyloxy)
propyl 2-(trimethylammonio)
cks 0
ethyl phosphate
4 Cholesterol
14,c
10,13-dimethy1-17- Hs
(6-methylheptan-2-y1)-
2,3,4,7,8,9,10,11,12,13,14,15,1 11111111 " H,
6,17-tetradecahydro-1H- 4=40 H
cyclopenta[a]phenanthren-3-ol
)40
These molecules can be attached to a phospholipid molecule using a variety of
techniques to create lipid anchoring molecules that can be intercalated into a
lipid
construct. These hepatocyte receptor binding molecules comprise an anchoring
portion located in the proximal position to the lipid construct. The anchor
portion
comprises two lipophilic hydrocarbon chains that can associate and bind with
other
lipophilic hydrocarbon chains on phospholipid molecules within the lipid
construct.
In a preferred embodiment, a second group of hepatocyte receptor
binding molecules comprises a terminal biotin or iminobiotin moiety located in
the
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distal position from the lipid construct. The structures of such compounds are
given
in Table 2.
Table 2.
1 Biotin
0
5-((3aS,6aR)-2-
HNrjNNH
oxohexahydro-1H-
thieno[3,4-d]imidazol-
4-yl)pentanoic acid
HOOC
2 lminobiotin NH
5-((3aS,6aR)-2- HNVNNH
imitiohexahydro-1H-
thieno[3,4-d] imidazol- Him.... ...Him
4-y1) pentanoic acid
HOOC
3 Carboxybiotin
5-((3aS,6aR)-1-
(carboxymethyl)-2-
oxohexahydro-1H- Him.. ¨film
thieno[3,4-d] imidazol-
4-y1) pentanoic acid HOOC
4 Biocytin
2-amino-6-(5- RANH
((3aS,6aR)-2-
mit. ....HUH
oxohexahydro-1H-
thieno[3,4-d] imidazol-
4-y1) pentanatnido)
hexanoic acid
NH, 0
Both biotin and iminobiotin contain a mildly lipophilic bicyclic ring
structure attached to a five-carbon valeric acid chain at the 4-carbon
position on the
bicyclic ring. In an embodiment, L-lysine amino acid may be covalently bound
to the
valeric acid C-terminal carboxyl functional group by reacting the carboxyl
group on
valeric acid with either the N-terminal a-amino group or the E-amino group of
L-
lysine. This coupling reaction is performed using carbodiimide conjugation
methods
and results in the formation of an amide bond between L-lysine and biotin, as
illustrated in Figure 2.
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A third group of hepatocyte receptor binding molecules comprise
iminobiotin, carboxybiotin and biocytin with the valeric acid side chain
attached via
an amide bond to either the a-amino group or the s-amino group of the amino
acid L-
lysine. A preferred embodiment uses irninobiotin in forming an iminobiocytin
moiety
as shown in Figure 3. During synthesis of the hepatocyte receptor binding
molecule,
the a-amino group of iminobiocytin can react with the activated ester benzoyl
thioacetyl triglycine-sulfo-N-hydroxysuccinimide (BTA-3gly-sulfo-NHS) to form
the
active hepatocyte binding molecule (BTA-3gly-iminobiocytin) as shown in Figure
4.
BTA-3gly-iminobiocytin functions as a molecular spacer that ultimately
expresses an
active nucleophilic sulfhydral functional group that can be used in subsequent
coupling reactions. The spacer is located in the medial position in relation
to the lipid
construct and allows the terminal iminobiocytin moiety to extend approximately
thirty
angstroms from the surface of the lipid construct to develop an optimal and
non-
restricted orientation of iminobiocytin for binding to the hepatocyte
receptor. The
medial spacer can include other derivatives that provide the correct stereo-
chemical
orientation for the terminal biotin moiety. The main function of the medial
spacer is
to properly and covalently connect the proximal and distal moieties in a
linear array.
The BTA-3gly-sulfo-NHS portion of the hepatocyte receptor binding
molecule can be synthesized by a number of means and in subsequent steps be
linked
to biocytin or iminobiocytin. The initial step comprises adding benzoyl
chloride to
thioacetic acid to form by nucleophilic addition a protective group for the
active thio
functionality. The products of the reaction are the benzoyl thioacetic acid
complex
and hydrochloric acid, as shown in Figure 5. Additional steps in the synthesis
involve
reacting benzoyl thioacetic acid with sulfo-N-hydroxysuccinimide using
dicyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as
a
coupling agent to form benzoyl thioacetyl sulfo-N-hydroxysuceinirnide (BTA-
sulfo-
NHS), as depicted in Figure 5. Benzoyl thioacetyl sulfo-N-hydroxysuccinimide
is
then reacted with the amino acid polymer (glycine-glycine-glycine). Following
nucleophilic attack by the a-amino group of triglycine, benzoyl thioacetyl
triglycine
(BTA-3gly) is formed while the sulfo-N-hydroxysuccinimide leaving group is
solubilized by aqueous media, as shown in Figure 5. Benzoyl thioacetyl
triglycine is
again reacted with dicyclohexylcarbodiimide or 1-ethy1-3-(3-
dimethylaminopropyl)
carbodiimide to form an ester bond with sulfo-N-hydroxysuccinirnide, as shown
in
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CA 02864366 2014-09-18
Figure 6. The sulfo-N-hydroxysuccinimide ester of activated benzoyl thioacetyl
triglycine (BTA-3gly-sulfo-NHS) is then reacted with the a-amino group of the
L-
lysine functionality of biocytin or iminobiocytin to form the hepatocyte
receptor
binding moiety, the extended amphipathic lipid molecule of benzoyl thioacetyl
triglycine-iminobiocytin (BTA-3gly-iminobiocytin) illustrated in Figure 7.
A second major coupling reaction for the synthesis of an hepatocyte
receptor binding molecule is illustrated where benzoyl thioacetyl triglycine
iminobiocytin is covaiently attached through a thioether bond to a N-para-
maleimidophenylbutyrate phosphatidylethanolamine, a preferred phosphoIipid
anchoring molecule. This reaction results in a molecule that provides the
correct
molecular spacing between the terminal iminobiocytin ring and the lipid
construct.
An entire reaction scheme for forming a hepatocyte receptor binding molecule
that
functions as an extended amphipathic lipid molecule is depicted in Figure 8.
Prior to
reacting benzoyl thioacetyl triglycine iminobiocytin with N-para-
maleimidophenylbutyrate phosphatidylethanolamine to form a thioether linkage,
the
benzoyl protecting group is removed by heating in order to expose the free
sulfhydral
functionality. The reaction should be performed in an oxygen free environment
to
minimize oxidation of the sulfhydrals to the disulfide. Further oxidation
could lead to
the formation of a sulfone, sulfoxide, sulfenic acid or sulfonic acid
derivative.
In an embodiment, the anchoring moiety of the molecule contains a
pair of acyl hydrocarbon chains that form a lipid portion of the molecule.
This
portion of the molecule is non-covalently bound within the lipid domainc of
the lipid
construct. In an embodiment the anchoring moiety is produced from is N-para-
maleimidophenylbutyrate phosphatidylethanolarnine. Other anchoring molecules
may be used. In an embodiment, anchoring molecules can include thio-
cholesterol,
cholesterol oleate, dicetyl phosphate; 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine, 1,2-dipahnitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl), 1,2-dipalmitoyl-sn-g,lycero-3-[phospho-rac-(1-glycerol)] (sodium
salt), and
mixtures, thereof. The entire molecular structure of the fully developed lipid
anchoring and hepatocyte receptor binding molecule designated LA-HRBM is shown
in Figure 8.
A fourth group of hepatocyte receptor binding molecule comprises
amphipathic organic molecules having both a water-soluble moiety and a water-
- 27 -
CA 02864366 2014-09-18
insoluble moiety. The water-insoluble moiety reacts with a medial or connector
moiety by coordination and bioconjugation chemical reactions, while the water-
insoluble moiety binds to the hepatocyte binding receptor in the liver. The
molecule
contains a distal component comprising either by a non-polar derivatized
benzene ring
structure, such as a 2,6-diisopropylbenzene derivative, or by a lipophilic
heterobicyclic ring structure. The entire hepatocyte receptor binding molecule
possesses fixed or transient charges, either positive or negative, or various
combinations thereof These molecules contain at least one carbonyl group
located
equal to or less than, but not greater than, approximately 13.5 angstroms from
the
terminal end of the distal moiety, and at least one carbamoyl moiety
containing a
secondary amine and carbonyl group. The presence of a carbamoyl moiety or
moieties enhances the molecular stability of the organic molecule. A plurality
of
secondary amines can be present within the molecule. These secondary amines
contain a pair of unshared electrons allowing for ion-dipole and dipole-dipole
bonding
interactions with other molecules within the construct. These amines enhance
molecular stability and provide a partially created negative charge that
interacts with
the distal moiety to promote hepatocyte receptor binding and specificity. An
example
of this group of receptor binding molecules is polychromium-poly(bis)-[N-(2,6-
(dfisopropylphenyl)carbamoyl methyl)imino diacetic acid]. hi an embodiment,
chromium III is located in the medial position of the hepatocyte receptor
binding
molecule. The proximal moiety of the hepatocyte specific binding molecule
contains
hydrophobic and/or non-polar structures that allow the molecules to be
intercalated
into, and subsequently bound within, the lipid construct. The medial and
proximal
moieties also allow for the correct stereo-chemical orientation of the distal
portion of
the hepatocyte receptor binding molecule.
The structure and properties of the lipid construct are governed by the
structure of the lipids and interaction between lipids. The structure of the
lipids is
governed primarily by covalent bonding. Covalent bonding is the molecular
bonding
force necessary to retain the structural integrity of the molecules comprising
the
individual constituents of the lipid construct. Through non-covalent
interactions
between lipids, the lipid construct is maintained in a three-dimensional
conformation.
The non-covalent bond can be represented in general terms by an ion-
dipole or induced ion-dipole bond, and by the hydrogen bonds associated with
the
various polar groups on the head of the lipid. Hydrophobic bonds and van der
Waal's
- 28 -
CA 02864366 2014-09-18
interactions can be generated through induced dipole associations between the
lipid
acyl chains. These bonding mechanisms are transient in nature and result in a
bond-
making and bond breaking process that occurs in a sub-femtoseeond time
interval.
For example, van der Waal's interaction arises from a momentary change in
dipole
moment arising from a brief shift of orbital electrons to one side of one atom
or
molecule, creating a similar shift in adjacent atoms or molecules. The proton
assumes
a 6+ charge and the single electron a 6- charge, thus forming a dipole. Dipole
interactions occur with great frequency between the hydrocarbon acyl chains of
amphipathic lipid molecules. Once individual dipoles are formed they can
momentarily induce new dipole formation in neighboring atoms containing a
methylenic (-CH2-) functionality. A plurality of transiently induced dipole
interactions are formed between acyl lipid chains throughout the lipid
construct.
These induced dipole interactions last for only a fraction of a femtosecond (1
x 10-15
sec) but exert a strong force when functioning collectively. These
interactions are
constantly changing and have a force approximately one-twentieth the strength
of a
covalent bond. They are nevertheless responsible for transient bonding between
stable covalent molecules that determine the three-dimensional statistical
structure of
the construct and the stereo-specific molecular orientation of molecules
within the
lipid construct.
As a consequence of these induced-dipole interactions, the structure of
the lipid construct is maintained by the exchange of lipid components between
constructs. While the composition of the individual components of the
construct is
fixed, individual components of lipid constructs are subject to exchange
reactions
between constructs. These exchanges are initially governed by zero-order
kinetics
when a lipid component departs from a lipid construct. After the lipid
component is
released from the lipid construct, it may be recaptured by a neighboring lipid
construct. The recapture of the released component is controlled by second-
order
reaction kinetics, which is affected by the concentration of the released
component in
aqueous media around the construct capturing the component and the
concentration of
the lipid construct which is capturing the released component.
Examples of extended amphipathic lipids, along with their respective
identifiers, shown in Table 3 along with their chemical names, are:
-29 -
CA 02864366 2014-09-18
N-hydroxysuccinimide (NHS) biotin [1); sulfo-NHS-biotin [2]; N-
hydroxysuccinimide long chain biotin [3], sulfo-N-hydroxysuccinimide long
chain
biotin [4]; D-biotin [5); biocytin [6]; sulfo-N-hydroxysuccinimide-S-S-biotin
[7];
biotin-BMCC [8]; biotin-HPDP [9]; iodoacetyl-LC-biotin (10]; biotin-hydrazide
[11);
biotin-LC-hydrazide [12]; biocytin hydrazide [131; biotin cadaverine [14];
carboxybiotin (15]; photobiotin [16]; p-aminobenzoyl biocytin
trifluoroac,etate [17];
p-diazobenzoyl biocytin [18]; biotin DITPE [19]; biotin-X-DHPE [20]; 12-
((biotinyl)amino)dodecanoic acid [21];12-((biotinyl)amino)dodecanoic acid
succinimidyl ester 122); S-biotinyl homocysteine [23]; biocytin-X [24];
biocytin x-
hydrazide [25]; biotinethylenediamine [26]; biotin-XL [27]; biotin-X-
ethylenediamine
[28); biotin-XX hydrazide [29]; biotin-XX-SE [30]; biotin-3(X, SSE [31];
biotin-X-
cadaverine [32]; a-(t-B0C)biocytin [33]; N-(biotiny1)-N'-
(iodoacetyl)ethylenediamine
[34]; DNP-X-biocytin-X-SE [35]; biotin-X-hydrazide [36]; norbiotinamine
hydrochloride [37); 3-(N-maleimidylpropionyl) biocytin [38]; ARP [39]; biotin-
I-
sulfoxide [40]; biotin methyl ester [41]; biotin-maleimide [42]; biotin-
poly(ethyleneglycol)amine [43]; (+) biotin 4-amidobenzoic acid sodium salt
[44];
Biotin 2-N-acety1amino-2-deoxy-P-D-g1ucopyranoside [45]; Biotin-a-D-N-
acetylneuraminide [46]; Biotin-a-L-fucoside [47]; Biotin lacto-N-bioside [48];
Biotin-
Lewis-A trisaccharide [49]; Biotin-Lewis-Y tetrasaccharide [50]; Biotin-a-D-
mannopyranoside [51]; biotin 6-0-phospho-a-D-mannopyranoside [52]; and
polychromium-poly(bis)-114-(2,6-(diisopropylphenyl) carbamoyl
methyl)imino]diacetic acid [53].
-30-
Table 3.
_
1 N- hydroxysuccinimide (NHS) o
_____________________________________
biotin
HN../ILNH
0
2,5-dioxopyrrolidin- 1-y1 5-
((3aS,6aR)-2-oxohexahydro-1H- N-0
=-,
thieno[3,4-d]imidazol-4-y1) c s
pentanoate II
o
o
2 sulfo-NHS-biotin o
o
HN,N.NH
t\.)
co
c3)
sodium 2,5-dioxo-3- 0
0.
w
(trioxidanylthio)pyrrolidin- 1-y1
c3)
w 5-((3aS,6aR)-2-oxohexahydro-
1.)
¨
N-0
o
1H-thieno[3,4-dlimidazo1-4-y1)
1-.
0.
1
pentanoate Na03S II
o
0
ko
0
1
i-,
.
_
3 N-hydroxysuccinimide long
o co
chain biotin
HN)LNNH
0
2,5-dioxopyrrolidin-1-y1 6-(5- _1( 0 H
((3aS,6aR)-2-oxohexahydro-1H- II I
thieno[3,4-djimiclazol-4-y1) .........1,,..oc....,õ.....õ--,,,..õ".,-
,..,"....N.,
S
pentanamido)hexanoate II
0
µ
4 1-sulfo-N-hydroxysuccinimide
long chain biotin
HN)INNH
sodium 2,5-dioxo-3-
(trioxidanyithio)
6-(5-((3aS,6aR)-2-
oxohexahydro-1H-thieno[3,4-d] Nao
imidazol-4-yl)pentanamido)
hexanoate
D-biotin
0ci
0
co
HNVNN
5-((3aS,6aR)-2-oxohexahydro-
H
1H-thieno[3,4-d]imidazo1-4-y1)
1-111111,,,
pentanoic acid
Hooc
0
6 Biocytin 0
co
2-amino-6-(5-((3aS,6aR)-2- HNZNNH
oxohexahydro-1H-thieno[3,4-
d]imidazol-4-y1) pentanamido) H
II.IIIIIH
hexanoic acid
II
NH2 0
7 sullo-N-hydroxyguccinimide-S-
o
S-biotin
sodium 2,5-dioxo-3- o
Hr/NH
(trioxidanylthio) pyrrohdin-1 -y1
Mine". =.+111i1H
3-((2-(443aS,6aR)-2-
oxohexahydro-1H-thieno [3,4-d]
õ.....e.........<N...õ....eõc.õ,........./..õ,s.,,,,s..õ........"/-,,,
imidazol-4-Abutylamino) NaO3S
Iii
S
ethyl)disulfanyl)propanoate o
8 biotin-BMCC
o
HNNH
4-((2,5-dioxo-2,5-dihydro-1H-0
pyrrol-l-Amethyl)-N-(4-(5- o CI Ili
Him- onim ,
0
(0 aS,6aR)-2-oxohexahydro-1H-
0
L.., thieno[3,4-dlimidazol-4-y1)
F,
s .,
,..,
,p.
pentanarnido)butyl) i N
w
c3)
cyclohexanecarboxamide
-----<
0,
IV
0
I¨,
0
aa
I
0
9 biotin-HPDP
i
I-,
co
HNVLNH
4(3 aS,60.R)-2-oxohexahydro- o H
1H-thieno[3,4-djimidazo1-4-y1)- II I
Hlitil,. .b.1111H
N-(6-(3-(pridin-2-y1disulfanyl)
propanamido)hexyl)pentanamide < `=-=õ--., s
1 III
H 0
$
_
iodoacetyl-LC-biotin 0
HN/j'µNH
N-(6-(2-iodoacetamido)hexyl)-5- 0 H
((3aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-...11aH
yl)pentanamide i c\zN\
IN( -\=7 -N7 C
I II
S
H 0
11 biotin-hydrazide o
0
o
HNNH
tv
54(3aS,6aR)-2-oxohexahydro-
0
0,
0.
1H-thieno[3,4-d]irnida7o1-4-
w
H lillititu
mitillH cn
yl)pentanehydrazide
I
0,
w
1..)
4.
0
.."-= N....,
1-,
H2N C S
o.
II
1
o
0
ko
1
1-,
12 biotin-LC-hydrazide
9 0
HNNH
N-(6-hydraziny1-6-oxohexyl)-5-
((3aS,6aR)-2-oxohexahydro-1H-
0 H
Filliim. miiiIIH
thieno[3,4-djimidazol-4-y1) III
pentanamide
,....-_,õ....,_,..N.,
I II
H 0
13 ¨biocytin hydrazide
0
N-(5-amino-6-hydraziny1-6-
HN,NNH
oxohexyl.)-54(36,6aR)-2- o H
Kiwi. õmini-(
oxohexalaydro-1H-thieno[3,4- II I
d]imidazol-4-yl)pentanatnide H,N,,......
,,,,C.,..õ.õ....",,....õ....õ,......õ."....,..,õ/".N.,...,
N
S
i 11
cl
H NH2 0
o
rs)
14 biotin. cadaverine
9 co
01
4.
U.)
01
HN"1/'NNH o,
N-(5-aminopenty1)-5-
N3
0
((3aS,6aR)-2-oxohexahydro-1H-
c,4 H
"Mutts" ...niltH 41.
...A thieno{3,4-d]imidazol-4-y1)
pentanamide I
0
,.0
N,,..õ
'
1->
It
0
15 Carboxybiotin o
r=-=.N .,,õCOOH
(385)6aR)-4-(4-carboxybuty1)-2- HN N
oxollexahydro-111-thieno[3,4-
d]imidazole-1-carboxylic acid Hirsis,.. "tlisiSH
HOOC S
16 Photobiotin
o ___________________ _
HV1NNH
N-(343-(4-azido-2- H CH3 H
nitrophenylamino)propyl)(methy 1 1 I
Hums, .fifinH
1)amino)propy1)-5-((3aS,6aR)-2- 0 N\z,"\/N\
\c
oxohexahydro-1H-thieno[3,4-
s
d]imidazol-4-yppentanamide II
o
N3 No2
17 p-atninobenzoyl biocytin
0 CI
P
trifluoroacetate
2
co
P3CC00- H2N+
HNVNNH cn
o=
2-(4-aminobenzamido)-6-(5- H
w
cn
,.. ((3a8,6aR)-2-oxohexahydro-1H-
y1)pentanamido)hexanoic acid 1
N
HIIits., 0'MM 01
NJ
CT
thieno[3,4-d]imidazol-4- 0 \C
0
1-`
C
0.
s
1
2,2,2-trilluoroacetate II
11 0
ko
0 COON
0 I
1-`
CO
18 p-diazobenzoyl biocytin
. 0
4-(1-carboxy-5-(54(3a8,6aR)-2- Cr N
HN"NNH-_-:-77.¨N+
oxohexahydro-1H-thieno [3,4- H H
1 , L4õ
diimidazol-4-y1)pentanamido) ( 1
NW
pentylcarbamoyl) C/NM\rI/\/\s2
benzenediazonium chloride
11
0 COOH
0
,
.
19 biotin DHPE
o
o
HN"INNH
triethy1ammonium II
cH3-(C1-12)14¨c--0-----Fia H
2,3-diaeetoxypropyl 2-(5-
1 o I
How.. .giiiim
((3aS,6aR)-2-oxohexahydro-1H- CH3-(CH2)14
\
thieno[3,4-d]imidazol-4-y1) II N.
cm2-0¨P--0
C S
pentanamido)ethyl phosphate o
I il
(CH3CH2)3NH+ 0- o
20 biotin-X-DHPE
0
0
o
0
II
tiN)(NH tv
co
H
triethylammonium car(cH2)1 0 H 4¨C-
0¨CHz ) H
((3aS,6aR)-2-oxohexahydro-1H-
Ilii.
.1111H cn
2,3-diacetoxypropy1 2-(6-(5- l
CH34 ''''''''012)14 C ¨0¨CH
w
0cn
il 'cH2¨o¨P-0' C
S
w
,,,,,,,.
II "
--1 thieno[3,4-d]imidazol-4-y1) 0 II
I 0 0
(CH3cH2)3nui 0- o 1-.
pentammido)hexanamido)ethyl
0.
1
phosphate
2
i
21 12-((biotinyl)amino)dodecanoic
9
IT:O.
acid
HNVNNH
12-(54(36,6aR)-2- H
oxohexahydro-1H-thieno[3,4-d]
1
Hlitim. witt(IH
imidazo1-4-y1) pentanamido) H000 N
dodecanoic acid \c
11
s
0
_______________________________________________________________________________
______________________ _
22 12-((biotinyl)amino)dodecanoic
fi
acid succinimidyl ester
HNA.NH
0 0 H
2,5-dioxopyrrolidin-1-y112-(5- 11 I
Fon.. ...iiitH
((3aS,6aR)-2-oxohexahydro-1H- c N
thieno[3,4-djimidazol-4-y1) N-0/
\
pentanamido)dodecanoate
----A(
11
o s
o
0
23 S-biotinyl homocysteine o
0
1..)
0
HNZN cn
o.
4-mercapto-2-(5-((3aS,6aR)-2- NH
w
0,
oxohexahydro-1H-thieno[3,4-
01
f o) H Wm'.
..ffitt1H
co d]dazol-4-y1) pentanamido)
I
1..)
0
1-.
butanoic acid
0.
1
0
ii ko
1
1-,
COON 0
co
24 biocytin-X
9
2-amino-6-(6-(5-((3aS,6aR)-2-
HN'INNH
COOH 0
oxohexahydro-1H-thieno[3,4- H
dlimidazo1-4-y1)pentanamido)...film
hexanamido)hexanoic acid /\/\/\ \
H2N N C
S
I II
H 0
25 biocytin x-hydrazide
0
NH2
1
)N
.; ,.....,NH
HN
N-(5-amino-6-hydraziny1-6- 0
NH
H
oxohexyl)-6-(54(3aS,6aR)-2- 11 I
Hum.. .i.ioiH
oxohexahydro-1H-thieno[3,4-
d)inidazol-4-yl)pentanamido)
hexanamide H2NWN/c"\
I II
s
H 0
26 Biotinethylenediamine 0
0
0
1..)
HN'N.NH
co
0,
N-(2-aminoethyl)-5((3aS,6aR)-
0.
w
2-oxohexahydro-1H-thieno[3,4-
0,
w d]imidazol-4-yl)pentanamide H Wm..
..iiiiiIH c3)
v:.
I
tv
o
..====.õ/õN,...,õ.
1-,
o.
1
H2N" '`-'" C S
o
II
ko
1
1-,
0 0
27 biotin-X 0
)NN.
6-(5-((3aS,6aR)-2- HN
NH
oxohexahydro-1H-thieno [3,4- H
dlinfidazo1-4-y1)pentanamido) I Him...
mum
hexanoic acid HOOC
C
II s
0
28 biotin-X-ethylenediamine
0
ZI
N-(2-aminoethy1)-6-(5-
0
((3aS,6aR)-2-oxohexahydro-1H- H
HNNNH
thieno(3,4-dlimidazo1-4-y1) II I
Hum.. oti1111-1
pentanamido)hexanamide H2N /CzN \
N M C
I II
S
H 0
29 biotin-XX hydrazide
0 0
0
1..)
0
HN'INNH
cn
N-(6-hydraziny1-6-oxohexy1)-6- H 0
w
H
o.
(54(3 aS,6aR)-2-oxohexahydro-
I I WTI. .iiiIIH
cn
cn
.6. 1H-thieno[3,4-dlimidazol-4-
N "
o
0
yl)pentanamido)hexanamide
H2N/N\CWN \c 1-,
S
o.
1
II I
ll o
ko
0 H
0 1
1-,
0
30 biotin-XX-SE
0
0
HN'INNH
2,5-dioxopyrrolidin-1-y1 6-(6-(5- 0
H
((3aS,6aR)-2-oxohexahydro-1H- II
I Hni,.= miiIH
thieno[3limidazol-4-y1) N-0\ /\ /\ /\ dz,Cµ
pentanamido)hexanamido) '"-----<
Cr \f \f M C s
hexanoate II I
II
0 0 H 0
,
_______________________________________________________________________________
_______________________________________________________________________________
_____
1 31 biotin-XX,SSE
0
0
HN)NNH
Sodium 2,5-dioxo-1-(6-(6-(5- 0
H
((3aS,6aR)-2-oxohexahydro-1H- N 0 II
ili Hltito .411i1H
thieno[3,4-d]imidazol-4-
C N
C S
yl)pentanamido)hexanamido)hex Nao3s II I
H
anoyloxy)pyrrohdine-3-sulfonate o 0 H
o
32 biotin-X-cadaverine
F
HN,')NNNH
0
o
tv
co
c3)
5-(6-(5-((3aS,6aR)-2-
0
H o.
w
oxohexahydro-1H-thieno(3,4-d] 11
I Hill"filiiIIH 0)
0)
4. imidazol-4-yl)pentanamido)
N
"
,...
o
hexonsroido)pentan-1-aminium
1.42N/ \/ \/ \ \
N
C I-'S 1
2,2,2-trifitIOrOaCetate
1 II 0
ko
F3C¨000"
0 1
H
1-,
co
33 a-(t-B0C)biocytin
0
HN,"INNH
2-(tert-butoxycarbonylamino)-6-
CH
0
(5-((3aS,6aR)-2-oxohexahydro-
1 11 11
Him'. ..ilillH
1H-thieno[3,4-d]imidazol-4-Y1) H3c --- c----0¨C---NH N
pentonamido)hexanoic acid
I \c
s
CH3 H 11
COOH 0
i
CA 02864366 2014-09-18
r
r -
ozI:-:. co
Z r:
r =
x
0=_-0
I
rz =
1 2 =
A Z 7
B
C-)
, 0 u)
I
0.---0
x Z E
s
i . I -
I --E 1
1
4
xo-0-0¨z
!
0 00
.-,---xoz
1
I
o
xf
0.--=0 :
/
2¨Z =
z 1:
s =
0=c) 1
../ ;
I ¨z o
64
z zz
0
z_m
iz
0=-...0
0 \
z
N
2
1 1 v!) I A IA th
µ0.'8 I
0) evA I
,,, .4, . .--.
e
...... 6 ,
.g--0-
-, 0
0
¨ 1 1 .__Ni==
-8 :1:, oe 4-6. rn ¨ = U .4 '0 E k X 0
g 0 g
=,:i .5tt be' ? = >4 0
vb ,4 d
I 0 70
.v ., . = 1..., 4 ...õ._ ,-, .I-õ r-... ..t
I 0.)
0.-N /..... 0 t=-;1 7
k00 --,.-0 PI
0
... g ,..f ... 0 ;,5 10 = en g o =V -0 A
. 4 ''3 =:-..', 13
tr) " /.4 ' 8 ti .9. cn:=:g R.
'9
, '4 ,===== 0¨I N...., r-4 P., ,.1.4 õC)
.Zi. to
en en en
42
37 norbiotinamine hydrochloride 0
HN/N\NH
4-((34,6aR)-2-oxohexahydro-
111-thieno[3,4-dlimidazol-4-y1) Hi11,,. -1111H
butan-l-aminium chloride
-Cl+H3N S
38 3-(N-maleimidylpropionyl)
0
biocytin
0
H
II HN'ILNH
2-(3-(2,5-dioxo-2,5-dihydro-1H-
-----<
o
Pv
pyLio1-1-yl)propanamido)-6-(5- I C/
4. paSfiaR)-2-oxohexahydro-11-1- ...........<1 N
S
0
1.)
I.,
0
thieno[3,4-d]imidazol-4-y1) II 000H 0
0
0
41.
pentanamido)hexanoic acid
w
0
0
0
1.)
39 ARP; 0
0
1--.
4.
1
0
Nt-(2-(aminooxy)acety1)-5-
NH
HNZN, 0
1
((3a8,6aR)-2-oxohexahydro-1H-
1--.
0 H
co
thieno[3,4-d]imidazo1-4-y1) II i RINI. itH
pentanehydrazide 0 C N
I 11 S
H 0
'
40 biotin-l-sulfoxide 0
HINI)N.µ11H
5-((3aS,6aR)-2-oxohexahydro-
1H-thieno[3,4-d]imidazol-4-y1)
pentanoic acid sulfoxide
HOOC S
II
0
_
41 biotin methyl ester 0
Cl
methyl 5-((3aS,6aR)-2-
/1
o
tv
oxohexahydro-1H-thienop HN NH
,4-d]
co
0,
0.
imidazol-4y1)pentanoate
w
will, ...Hum 0,
0,
4.
4.
IV
H3C0
0
====,..c I-
II 0
l0
I
0 1-
`
CO
42 biotin-maleimide
0
6-(2,5-dioxo-2,5-dihydro-1H- 0 0
HN)(NH
pyrrol-1-y1)-N'-(543aS,6aR)-2- 11 H
Hist.. .iiiIH
oxohexahydro-1H-thieno C hil
[3,4-d)imidazol-4-yl)pentanoyl).---.
N N
hexanehydrazide
------< H II
o s
o
43 Biotin-poly(ethyleneglycol) 0
amine
HN)NNH
aminomethyl polyethylene 5-
((3aS,6aR)-2-oxohexahydro-1H- Hum.
milt H
thieno[3,4-d]imidazo1-4-y1) NH2¨CH2¨(0012CH2-0\
pentanoate c
11 S
0
44 (+) biotin 4-amidobenzoic acid o
0
sodium salt
HN)LNNH
o
tv
oxohexahydro-1H-thieno
sodium 4-(5-((3aS,6aR)-2- o
0
0,
0.
[3,4-dlimidazol-4-y1) '1a0-1 41 NH
\c
w
cn
cn
4, S
IV
,... pentanamido) benzoate ii
0
o
0.
1
45 Biotin 2-N-acetylaraino-2- 0
0
ko
deoxy-13-D-glucopyranoside
1
1-.
0
HN,N.NH
((2R,5S)-3-acetamido-4,5-
dihydroxy-6-(hydroxymethy1)- CH2OH Him..
mitIH
2,3,4,5,6-pentamethyltetrahydro-
2H-pyran-2-yl)methyl 5-
((3a8,6aR)-2-oxohexahydro-1H- S
thieno[3,4-d]imidazol-4-y1) OH 0
pentanoate 1
HN¨C¨CH3
11
0
46 Biotin-a-D-N-acetylneurarainide 0
0
(2S,5R)-5-acetamido-4-hydroxy- H3c ¨CII
¨NH 0COOH
HN)\.NH
3,3,4,5,6-pentamethy1-245- (cHQH)2
((3aS,6aR)-2-oxohexahydro-11-1- cH2oH
Hlw..
..,IIIH
thieno[3,4-d]imidazol-4-y1) 0
pentanoyloxy)methyl)-6-(1,2,3-
trihydroxypropyl) tetrahydro- OH
S
2H-pyran-2-carboxylic acid 0
47 Biotin-a-L-fucoside 0
0
cH3
0
HN)NNH
((2R,5S)-3,4,5-trihydroxy-
"
c
2,3,4,5,6,6-
0,
0.
w
hexamethyltetrahydro-2H-pyran-71> Him.. mum
0,
0,
en 2-yl)methyl 5-((3aS,6aR)-2-
oxohexahydro-1H-tbieno[3,4- (\c
0
1-.
d]imidazol-4-yl)pentanoate OH ii s
0.
1
0 0
ko
1
,
48 Biotin lacto-N-bioside
o
co
HN)LNH
CH2OH
See end of table for name cH2oH
o --
OH 0 ss---0
OH
sl.......
....>
OH
OH
HN¨C---CH3
li 0
S
0
49 Biotin¨Lewis-A tfisaccharide
HN1NH
cH201-i
CH2OH CH3
See end of table for name
S
OH
HN1¨CH3 OH 0
0
=
50 Biotin¨Lewis-.Y tetrasac,charide o
HNANH
0
P
See end of table for name
H1110. mit1H o
CH2OH
rs.)
CH2OH CHa
co
*i........0)s.....i> 1
cn
o=
Lo
OH OH 0 0H
cn
OH HN¨C--CH3
01
41. OH 11
rs.)
0 0
1-`
CH3 \
0.
I
...L.1>Of.i \
0
tC)
0
I
1-`
OH
CO
OH
51 Biotin-a-D-mannopyranoside 0
CH2OH
((1R,4R)-2,3,4-trihydroxy-5- HNV\H
(hydroxymethyl)-1,2,3,4,5-
HUH,.
..itilH =
pentamethylcyclohexyl)methyl OH OH
5-((3a3,6aR)-2-oxohexahydro- OH 0\C
1H-thieno[3,4-d]imidazol-4-y1) s
ii
pentanoate 0
,
_
52 biotin 6-0-phospho-a-D- o
mannopyranoside cH20P03112
0 HN'ANH
((2R,5S)-3,4,5-trihydroxy-
2,3,4,5,6-pentamethy1-6- 0H H
(phosphonooxymethyptetrahydr OH \
o-2H-pyran-2-yl)methyl 5- ii s
((3aS,6aR)-2-oxohexahydro-1H- o
thieno[3,4-d]imidaw1-4-y1)
pentanoate
53 polychroraium-poly(bis)- .....
___
0
EN-(2,6-(ciiisopropy1pheny1) H,
cH,
0
carbamoyl methyl)imino diacetic cH,
acid] L fj
co
.,
CH2¨ 0.õ..
.../.0-- ¨CH2\
li H
W
0)
. 11 11 CH2 ../ -',C1**3
,N¨CH2¨C¨N . 0)
F. N \
Ce
CH2¨C-0 0 C CH'
"
iji il
0
I - `
0 =
I
CH 3
113 0
tO
CH 3
Cl-I3 I
I-,
n
co
¨
_
Names of Compounds 48-50.
48. ((2R,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,3,4,6-tetramethy1-4-
((a2S,5R)-3,4,5-trihydroxy-6-(hyciroxymethy1)-2,3,4,5,6-
pentamethyltetrahydro-2H-pyran-2-yl)methoxy)methyl) tetrahydro-2H-pyran-2-
yl)methyl 54(3aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-4]imidazol-4-yl)pentanoste ((2R,5S)-3-acetamido-5-hydroxy-6-
(hydroxymethyl)-2,3,4,6-tetramethyl-4-(a2S,5R)-3,4,5-
trihydroxy-6-(hydroxymethyl)-2,3,4,5,6-pentamethyltetrahydro-2H-pyran-2-
y1)methoxy)methyl) tetrahydro-2H-pyran-2-yl)methyl 5-
((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate
49. (2R,3R,5S)-5-((((2S,3S,5S)-3-acetamido-5-hydroxy-6-(hydroxymethyl)-2,4,6-
trimethy1-4-(0(2S,5R)-3,4,5-trihydroxy-6-
(hydroxymethyl)-2,3,4,5,6-pentamethyltetrabydro-2H-pyran-2-y1)methoxy)
metbyptetrahydro-2H-pyran-2-yl)methoxy)methyl)-3,4-
dihydroxy-2,4,5,6,6-pentamethyltetrahyciro-2H-pyran-2-yi 5-03aS,6aR)-2-
oxohexahydro-11-1-thieno[3,4-d]iraidazol-4-yl)pentanoate
50. (2S,5S)-3-acetamido-4-((a2R,5S)-5-((a2R,5S)-4,5-dihydroxy-6-
(hydroxymethyl)-2,3,4,5,6-pentamethy1-3-((((25,5S)-3,4,5-
trihydroxy-2,3,4,5,6,6-hexamethyltetrahydro-2H-pyran-2-
yl)methoxy)methyptetrahydro-2H-pyran-2-yl)methoxy) methyl)-3,4-
= dihydroxy-2,3,4,5,6,6-hexametbyltetrahydro-2H-pyran-2-yl)methoxy)methyl)-
5-hydroxy-6-(hydroxymethyl)-2,3,4,5,6-
pentamethyltetrahydro-2H-pyran-2-y15-((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-
djimidazol-4-y1)pentanoate
0
Structure of iminobiotin compounds are not shown in Table 3. The iminobiotin
structures are analogs of the biotin structure where the
biotin group is replaced by a an iminobiotin group. An example is shown below
with the analogs N-hydroxysuccinimide biotin and N- 0
1..)
bydroxysuccinimide iminobiotin.
co
0,
0.
w
0
NH 0)
4.
vz
A
0 13
HNVNNH
HN,NNH 0)
0"
I-.
0.
r------( HUM.. __ ...IIIIIH
"-----'\
\
Hlitokt outilfH I
to
I
I-.
,........yN¨
co
N-...
S
g
TI:.../ss.'N'''''N.N.CS
\\
1 0
N-hydroxysuccinimide biotin N-
hydroxysuccinimide iminobiotin
CA 02864366 2014-09-18
In an embodiment, a cellulose acetate hydrogen phthalate polymer is
incorporated into the lipid construct where it can bind to hydrophilic
functional groups on
the insulin molecule and protect insulin from hydrolytic degradation.
Cellulose acetate
hydrogen phthalate comprises two glucose molecules linked beta (1-4) in a
polymeric
arrangement in which some of the hydrogen atoms on the hydroxyl groups of the
polymer
are replaced by an acetyl functionality (a methyl group bound to a carbonyl
carbon) or a
phthalate group (represented by a benzene ring with two carboxyl groups in the
first and
second positions of the benzene ring). The structural formula of cellulose
acetate
hydrogen phthalate polymer is shown in Figure 9. Only one carboxyl group on
the
phthalate ring structure is involved in a covalent ester linkage to the
cellulose acetate
molecule. The other carboxyl group, which contains a carbonyl carbon and a
hydroxyl
functionality, participates in hydrogen bonding with neighboring negative and
positive
charged dipoles residing on insulin and various lipid molecules.
In an embodiment, cellulose acetate hydrogen phthalate polymer interacts
with the lipids through ion-dipole bonding with 1,2-distearoyl-sn-glyc,ero-3-
phosphocholine phosphate and dicetyl phosphate molecules. The ion-dipole
bonding
occurs between the 8+ hydrogen on the hydroxyl groups of cellulose and the
negatively
charged oxygen atom on the phosphate moiety of the phospholipid molecules. The
functional groups with the largest role in the ion-dipole interaction are the
negatively
charged oxygen atoms on the phosphate groups of the phospholipid molecules,
hydrogen
atoms on the hydroxyl groups and the hydrogen atoms on amide bonds of the
insulin
molecules. Negatively charged functional groups form sites for ion-dipole
interactions
and for reacting with the 8+ hydrogen atom on individual hydroxyl groups and
the
hydroxyl groups of the carboxyl functionalities on cellulose acetate hydrogen
phthalate.
Ion-dipoles can be formed between the positively charged quaternary amines on
the
phosphocholine functionalities and the 8-carbonyl oxygen found on cellulose
acetate
hydrogen phthalate and insulin. Sugar molecules comprising branched
hydrophilic
structures in insulin can participate in hydrogen bonding and ion-dipole
interactions.
The molecular configuration and the size of the polymer (with an
approximate molecular weight of 15,000 or more) enables cellulose acetate
hydrogen
- 50 -
CA 02864366 2014-09-18
phthalate to coat individual phospholipid molecules of the lipid construct in
the region of
the hydrophilic head group. This coating protects insulin within the lipid
construct from
the acid milieu of the stomach. There are several ways that cellulose acetate
hydrogen
phthalate can be attached to the surface of molecules within the lipid
construct. A
preferred means of linking cellulose acetate hydrogen phthalate to the surface
of the lipid
construct is to attach the polymeric cellulosic species to a tail of an
insulin molecule that
presents a sugar that projects from the surface of the lipid construct. This
protects the
insulin proteinaceous tails from enzymatic hydrolysis.
An extended amphipathic lipid comprises a variety of multi-dentate
binding sites for attachment to the receptor. Multi-dentate binding, as
defined herein,
requires a plurality of potential binding sites on the surface of insulin and
its
accompanying sugar moieties, as well as on the lipid construct that can
interface with
carbonyl, carboxyl and hydroxyl functional groups on the cellulose acetate
hydrogen
phthalate polymer. This enables the cellulose acetate hydrogen phthalate
polymer to bind
to a plurality of hydrophilic regions not only on the lipid construct but also
on molecules
of insulin in order to establish a shield of hydrolytic protection for the
lipid construct. In
this manner both insulin and the lipid construct are protected from the acid
environment
of the stomach following oral administration of the insulin dosage form. Even
though
cellulose acetate hydrogen phthalate covers or shields individual lipid
molecules within
and on the surface of the lipid construct while passing through the stomach,
once the
construct migrates to the alkaline region of the small intestine, cellulose
acetate hydrogen
phthalate is hydrolytically degraded. After cellulose acetate hydrogen
phthalate is
removed from the surface of the molecules of the lipid construct, a lipid
anchoring-
hepatocyte receptor binding molecule, such as 1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine-N-(Cap Biotinyl), becomes exposed and then is available to
bind
with the receptor. The employment of a cellulose acetate hydrogen phthalate
coating on
insulin and the lipid construct is needed to ensure that a greater
bioavailability of insulin
is achieved.
- 51 -
CA 02864366 2014-09-18
Target Molecule Complex
In an embodiment, the lipid construct comprises a target molecule
complex comprising multiple linked individual units formed by complexing a
bridging
component with a complexing agent. The bridging component is a water soluble
salt of a
metal capable of forming a water-insoluble coordinated complex with a
complexing
agent. A suitable metal is selected from the transition and inner transition
metals or
neighbors of the transition metals. The transition and inner transition metals
from which
the metal are selected from: Sc (scandium), Y (yttrium), La (lanthanum), Ac
(actinium),
the actinide series; Ti (titanium), Zr (zirconium), Hf (hafithun), V
(vanadium), Nb
(niobium), Ta (tantalum), Cr (chromium), Mo (molybdenum), W (tungsten), Mn
(manganese), Tc(technetium), Re (rhenium), Fe (iron), Co (cobalt), Ni
(nickel), Ru
(ruthenium), Rh (rhodium), Pd (palladium), Os (osmium), Ir (iridium), and Pt
(platinum).
The neighbors of the transition metals from which the metal can be selected
are: Cu
(copper), Ag (silver), Au (gold), Zn (zinc), Cd (cadmium), Hg (mercury), Al
(aluminum),
Ga (gallium), In (indium), Ti (thallium), Ge (germanium), Sn (tin), Pb (lead),
Sb
(antimony) and Bi (bismuth), and Po (polonium). Examples of metal compounds
useful
as bridging agents include chromium chloride (III) hexahydrate; chromium (III)
fluoride
tetrahydrate; chromium (III) bromide hexahydrate; zirconium (IV) citrate
anunonium
complex; zirconium (IV) chloride; zirconium (IV) fluoride hydrate; zirconium
(IV)
iodide; molybdenum (III) bromide; molybdenum (III) chloride; molybdenum (IV)
sulfide; iron (III) hydrate; iron (III) phosphate tetrahydrate, iron (III)
sulfate
pentahydrate, and the like.
The complexing agent is a compound capable of forming a water insoluble
coordinated complex with a bridging component There are several families of
suitable
complexing agents.
A complexing agent can be selected from the family of iminodiacetic
acids of the formula (1) where R1 is loweralkyl, aryl, arylloweralkyl, and a
heterocyclic
substituent.
- 52 -
CA 02864366 2014-09-18
0 0
II II
H0¨C¨CH2¨N¨CH2¨C¨OH
I
Lowerailkylene (1)
ril¨R1
OH
Suitable compounds of the formula (1) include:
N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,6-diethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(4-isopropylphenylcarbamoylmethyl) iminodiacetic acid;
N-(4-butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,3-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(3,4-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N- (3,5-dimethylphenylcarbamoylmethyl) iminodiacetic acid;
N-(3-butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(2-butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(4-tertiary butylphenylcarbamoylmethyl) iminodiacetic acid;
N-(3-butoxyphenylcarbamoylmethyl) iminodiacetic acid;
N-(2-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;
N-(4-hexyloxyphenylcarbamoylmethyl) iminodiacetic acid;
aminopyrrol iminodiacetic acid;
N-(3-bromo-2,4,6-trimethylphenylcatbamoylmethyl) iminodiacetic acid;
benzimidazole methyl iminodiacetic acid;
N-(3-cyano-4,5-dimethy1-2-pyrrylcarbamoylmethyl) iminodiacetic acid;
N-(3-cyano-4-methyl-5-benzy1-2-pyrrylc,arbamoylmethyl) iminodiacetic acid; and
N-(3-cyano-4-methyl-2-pynylcarbamoylmethyl) iminodiacetic acid and other
derivatives of N-(3-cyano-4-methyl-2-pyrrylcarbanaoylmethyl) iminodiacetic
acid of formula (2),
- 53 -
CA 02864366 2014-09-18
CH3 /CN
,,,CH2COOH
R3 N (2)
1 CH2COOH
R2
where R2 and R3 are the following:
Ra.
iso-C41-19
H CH2CH2SCH3
C112C6H4-p-OH
CH3 CH3
CH3 iso-C4H9
CH3 CH2CH2SCH3
CH3 C61-15
CH3 CH2C61-15
CH3 CH2C6H4-p-OCH3
A complexing agent is selected from the family of imino diacid
derivatives of the general formula (3), where R4, R3, and R6 are independent
of each other
and can be hydrogen, loweralkyl, aryl, aryllowerallcyl, alkoxyloweralkyl, and
heterocyclic.
0
11
R4-0¨C¨loweralkylene (3)
Suitable compounds of the formula (3) include: N'(2-acetylnaphthyl)
iminodiacetic acid (NAIDA); N'-(2-naphthylmethyl) iminodiacetic acid (NMIDA);
iminodiearboxymethy1-2-naphthylketone phthalein complexone; 3 (3: 7a: 12a:
tihydroxy-24-norchol any1-23-iminodiacetic acid; benzimidazole methyl
iminodiacetic
acid; and N- (5,pregnene-3-p-o1-2-oyl earbamoylmethyl) iminodiacetic acid.
A complexing agent is selected from the family of amino acids of formula
(4),
- 54-
CA 02864366 2014-09-18
0
II
(4)
where R7 is an amino acid side chain, Rs is loweralkyl, aryl,
aryllowerallcyl, and R9 is pyridoxylidene.
Suitable amino acids of the formula (4) are aliphatic amino acids,
including, but not limited to: g,lycine, alanine, valine, leucine, isoleucine;
hydroxyamino
acids, including serine, and threonine; dicarboxylic amino acids and their
amides,
including aspartic acid, asparagine, glutarnic acid, glutamine; amino acids
having basic
functions, including lysine, hydroxylysine, histidine, arginine; aromatic
amino acids,
including phenylalanine, tyrosine, tryptophan, thyroxine; and sulfur-
containing amino
acids, including cystine, methionine.
A complexing agent is selected from amino acid derivatives including, but
not necessarily limited to (3-alanine-y-amino) butyric acid, 0-
diazoacetylserine
(azasezine), homoserine, omithine, citrulline, penicillamine and members of
the
pyridoxylidene class of compounds including, but are not limited to:
pyridoxylidene
glutamate; pyridoxylidene isoleucine; pyridoxylidene phenylalanine;
pyridoxylidene
tryptophan; pyridoxylidene-5-methyl tryptophan; pyridoxylidene-5-
hydroxytryptamine;
and pyridoxylidene-5-butyltryptamine.
A complexing agent is selected from the family of diarnines of the general
formula (6),
RiiCOORio
R12¨N¨loweralkylene¨N (6)
RiiC001210
Ri3
where Rio is hydrogen, loweralkyl, or aryl; R11 is lowerallcylene or
arylloweralky; R12
and Ri3 independently are hydrogen, loweralkyl, alkyl, aryl, arylloweralkyl,
acylheterocyclic, toluene, .sulfonyl or tosylate.
Some suitable diamines of the formula (6) include, but are not limited to,
ethylenediamine-N, N diacetic acid; ethylenediamine-N,N-bis (-2-hydroxy-5-
- 55 -
CA 02864366 2014-09-18
bromophenyl) acetate; N'aoetylethylenediamine-N,N diacetic acid; N'-benzoyl
ethylenediamine-N,N diacetic acid; N'-(p-toluenesulfonyl) ethylenediamine-N, N
diacetic
acid; N'-(p-t-butylbenzoyl) ethyhmediamine-N, N diacetic acid; N'-
(benzenesulfonyl)
ethylenediamine-N, N diacetic acid; N'- (p-chlorobenzenesulfonyl)
ethylenediamine-N, N
diacetic acid; N'(p-ethylbenzenesulfonyl ethylenediamine-N,N diacetic acid; N'-
acyl and
N'-sulfonyl ethylenediamine-N, N diacetic acid; N'- (p-n-
propylbenzenesulfonyl)
ethylenediamine-N, N diacetic acid; N'- (naphthalene-2-sulfonyl)
ethylenediamine-N, N
diacetic acid; and N'- (2, 5-dimethylbenzenesulfonyl) ethylenediamine-N, N
diacetic acid.
Other suitable complexing compounds or agents include, but are not
limited to: penicillamine; p-mercaptoisobutyric acid; dihydrothioctic acid; 6-
mercaptopurine; kethoxal-bis(tbiosemicarbazone); Hepatobiliary Amine
Complexes, 1-
hydrazinophthalazine (hydralazine); sulfonyl urea; Hepatobiliary Amino Acid
Schiff
Base Complexes; pyridoxylidene glutamate; pyridoxylidene isoleucine;
pyridoxylidene
phenylalanine; pyridoxylidene tryptophan; pyridoxylidene 5-methyl tryptophan;
pyridoxylidene-5-hydroxytryptamine; pyridoxylidene-5-butyltryptamine;
tetracycline; 7-
carboxy-p-hydroxyquinoline; phenolphthalein; eosin I bluish; eosin I
yellowish;
verograffin; 3-hydroxyl-4-formyl-pyridene glutamic acid; Azo substituted
iminodiacetic
acid; hepatobiliary dye complexes, such as rose bengal; congo red;
bromosulfophthalein;
bromophenol blue; toluidine blue; and indocyanine green; hepatobiliary
contrast agents,
such as iodipamide; and ioglycamic acid; bile salts, such as bilirubin;
cholgycyliodohistamine; and thyroxine; hepatobiliary thio complexes, such as
penicillamine; p-mercaptoisobutpie acid; dihydrothiocytic acid; 6-
mercaptopurine; and
kethoxal-bis (thiosemicarbazone); hepatobiliary amine complexes, such as 1-
hydrazinophthalazine (hydralazine); and sulfonyl urea; hepatobiliary amino
acid Schiff
Base complexes, including pyridoxylidene-5-hydroxytryptainine; and
pyridoxylidene-5-
butyltryptamine; hepatobiliary protein complexes, such as protamine; faritin;
and asialo-
orosomucoid; and asialo complexes, such as lactosaminated albumin;
immunoglobulins,
G, IgG; and hemoglobin.
The three-dimensional target molecule complex made from combining
bridging agents and complexing agents is described in WO 99/59545.
In an embodiment, the bridging agent is a metal salt,
- 56 -
CA 02864366 2014-09-18
such as chromium chloride hexahydrate, capable of forming a coordinated
complex with
complexing agents, such as N-(2,6-diisopropylphenylcarbamoylmethyl)
hninodiacetic
acid. The bridging agent and the complexing agents are combined to form a
complex
composed of multiple linked units in a three-dimensional array. In a preferred
embodiment, the complex is composed of multiple units of chromium (his) (N-
(2,6-
(diisopropylphenyl)carbamoyl methyl)imino diacetic acid] linked together. In
an
embodiment, the chromium target molecule complex substance is soluble in a
mixture of
lipids containing 1,2-distearoyl-sn-glycero-3-phosphocholine, dicetyl
phosphate and
cholesterol. The complex is incorporated within a lipid construct formed from
the groups
of lipids previously described.
Modification of the Isoelectric Point of Insulin
The isoelectric point of a protein can affect the release and distribution of
the protein in the body of a patient treated with the protein. By changing the
isolectric
IS point of a protein, the rate of release of the protein from the site of
administration may be
altered and the pharmacoldnetics of the protein can be changed.
One method of altering the isolectric point of insulin is to alter its
molecular structure by substituting or adding various amino acids. Two
examples of
altering the structure of insulin to obtain different properties are glargine
insulin and
insulin aspart. Both of these insulins differ in amino acid composition from
recombinant
human regular insulin. Recombinant human regular insulin has an isoelectric
point at
5.30 ¨ 5.35. Glargine insulin substitutes glycine for asparagine at position
A21 and two
arginines are added at the C-terminus of the B chain. The isoelectric points
of glycine
and asparagine are 5.97 and 5.41, respectively. The substitution of glycine
for asparagine
has little or no effect on the isoelectric point of glargine insulin. However,
the addition of
two highly basic arginine amino acid residues, with isoelectric points of
10.76,
significantly raises the isoelectric point of glargine insulin to pH 5.8 ¨
6.2.
Insulin aspart substitutes aspartic acid for praline at position B-28. The
isoelectric points of aspartic acid and praline are 2.97 and 6.10,
respectively. With this
single acidic amino acid substitution, the isoelectric point of insulin aspart
is shifted
significantly toward a lower, more acidic, pH.
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These two examples of commercially available insulins illustrate how a
relatively small number of amino acid substitutions can significantly either
raise or lower
the isoelectric points of insulin glargine or insulin aspart with respect to
recombinant
human regular insulin. By altering the chemical properties of insulin, the
bioavailability
__ and pharmacodynamic profiles are also changed. When an insulin with a
modified
structure is administered to a diabetic patient in order to improve
bioavailability, the new
phamiacological responses provide new therapeutic benefits.
The isoelectric point of insulins can be modified not only by internal
molecular restructuring of the primary amino acid sequences of insulin, but
also by
__ binding charged organic molecules to insulin. The charged organic molecules
can be
bound to the surface, or within the insulin structure. The isoelectric point
of native
insulin can be changed from pH 5.3 to pH 7.2 by adding between 1.0 and 1.5 mg
of a
mixture of highly basic proteins to 1.0 ml of an insulin solution containing
100 units or
3.65 mg of insulin/ml. Protamines are an example of a group of simple, highly
basic
__ proteins that can be can used to alter the isoelectric point of insulin.
Protarnines yield
numerous basic amino acids on hydrolysis, possess a high nitrogen content and
occur
naturally, combined with nucleic acid, in the sperm of fish. For example, the
protamines
salmine, clupeine, iridine, sturine and scombrine are isolated from salmon,
herring, trout,
sturgeon and mackerel sperm, respectively. These basic proteins, either
individually or
__ as a mixture, associate with insulin and increase the isoelectric point of
insulin.
Compounds that alter the surface charge of insulin include derivatives of
polylysine and other highly basic amino acid polymers, such as polyomithine,
polyhydroxylysine, polyarginine and polyhistidine or combinations thereof.
Other
polymers include poly (arg-pro-thr)õ in a mole ratio of 1:1:1 with a molecular
weight
__ range of a few hundred to several thousand or poly (DL-Ala-poly-L-lys)õ in
a mole ratio
of 6:1 with a molecular weight range of a few hundred to several thousand.
Histones,
basic proteins that exist in several subtypes that contain different and
varying amounts of
arginine, lysine and other basic amino acids that can bind ionically to
carboxyl groups of
insulin, and fragments of histones, are also used to provide a positive
charge. Also
__ included are polymers such as polyglucosarnine, polygalactosamine and
various other
sugar polymers that contain a positive charge contributed by a primary amino
group.
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Polynucleotides such as polyadenine, polycytosine or polyguanine that provide
a positive
charge through the ionization of their primary amino group are also used. All
the above
polymeric species when bound to insulin provide an increase in positive charge
that is
accompanied by an increase in the isoelectric point of insulin. Small amounts
of these
polymeric compounds, such as a few micrograms of polymer/ml of insulin,
areadded to
change the isoelectric point of insulin a minimal amount, generally less that
one pH unit.
Larger amounts, generally greater than a milligram or two, of basic organic
compounds
can be added per ml of insulin at 100 units/mi. to progressively increase the
isoelectric
point of insulin to more than two pH units beyond its native isoelectric
point.
Conversely, the isoelectric point of insulin can be lowered in a similar
fashion by adding carboxylated polymers and polymeric amino acids such as
polyaspartic
acid, polyglutamic acid, proteins or fragments of proteins that contain large
amounts of
amino acid residues with carboxyl (COO-) or sulfhydral (S.) functional groups.
Highly
basic proteins can be changed to highly acidic proteins by reacting them with
an
appropriate anhydride, such as acetic anhydride, to form a negatively charged
terminal
acidic carboxyl group in place of a positively charged basic primary amino
group. Other
acidic polymers, such as sulfate-laden polymers, may be added to insulin to
lower the
isoelectric point of insulin. Sugar polymers such as polygalacturonic acid,
polyglueonic
acid, polyglucuronic acid or polyglucaric acid that contain negatively charged
carboxyl
groups can be used to lower the isoelectric point of the protein.
Changing the isoelectric point of an insulin alters not only the ionic
character of the native insulin molecule, but also the nature of the ionic
envelope, known
as the Hemholtz double layer, that surrounds insulin and extends into the bulk
phase
aqueous media around the insulin. The ionic environment surrounding insulin
tends to
exist in layers with a layer of counter-ions associated with the participating
charged
organic molecules that are bound to insulin. An electric potential exists on
modified
insulin molecules that are maintained in a colloidal suspension in bulk phase
media
because of the presence of ions on the surface of insulin. That part of the
electric
potential existing between the layer of fixed counter ions associated with the
bound
organic molecules and that of the bulk phase media is know as the
electrokinetic or zeta (
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4 ) potential. The zeta potential contributes significantly to the electrical
properties and
stability of colloidal systems such as insulin in aqueous media.
As a result of forming a different chemical structure by the addition of
material to change the isoelectric point , the stability of the protein
insulin in colloidal
suspension is inherently altered. Insulin experiences a shift in stability at
the newly
modified isoelectric point due to a lower zeta potential. Insulin is least
stable when it is
in the zwitterionic, or hybrid form, where the negatively charged functional
groups
precisely balance the positively charged functional groups and create an
overall net zero
charge on the protein. Even though the overall net charge is zero, there
remain pockets of
negative charge and pockets of positive charge throughout the protein
structure. As the
pH of a solution of insulin reaches its isoelectric point, its solubility
decreases and insulin
may precipitate from solution. During the isoelectric precipitation of
insulin, the
insulating and dielectric properties of the bulk phase aqueous buffer media
are overcome
and the ion atmosphere of the Hemholtz double layer is fractured so that
dissimilar
charges between colloidal particles can associate which leads to a protein
colloidal
suspension with increasing instability. These effects eventually result in the
coagulation
and subsequent precipitation of the protein at the isoelectric point. The
ideal range for
isoelectic precipitation is two or three pH units above or below the
isoelectric point of
insulin at pH 5.3. However, isoelectric points extending beyond this pH range
may be
formulated through the use of information by one skilled in the art.
As the pH changes from the isoelectric point, solubility increase and
insulin that precipitated at the isoelectric point can be resolubilized. This
occurs because
as the pH is increased or decreased from the isoelectric point, there is a
respective
accumulation of negative charge (above the isoelectric point) or an
accumulation of
positive charge (below the isoelectric point) that is regulated by the pKa of
the
representative functional groups. Resolubilization occurs as the protein
develops a
greater disparity of charge thereby increasing the zeta potential of the
protein which in
turn improves protein stability. These effects result in the redevelopment of
an ionic
envelope which surrounds the protein which facilitates greater colloidal
dispersion of the
insulin molecules.
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The isoelectric point of native insulin, which occurs at pH 5.3, can be
progressively raised by adding proteins, peptide fragments, polymers or
polymer
fragments that bind to insulin and alter the ionic character of insulin. The
overall affect
of adding basic functional groups is to raise the isoelectric point of insulin
and create an
insulin that has a slower onset of pharmacological action by having the
insulin transition
between a soluble from, to an insoluble form, and then to a new soluble form.
By
modifying the isoelectric point of native insulin, especially in the presence
of HDV
insulin, the bioavailability of both insulin forms can be regulated.
Insulins in which the isoelectric point was altered by changing the amino
acid sequence can be incorporated into a lipid construct. In an embodiment,
glargine
insulin is incorporated into a target molecule complex comprising a lipid and
multiple
linked individual units formed by complexing a bridging component with a
complexing
agent A description of the target molecule complex and its components was
previously
described herein. The structure of glargine insulin is provided in Figure 11.
Glargine
insulin differs from human insulin in that glargine insulin has a molecular
structure that
replaces asparagine with glycine at the C-terminal end of the A chain of human
insulin
and adds the dipeptide of arginine at the C-terminal end of the B chain of
human insulin.
The isoelectric point of a compound is the pH at which the overall charge of
the
compound is neutral. However, regions of negative and positive charges still
remain
within the compound. The isoelectric point of human insulin is at pH 5.3. The
isoelectric point of glargine insulin is higher than human insulin because the
amino acid
substitutions in glargine insulin raise the isoelectric point of glargine
insulin to pH 5.8-
6.2. Compounds are generally less soluble in aqueous solutions at ranges
around the
isoelectric point. A compound is generally more soluble in aqueous systems
where the
pH of the solution is approximately 1-2 pH units higher or lower than the
isoelectric
point. The higher isoelectric point allows glargine insulin to remain soluble
in a mildly
acidic environment over a broader pH range.
A commercial form of glargine insulin, LANTUS (insulin glargine
[rDNA origin] injection), is a sterile solution of glargine insulin for use as
an injectable
insulin for diabetic patients for subsequent management of glucose levels in
vivo.
Glargine insulin is a recombinant human insulin analog that is a long-acting
(up to 24-
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CA 02864366 2014-09-18
hour duration of action), parenteral blood-glucose-lowering agent. LANTUS is
produced by recombinant DNA technology utilizing a non-pathogenic laboratory
strain of
Escherichia colt (I(12) as the production organism. LANTUS consists of
glargine insulin
dissolved in a clear aqueous fluid. Each milliliter of LANTUS (insulin
glargine
injection) contains 100 TU (3.6378 mg) glargine insulin, 30 mcg zinc, 2.7 mg m-
cresol,
20 mg glycerol 85%, and water for injection. The pH of commercially available
LANTUS insulin can be adjusted by addition of aqueous solutions of acids,
bases or
buffers that are physiologically compatible. LANTUS has a pH of approximately
4.
A depiction of a pharmaceutical composition that combines free insulin
and insulin associated with a target molecule complex is shown in Figure 13.
In an
embodiment, a pharmaceutical composition may comprise two or more insulins.
The
target molecule complex comprises multiple linked individual units formed by
complexing a bridging component with a complexing agent. The bridging
component is
a water soluble salt of a metal capable of forming a water-insoluble
coordinated complex
with a complexing agent. A suitable metal is selected from the transition and
inner
transition metals or neighbors of the transition metals. A description of the
target
molecule complex and its components was previously described herein. In an
embodiment, a pharmaceutical composition comprises a mixture of free insulin
and
insulin associated with a water insoluble target molecule complex. Free
insulin is not
associated with the target molecule complex and is soluble in water. The other
form of
insulin in the composition is associated with a water insoluble target
molecule complex.
Adjustment of the pH of an aqueous solution surrounding the lipid
construct containing the target molecule complex, by the addition of acids,
bases, or
buffers, results in a negative charge in the lipid construct structure. The pH
range at
which this occurs depends upon the composition of the lipids. A preferred
lipid system is
a mixture of 1,2-distearoyl-sn-g,lycero-3-phosphocholine, cholesterol and
dicetylphosphate. This mixture forms a negatively charged lipid construct
structure
under physiological conditions. The lipid construct exhibits hepatocyte
targeting
specificity, i.e. is specific for cellular hepatocytes, thereby allowing the
construct to be
targeted to the liver.
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It has been discovered in the present invention that when the appropriate
lipid components are formulated into a water insoluble target molecule complex
using
Sterile Water for Injection, US? (SWI) that has been terminally pH adjusted to
pH 3.95
0.2, the overall electronic charge on the target molecule complex is
predominately
negative. Glargine insulin has a net positive charge at pH 5.2 E 0.5, which is
below the
isoelectric point of the protein. The positive charge on glargine insulin at
pH 5.2 0.5
allows for interaction of the positively charged portion of glargine insulin
with the
negatively charge portion of the target molecule complex. This results in
positively
charged glargine insulin being attracted to the negatively charged target
molecule
complex. Portions of the charged glargine insulin become associated with
charges on the
lipids and the charged glargine insulin moves within the lipids, while other
charged
glargine insulin molecules are sequestered within the core volume of the lipid
construct
after partitioning through the various lipid moieties of the lipid construct.
There is an equilibrium between free glargine insulin in solution and
glargine insulin associated with the water insoluble target molecule complex.
Because
the interactions between glargine insulin and the target molecule complex
involve
equilibria, over time free glargine insulin is able to further bind and
partition into the lipid
domains and/or the central core volume of the water insoluble target molecule
complex.
In an embodiment, free glargine insulin can be transformed into transitory
lipid
derivatives by adsorbing onto, or reacting with, individual molecules of lipid
that are in
equilibrium with the water insoluble target molecule complex. These
derivatives
associate with the lipids of the water insoluble target molecule complex and
enter the
core-volume of the complex, thus affecting the pharmacological activity of the
product.
In.sulins in which the isoelectric point was altered by binding charged
organic molecules to insulin can be incorporated into a lipid construct. In an
embodiment, recombinant human insulin isophane is incorporated into a target
molecule
complex comprising a lipid and multiple linked individual units formed by
complexing a
bridging component with a complexing agent.
The structure of recombinant human insulin isophane and protamine are
provided in Figure 12. Recombinant human insulin isophane differs from human
insulin
in that recombinant human insulin has been treated with protamine such that
protamine
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CA 02864366 2014-09-18
forms a coating over the insulin. The isoelectric point of recombinant human
insulin
isophane (pH 7.2) is higher than human insulin (pH 5.3) because the addition
of
protamine to recombinant human insulin isophane raises the isoelectric point
of the
protein. The higher isoelectric point allows recombinant human insulin
isophane to
__ remain insoluble at physiological pH. The Humulin NPH insulin product
currently
marketed exists as a milky suspension where recombinant human insulin isophane
settles
to the bottom of the vial.
In an embodiment, a pharmaceutical composition comprises a mixture of
free recombinant human insulin isophane and free recombinant human regular
insulin
__ and recombinant human insulin isophane and recombinant human regular
insulin that is
associated with a water insoluble target molecule complex. Free recombinant
human
insulin isophane is the material depicted in Figure 12. Free recombinant human
insulin
isophane is not associated with the target molecule complex and is insoluble
at
physiological pH of approximately 7.2, the isoelectric point of NPH insulin.
__ Recombinant human regular insulin is soluble at pH 7.2.
For each of the insulins, there is an equilibrium between the free form of
insulin in solution or suspension and the forms of the insulin associated with
the water
insoluble target molecule complex. Because the interactions between each form
of
insulin and the target molecule complex involve equilibria, over time the free
forms of
__ the insulins bind and partition into the lipid domains and/or the central
core volume of the
water insoluble target molecule complex. In an embodiment, free recombinant
human
insulin isophane and recombinant human regular insulin can be transformed into
transitory lipid derivatives by adsorbing onto, or reacting with, individual
molecules of
lipid that are in equilibrium with the water insoluble target molecule
complex. These
__ derivatives associate with the lipids of the water insoluble target
molecule complex and
enter the core-volume of the complex, thus affecting the pharmacological
activity of the
product.
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Description of the Invention. Method of Manufacturing the Lipid Construct
Figure 14 demonstrates an outline for the process for manufacturing a
lipid construct comprising an amphipathic lipid, an extended amphipathic lipid
and
" insulin. The manufacture of the composition comprises three overall
steps: preparing a
mixture of an amphipathic lipid and an extended amphipathic lipid, preparing a
lipid
construct from the mixture of an amphipathic lipid and an extended amphipathic
lipid,
and combining insulin into the lipid construct.
Lipids are produced and loaded by the methods disclosed herein, and those
methods described in U. S. Patent Nos. 4,946,787; 4,603,044; and 5,104,661,
and the
references cited therein. Typically, the aqueous lipid construct formulations
of the
invention comprise 0.1% to 10% active agent by weight (i.e. 1-10 mg drug per
ml), and
0.1% to 4% lipid by weight in an aqueous solution, optionally containing salts
and
buffers, in a quantity to make 100% by volume. Preferred are formulations
which
comprise 0.1% to 5% active agent. Most preferred is a formulation comprising
0.01% to
5% active agent by weight and up to 2% by weight of a lipid component in an
amount of
aqueous solution sufficient (q. s.) to make 100% by volume.
In an embodiment, the lipid construct is prepared by the following
procedure. Individual lipid constituents are mixed together in an organic
solvent system
where the solvent had been dried over molecular sieves for approximately two
hours to
remove any residual water that may have accompanied the solvent. In an
embodiment,
the solvent system comprises a mixture chloroform and methanol in the ratio
2:1 by
volume. Other organic solvents that can be easily removed from a mixture of
dried lipids
also can be used. Use of a single-step addition of the lipid constituents in
the initial
mixing procedure obviates the need for introducing any additional coupling
reactions
which would unnecessarily complicate the structure of the lipid construct and
require
additional separation procedures. The lipid components and the hepatocyte
receptor
binding molecule are dissolved in the solvent, then the solvent is removed
under high
vacuum until a dried mixture of the lipids forms. In an embodiment, the
solvent is
removed under vacuum using a rotoevaporator, or other methods known in the
art, with
slow turning at approximately 60 C for approximately two hours. This mixture
of lipids
can be stored for further use, or used directly.
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CA 02864366 2014-09-18
The lipid construct is prepared from the dried mixture of amphipathic
lipids and an extended amphipathic lipid. The dried mixture of lipids are
added to an
appropriate amount of aqueous buffered media, then the mixture is swirled to
form a
homogeneous suspension. The lipid mixture is then heated with mixing at
approximately
80 C for approximately 30 minutes under a dry nitrogen atmosphere. The heated
homogeneous suspension is immediately transferred to a micro-fluidizer
preheated to
approximately 70 C. The suspension is passed through the microfluidizer. The
suspension may require additional passes through the microfluidizer to obtain
a
homogeneous lipid micro-suspension. In an embodiment a Model #M-110 EHI micro-
fluidizer was used where the pressure on the first pass was approximately
9,000 psig. A
second pass of the lipid suspension through the micro-fluidizer may be needed
to produce
a product that exhibits the properties of a homogeneous lipid micro-
suspension. This
product is defined structurally and morphologically as a three-dimensional
lipid construct
which contains a hepatocyte receptor binding molecule.
Insulin is loaded into the lipid constructs using one of two methods:
equilibrium loading and non-equilibrium loading. Equilibrium loading of
insulin begins
when insulin is added to a suspension of the lipid constructs. Over time,
insulin
molecules move into and out of the lipid construct. The movement is governed
by
partitioning equilibrium, where movement into the lipid construct after the
initial
introduction of insulin to the suspension.
Non-equilibrium loading of insulin into the lipid constructs localizes
insulin within the lipid construct. Following equilibrium loading of free
insulin into the
lipid construct, the bulk phase media that contains free insulin is removed.
The non-
equilibrium loading procedure is a vector-driven process that begins the
instant the
external bulk phase media is removed. The gradient potential for insulin to
migrate out
of the lipid constructs is eliminated when the aqueous phase containing
insulin has been
removed. The overall process results in a greater concentration of insulin
within the final
lipid construct because movement of insulin from within the construct is
eliminated, The
equilibrium loading of insulin is a time-dependent phenomenon whereas the non-
equilibrium loading procedure is practically instantaneous. Non-equilibrium
loading can
be initiated by a variety of processes where the material in solution is
separated from the
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CA 02864366 2014-09-18
lipid construct. Examples of such processes include, but are not limited to:
filtration,
centricon filtration, centrifugation, batch style affinity chromatography,
streptavidin
agarose affinity-gel chromatography or batch style ion-exchange
chromatography. Any
means that eliminates the gradient potential for insulin diffusion and leakage
and causes
the insulin to be retained by the lipid construct can be utilized.
When using batch-style chromatography, the affinity or ion-exchange gel
is mixed rapidly with the mixture of insulin and the construct Binding to the
chromatography medium occurs rapidly and the chromatography medium is removed
from the aqueous media by decanting of the aqueous phase or by using classic
filtering
techniques such as the use of filter paper and a Buchner funnel.
The lipid construct contains a discrete amount of loaded insulin located
not only inside, but also within and on the surface of the lipid construct The
lipid
construct created is a new and novel composition of matter and becomes a
composition
for delivering an effective amount of insulin as a result of non-equilibrium
loading. The
loading of insulin into this lipid construct and the subsequent removal of
bulk phase
insulin results in a high concentration of insulin in a lipid construct by
shortening the
length of time needed for removal of the external phase media. It would be
difficult to
achieve this level of loading insulin into the construct using time-dependent
procedures,
such as ion-exchange or gel-filtration chromatography, since these procedures
require a
constant infusion of buffer comprising high concentrations of insulin. For
example,
loading insulin into the construct using small scale column chromatography
requires
approximately twenty minutes to remove the external bulk phase media
containing
insulin from the construct containing insulin. Equilibrium conditions are
reestablished
during this time period by movement of insulin from the construct. Maintaining
a high
concentration of insulin in and on the lipid construct is one of the positive
benefits of
using non-equilibrium loading.
In an extension of the non-equilibrium loading process, cellulose acetate
hydrogen phthalate is added to the lipid construct during the step of loading
insulin to the
lipid construct after the insulin has undergone equilibrium loading but before
the non-
equilibrium loading process is initiated. The nature and structure of the
insulin molecule
allows it to be intercalated into the lipid construct were insulin is
dispersed throughout
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the lipid construct. Hydrophilic portions of insulin, as well as branched
complex sugars
and additional functional groups, extend into the bulk phase media from the
surface of
the lipid construct. These extended hydrophilic portions of insulin can
participate in
hydrogen bonding, dipole-dipole and ion-dipole interactions at the surface of
the lipid
construct with the hydroxyl groups, carboxyl groups and carbonyl
functionalities of
cellulose acetate hydrogen phthalate as illustrated in Figure 9. Cellulose
acetate
hydrogen phthalate offers a unique means of combining with the molecules of
the lipid
construct to provide an excellent shield for masking the contents of the lipid
construct
from the digestive milieu of the stomach. The digestive processes in the
stomach result
from the hydrolytic cleavage of proteinaceous substrates by the enzyme pepsin
as well as
cleavage by acid hydrolysis. The acidic environment of the stomach degrades
free
insulin and can hydrolyze the ester bonds that hold the acyl hydrocarbon
chains to the
glycerol backbone in the phospholipid molecules. Hydrolytic cleavage can also
occur on
either side of the phosphate functionality in the phosphocholine group. The
digestive
system changes from the acid region of the stomach to an alkaline region of
the small
intestine were enzymatic action of trypsin and chymotrypsin occurs. Amino acid
lysing
enzymes, such as alpha amino peptidases, can degrade proteins such as insulin
from the
N-tenninal end. The presence of cellulose acetate hydrogen phthalate in the
lipid
construct protects insulin from hydrolytic degradation. As the alkaline
environment of
the small intestine hydrolytically degrades the cellulose acetate hydrogen
phthalate shield
of the lipid construct the hepatocyte receptor binding molecule becomes
available to
direct binding of the construct to the hepatocyte binding receptor. While not
wishing to
be bound by any particular theory, there is a synergy of hydrolytic protection
upon the
addition of cellulose acetate hydrogen phthalate at the end point of non-
equilibrium
loading. The protection is distributed not only to insulin and individual
lipid molecules,
but also to the entire lipid construct. This synergy provides collective as
well as
individual molecular protection from enzymatic and acid hydrolysis.
In an embodiment, cellulose acetate hydrogen phthalate is covalently
bound to either insulin or the lipid construct using a variety of methods. For
example,
one method involves coupling the hydroxyl groups on cellulose acetate hydrogen
phthalate with the amine functionalities on either 1,2-diacyl-sn-glycero-3-
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CA 02864366 2014-09-18
phosphoethanolamine or the &amino group of the ten L-lysines in the insulin
molecule
utilizing the Mannich reaction.
In an embodiment, cellulose acetate hydrogen phthalate is loaded into the
lipid construct during equilibrium loading of insulin into the construct. The
hydroxyl and
carbonyl functionalities of the cellulose acetate hydrogen phthalate hydrogen
bond with
lipid molecules in a lipid construct. Hydrogen bonds between cellulose acetate
hydrogen
phthalate and the construct are formed concurrently as insulin is loaded under
equilibrium
conditions into the lipid construct creating a shield around insulin and
around the
construct.
HDV-Insulin is recovered and recycled from aqueous media by binding it
to streptavidin-agarose iminobiotin. Streptavidin covalently bound to cyanogen
bromide
activated agarose provides a means to separate an iminobiotin-based lipid
construct from
insulin in the aqueous media at the end of non-equilibrium loading of insulin
into the
construct. In an embodiment, an iminobiotin derivative forms the hepatocyte
receptor
binding portion of the phospholipid moiety within the lipid construct. The
water-soluble
portion of the lipid anchoring molecule extends approximately 30 angstroms
from the
lipid surface to facilitate binding of the hepatocyte receptor binding portion
of the
phospholipid moiety with a hepatocyte receptor and to aid in the attachment of
the lipid
construct to streptavidin.
Streptavidin reversibly binds to iminobiotin at pH values of 9.5 and
greater, where the uncharged guandino functional group of iminobiotin strongly
binds to
one of the four binding sites on streptavidin located approximately nine
angstroms below
the surface of the protein. A lipid construct containing iminobiotin is
removed from
buffered media by raising the pH of an aqueous mixture of the construct to pH
9.5 by the
addition of a 20 mM sodium carbonate-sodium bicarbonate buffer. At this pH,
the bulk
phase media contains free insulin which is reclaimed and separated from the
lipid
construct using a variety of procedures including to, but not limited to
filtration,
centrifugation or chromatography.
The mixture at pH 9.5 is then mixed with streptavidin-agarose cross-
linked beads, where the construct is adsorbed onto the streptavidin. The
beads, which are
approximately 120 microns in diameter, are separated from the solution by
filtration. The
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lipid construct is released from the streptavidin-agarose affinity-gel by
reducing the pH
from pH 9.5 to pH 4.5 by the addition of a 20 mM sodium acetate-acetic acid
buffer at pH
4.5. At pH 4.5 the guandino group of iminobiotin becomes protonated and
positively
charged, as shown in Figure 10. The lipid construct is released and separated
from the
streptavidin-agarose bead by filtration. The streptavidin-agarose bead are
reclaimed for
additional usage. Thus both free insulin and streptavidin¨agarose are
conserved and can
be re-used.
In an embodiment, a composition that provides for the extended release of
insulin is produced when iminobiotin or iminobiocytin lipid constructs are
loaded with
insulin using streptavidin-agarose beads. When the pH of the forementioned
construct is
adjusted from pH 9.5 to pH 4.5 insulin will precipitate within the lipid
construct at
approximately pH 5.9. The isoelectric point of insulin is at pH 5.9 and
represents the pH
at which insulin has its lowest water-solubility. Over a pH range from pH 5.9
to pH 6.7
insulin remains essentially insoluble and exhibits properties that are
commonly attributed
to particulate matter. The insolubiliz,ed insulin within a lipid construct
creates a novel
insulin formulation that provides for the time-release of insulin molecules
when
administered by subcutaneous injection or through oral dosing. Solubilization
of insulin
is initiated as the pH of the lipid construct approaches pH 7.4.
The lipid construct is freeze-dried or kept in a non-aqueous environment
prior to dosing. In an aqueous dosage form of insulin, the pH of the insulin
solution is
maintained at approximately pH 6.5 in order to maintain insulin in the
insoluble form.
When insulin is exposed to an external pH gradient in vivo insulin is
solubilized and
move from the lipid construct, thereby supplying insulin to other virus-
harboring tissues.
Insulin remaining with the lipid construct maintains the capability of being
directed to the
hepatocyte binding receptor on the hepatocytes in the liver. Therefore two
forms of
insulin are produced from this particular lipid construct. In an in vivo
setting, free and
lipid associated insulin are generated in a time-dependent manner. It is
anticipated that
the solubilization of insulin that is lipid associated, as previously
described, can be
manufactured to release of insulin over a designated time-release period. This
could lead
to less frequent dosing schedules for patients afflicted with diabetes.
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CA 02864366 2014-09-18
In a preferred embodiment, insulin molecules move into the lipid construct
and become sequestered within the lipid domains of the loaded lipid construct.
A vector-
driven process is employed to move insulin molecules in one direction during
the final
phase of the insulin loading procedure when the chemical equilibrium is
disrupted.
During the final phase of insulin loading, the buffer or aqueous media is
rapidly removed
so that the insulin molecules associated with the lipid construct are deprived
of an
external media into which to migrate. Removal of the external media
effectively
quenches the equilibrium between insulin associated with the lipid construct
and insulin
solubilized in the external media. This process is termed non-equilibrium
loading, as
decribed elsewhere herein.
In an embodiment, a lipid construct is loaded with insulin using
equilibrium methods, an insulin concentration of 273,000 units of insulin per
microgram
of protein is selected to initiate the loading procedure. Equilibrium loading
continues
until the lipid construct is saturated with insulin.
The end process of non-equilibrium loading of insulin into the lipid
construct requires using a procedure that separates the solid lipid construct
from the
buffered media containing free insulin. In an embodiment, a filtration
procedure with a
very fine micro-pore synthetic membrane is used to separate the lipid
construct from the
external media. In another embodiment, a centricon device equipped with an
appropriate
filter with a 100,000 molecular weight cut off membrane, such as NanoSep
filter is used
to remove the lipid construct from the buffered media containing free insulin.
The
concentration of insulin in the lipid construct is maintained because
associated insulin is
no longer in equilibrium with the free insulin molecules located in the bulk
phase media
that had been removed from the construct. Free insulin which was in solution
is available
to load other lipid constructs. Thus, the vector-driven process of
concentrating insulin
within the lipid construct is achieved in one-step in essentially a time-
independent
procedure.
After the lipid construct is isolated from the bulk phase media, it can range
in size from approximately 0.0200 microns to 0.4000 microns in diameter. Lipid
constructs comprise different particle sizes that generally follow a Gaussian
distribution.
The appropriate size of the lipid construct needed to achieve the intended
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pharmacological efficacy can be selected from lipid constructs that comprise
particle
sizes in a Gaussian distribution by the hepatocyte binding receptor.
The lipid construct comprising insulin, lipids and the hepatocyte receptor
binding molecule is prepared by using a micro-fluidization process that
provides a high
shear force which degrades larger lipid constructs into smaller constructs.
The
amphipathic lipid constituents of the lipid construct are 1,2-distearoyl-sn-
glycero-3-
phosphocholine, cholesterol, dicetyl phosphate, 1,2-dipahnitoyl-sn-glycero-3-
phosphoethanolamine-N-(Cap Biotinyl), 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl),
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)) (sodium salt),
triethylarrunonium 2,3-diacetoxypropyl 2-(5-((3aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-
d]imidazol-4-y1) pentanamido)ethyl phosphate and appropriate derivatives
thereof whose
representative structures are depicted in Table 1.
In an embodiment, a construct comprises a target molecule complex
comprising multiple linked individual units formed by complexing a bridging
component
with a complexing agent. Typically the target molecule complex is formed by
combining
the selected metal compound, e. g. chromium chloride (HI) hexahydrate, with an
aqueous
buffered solution of the complexing agent. In an embodiment, an aqueous
buffered
solution of the complexing agent is prepared by dissolving the complexing
agent, e.g.,
N-(2,6-diisopropylphenylcarbamoyl methyl)iminodiacetic acid, in an aqueous
buffered
solution, e.g., 10 mM sodium acetate buffer at a final pH of 3.2-3.3. The
metal
compound is added in excess in an amount sufficient to complex with an
isolatable
portion of the complexing agent, and the reaction is conducted at a
temperature of 20 C
to 33 C for 24 to 96 hours, or until the resultant complex precipitates out of
aqueous
buffered solution. The precipitated complexing agent, which demonstrates
polymeric
properties, is then isolated for future use. This complex is added to the
mixture of
amphipathic lipid molecules and an extended amphipathic lipid prior to
preparing a lipid
construct.
Methods of manufacturing a composition of an insulin in which the
isoelectric point was altered by changing the amino acid sequence can be
incorporated
into a water insoluble target molecule complex are given below. In an
embodiment,
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glargine insulin is incorporated into a water insoluble target molecule
complex. Figure
15 demonstrates an outline for a process for manufacturing a mixture of free
glargine
insulin and glargine insulin associated with a water insoluble target molecule
complex.
In an embodiment, the manufacture of the composition involves three overall
steps:
preparing a target molecule complex, incorporating the target molecule complex
into a
lipid construct, and combining the target molecule complex with glargine
insulin to form
a pharmaceutical composition.
The target molecule complex comprises multiple individual units linked
together in a polymeric array. Each unit comprises a bridging component and a
complexing agent. In an embodiment, the target molecule complex is formed by
combining the selected metal compound, e. g. chromium chloride (III)
hexahydrate, with
an aqueous buffered solution of the complexing agent. In an embodiment, an
aqueous
buffered solution of the complexing agent is prepared by dissolving a
complexing agent,
e.g., N-(2,6-diisopropylphenylcarbamoyhnethyl) iminodiacetic acid, in an
aqueous
is buffered solution, e.g., I 0 mM sodium acetate buffer at a final pH of
3,2-3.3. A metal
compound is added in excess in an amount sufficient to complex with an
isolatable
portion of the complexing agent, and the reaction is conducted at a
temperature of
approximately 20 C to 33 C for approximately 24 to 96 hours, or until the
resultant
complex precipitates out of the aqueous buffered solution. The precipitated
complex is
then isolated for future use.
The precipitated complex is then mixed with the selected lipids or the
lipids of the lipid construct and dissolved in an organic solvent. In an
embodiment, the
organic solvent is chloroform:methanol (2:1 v/v). The lipids are in a
concentration
sufficient to dissolve and incorporate either all or a portion of the metal
complex therein.
The mixture of the complex and the selected lipids that form the lipid
construct are
maintained at a temperature of approximately 60 C when a high transition
temperature
lipid, such as 1,2-distearoyl-sn-glycero-3-phosphocholine, is employed. Lower
temperatures may be used depending upon the transition temperature of the
lipids
selected for incorporation into the lipid construct. A time period from 30
minutes to 2
hours under vacuum is generally required to dry the lipids and remove any
residual
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organic solvent from the lipid matrix in order to form the target molecule
complex
intermediate.
Lipids are produced and loaded by the methods disclosed herein, and those
methods described in U. S. Patent Nos. 4,946,787; 4,603,044; and 5,104,661,
and the
references cited therein. Typically, the aqueous lipid construct formulations
of the
invention will comprise 0.1% to 10% active agent by weight (i.e. 1 -100 mg
drug per ml),
and 0.1% to 4% lipid by weight in an aqueous solution, optionally containing
salts and
buffers, in a quantity to make 100% by volume. Preferred are formulations
which
comprise 0.01% to 5% active agent. Most preferred is a formulation comprising
0.01%
to 5% active agent by weight and up to 2% by weight of a lipid component in an
amount
of aqueous solution sufficient (q. s.) to make 100% by volume.
In an embodiment, glargine insulin was loaded into the target molecule
complex after the pH of a suspension of the target molecule complex and Water
for
Injection, USP was adjusted from approximately pH 4.89 0.2 to 5.27 0.5.
The pH of
a solution of glargine insulin was adjusted from pH 3.88 0.2 to
approximately pH 4.78
0.5, then the water insoluble target molecular complex was added. The
resulting
composition was a mixture of free glargine insulin and glargine insulin
associated with a
water insoluble target molecule complex. A portion of glargine insulin became
associated with the lipid construct matrix or entrapped in the core volume of
the lipid
construct. This pharmaceutical composition is also referred to as HDV-
glargine. In an
embodiment, an aliquot of the target molecule complex is introduced into a
vial of
Glargine Insulin containing 100 International units of insulin/ml to provide a
hepatocyte
specific delivery system containing both free glargine insulin and glargine
insulin
associated with the target molecule complex.
A pharmaceutical composition that combines free glargine insulin and
glargine insulin associated with a water insoluble target molecule complex was
prepared
by the following procedure. The p11 of a sample of Sterile Water for
Injection, USP, was
adjusted to pH 3.95 0.2. An aliquot of HDV suspension was taken and its pH
was
adjusted in a series of steps until the final pH was 5.2 0.5. An aliquot of
the Sterile
Water for Injection, USP, at pH 3.95 0.2 was mixed with the suspension of
the target
molecule complex. The pH of the resulting suspension was 4.89 0.2. The p11
of this
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suspension was then adjusted to pH 5.27 0.5. The pH of an aliquot of glargine
insulin
was adjusted from pH 3.88-1 0.2 to pH 4.78 0.5. This solution was then added
to the
suspension of the target molecule complex at pH 5.20 0.5. The resulting
pharmaceutical composition is a mixture of free glargine insulin and glargine
insulin
associated with a water insoluble target molecule complex. This pharmaceutical
composition is also referred to as HDV-glargine.
Methods of manufacturing a composition of an insulin in which the
isoelectric point was altered by binding charged organic molecules to insulin
can be
incorporated into a water insoluble target molecule complex are given below.
In an
embodiment, recombinant human insulin isophane is incorporated into a water
insoluble
target molecule complex. Figure 16 demonstrates an outline for a process for
manufacturing a mixture of free recombinant human insulin isophane, free
recombinant
human regular insulin and a mixture of recombinant human insulin isophane and
recombinant human regular insulin that are associated with a water insoluble
target
molecule complex. In an embodiment, the manufacture of the composition
involves three
overall steps: preparing a target molecule complex, incorporating the target
molecule
complex into a lipid construct that contains free and associated recombinant
human
regular insulin, and combining the target molecule complex with free and
associated
recombinant human insulin isophane to form a pharmaceutical composition.
The target molecule complex comprises multiple individual units linked
together in a polymeric array. Each unit comprises a bridging component and a
complexing agent. In an embodiment, the target molecule complex is formed by
combining the selected metal compound, e. g. chromium chloride (III)
hexahydrate, with
an aqueous buffered solution of the complexing agent. In an embodiment, an
aqueous
buffered solution of the complexing agent is prepared by dissolving a
complexing agent,
e.g., N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid, in an
aqueous
buffered solution, e.g., 10 mM sodium acetate buffer at a final pH of 3.2-3.3.
A metal
compound is added in excess in an amount sufficient to complex with an
isolatable
portion of the complexing agent, and the reaction is conducted at a
temperature of
approximately 20 C to 33 C for approximately 24 to 96 hours, or until the
resultant
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CA 02864366 2014-09-18
complex precipitates out of the aqueous buffered solution. The precipitated
complex is
then isolated for future use.
The precipitated complex is then mixed with the selected lipids or the
lipids of the lipid construct and dissolved in an organic solvent. In an
embodiment, the
organic solvent is chloroform:methanol (2:1 v/v). The lipids are in a
concentration
sufficient to dissolve and incorporate either all or a portion of the metal
complex therein.
The mixture of the complex and the selected lipids that form the lipid
construct are
maintained at a temperature of approximately 60 C when a high transition
temperature
lipid, such as 1,2-distearoyl-sn-glycero-3-phosphocholine, is employed. Lower
temperatures may be used depending upon the transition temperature of the
lipids
selected for incorporation into the lipid construct. A time period from 30
minutes to 2
hours under vacuum is generally required to dry the lipids and remove any
residual
organic solvent from the lipid matrix in order to form the target molecule
complex
intermediate.
Lipids can be produced and loaded by the methods disclosed herein, and
those methods described in U. S. Patent Nos. 4,946,787; 4,603,044; and
5,104,661, and
the references cited therein. Typically, the aqueous lipid construct
formulations of the
invention will comprise 0.1% to 10% active agent by weight (i.e. 1 -100 mg
drug per ml),
and 0.1% to 4% lipid by weight in an aqueous solution, optionally containing
salts and
buffers, in a quantity to make 100% by volume. Preferred are formulations
which
comprise 0.01% to 5% active agent. Most preferred is a formulation comprising
0.01%
to 5% active agent by weight and up to 2% by weight of a lipid component in an
amount
of aqueous solution sufficient (q. s.) to make 100% by volume.
In an embodiment, Humulin NPR insulin was added to a previously
formed mixture of recombinant human regular insulin and a lipid construct. The
resulting composition was a mixture of free recombinant human regular insulin
and free
recombinant human insulin isophane. Likewise a portion of recombinant human
regular
insulin and recombinant human insulin isophane is associated with the lipid
construct
matrix or entrapped in the core volume of the lipid construct. This
pharmaceutical
composition is also referred to as HDV-NPH insulin. In an embodiment, an
aliquot of
the target molecule complex is introduced into a vial of recombinant human
insulin
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CA 02864366 2014-09-18
isophane to provide a hepatocyte specific delivery system containing both free
recombinant human insulin isophane and recombinant human insulin isophane
associated
with the target molecule complex. In an embodiment, recombinant human insulin
isophane can be combined with other forms of insulin such as the rapid acting
Humalog
insulin and Novolog insulin, short acting Regular insulin, intermediate
acting Lente
insulin and long acting Ultralente insulin and Lantus insulin, or premixed
combinations
of insulin. An aliquot of recombinant human insulin isophane can be added to a
mixture
of the target molecule complex combined with an insulin that is not
recombinant human
insulin isophane.
Description of the Invention - Method of Use
Patients with Type I or Type II diabetes are administered an effective
amount of a hepatocyte targeted lipid construct comprising an amphipathic
lipid, an
extended amphipathic lipid and insulin. When this composition is administered
subcutaneously, a portion of the composition enters the circulatory system
where the
composition is transported to the liver and other areas where the extended
amphipathic
lipid binds the lipid construct to receptors of hepatocytes. A portion of the
administered
composition is exposed to an external gradient in vivo where insulin can be
solubilized
and then move from the lipid construct thereby supplying insulin to the muscle
and
adipose tissue. Insulin that remains with the lipid construct maintains the
capability of
being directed to the hepatocyte binding receptor on the hepatocytes in the
liver.
Therefore two forms of insulin are produced from this particular lipid
construct. In an in
vivo setting, free and lipid associated insulin are generated in a time-
dependent manner.
The lipid construct structure of the invention provides a useful agent for
pharmaceutical application for administering insulin to a host. Accordingly,
the
structures of the invention are useful as pharmaceutical compositions in
combination with
pharmaceutically acceptable carriers. Administration of the structures
described herein
can be via any of the accepted modes of administration for insulin that are
desired to be
administered. These methods include oral, parenteral, nasal and other systemic
or aerosol
forms.
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CA 02864366 2014-09-18
Oral administration of a pharmaceutical composition comprising insulin
associated with a target molecule complex is followed by intestinal absorption
of insulin
associated with the target molecule complex into the circulatory system of the
body
where it is also exposed to the physiological pH of the blood. The lipid
construct is
targeted for delivery to the liver. In an embodiment, the lipid construct is
shielded by the
presence of cellulose acetate hydrogen phthalate within the construct. In the
case of oral
administration, the shielded lipid construct transverses the oral cavity,
migrates through
the stomach and moves into the small intestine where the alkaline pH of the
small
intestine degrades the cellulose acetate hydrogen phthalate shield. The de-
shielded lipid
construct is absorbed into the circulatory system. This enables the lipid
construct to be
delivered to the sinusoids of the liver. A receptor binding molecule, such as
1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) or other
forementioned
hepatocyte specific molecules, provides a means for lipid construct to bind to
the receptor
and then be engulfed or endocytosed by the hepatocytes. Insulin is then
released from the
lipid construct where, upon gaining access to the cellular environment, it
performs its
designated function with regard to acting as an agent to control diabetes.
The amount of insulin administered will be dependent on the subject being
treated, the type and severity of the affliction, the manner of administration
and the
judgment of the prescribing physician. Although effective dosage ranges for
specific
biologically active substances of interest are dependent upon a variety of
factors, and are
generally known to one of ordinary skill in the art, some dosage guidelines
can be
generally defined. For most forms of administration, the lipid component will
be
suspended in an aqueous solution and generally not exceed 4.0% (w/v) of the
total
formulation. The drug component of the formulation will most likely be less
than 20%
(w/v) of the formulation and generally greater than 0.01% (w/v).
Methods of administering a composition of an insulin in which the
isoelectric point was altered by changing the amino acid sequence is
incorporated into a
water insoluble target molecule complex are given below. In an embodiment,
patients
with Type I or Type II diabetes are administered an effective amount of a
hepatocyte
targeted composition comprising a mixture of free glargine insulin and
glargine insulin
associated with a water insoluble target molecule complex. In an embodiment,
glargine
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CA 02864366 2014-09-18
insulin can be combined with other forms of insulin, such as insulin lispro,
insulin aspart,
regular insulin, insulin zinc, human insulin zinc extended, isophane insulin,
human
buffered regular insulin, insulin glulisine, recombinant human regular
insulin,
recombinant human insulin isophane or premixed combinations of any of the
aforementioned insulins, a derivative thereof, and a combination of any of the
aforementioned insulins. In an embodiment, the composition can be administered
by a
subcutaneous or oral route.
The lipid construct structure of the invention provides a useful agent for
pharmaceutical application for administering insulin to a host. Accordingly,
the
structures of the invention are useful as pharmaceutical compositions in
combination with
pharmaceutically acceptable carriers. Administration of the structures
described herein
can be via any of the accepted modes of administration for insulin that are
desired to be
administered. These methods include oral, parenteral, nasal and other systemic
or aerosol
forms.
After a composition is administered to a patient by subcutaneous injection,
the in situ physiological environment in the injection area, the morphology
and chemical
structures of free glargine insulin and the glargine insulin associated with
the water
insoluble target molecule complex begin to change. As the pH of the
environment
around the free glargine insulin and the glargine insulin associated with the
water
insoluble target molecule complex increases after being diluted with
physiological media,
the pH reaches the isoelectric point of glargine insulin, where flocculation,
aggregation
and precipitation reactions occur for both free glargine insulin and glargine
insulin
associated with the target molecule complex. The rates at which these
processes occur
differ between free glargine insulin and glargine insulin associated with the
target
molecule complex. The free glargine insulin is directly exposed to changes in
pH and
dilution. Exposure of glargine insulin associated with the target molecule
complex to
small changes in pH and dilution at physiological pH is delayed due to the
time required
for diffusion of physiological fluids or media through the lipid bilayer in
the water
insoluble target molecule complex. The delay in the release of insulin from
the lipid
construct as well as the delay of the release of lipid construct with
associated insulin
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CA 02864366 2014-09-18
within the precipitated free glargine matrix is an essential feature of the
invention since it
affects and augments the biological and pharmacological response in vivo.
Oral administration of a pharmaceutical composition that combines free
glargine insulin and glargine insulin associated with a target molecule
complex is
followed by intestinal absorption of glargine insulin associated with the
target molecule
complex into the circulatory system of the body where it is also exposed to
the
physiological pH of the blood. All or a portion of the lipid construct is
delivered to the
liver.
As the physiological dilution is increased in situ in the subcutaneous space
or upon entering into the circulatory system, the free glargine insulin and
glargine insulin
associated with the target molecule complex encounter a normal physiological
pH
environment of pH 7.4. As a result, free glargine insulin changes from a
soluble form at
injection, to a insoluble form at a pH near its isoelectric point of pH 5.8-
6.2, and then to a
soluble form at physiological pH. In the soluble form, glargine insulin
migrates through
the body to sites where it is capable of eliciting a pharmacological response.
Glargine
insulin associated with the water insoluble target molecule complex becomes
solubilized
and released from the complex at a different rate that is slower than that of
free glargine
insulin. This is because glargine insulin associated with the water insoluble
target
molecule complex has to traverse the core volume and lipid domains of the
water
insoluble target molecule complex before it contacts the bulk phase media.
The amount of glargine insulin administered will be dependent on the
subject being treated, the type and severity of the affliction, the manner of
administration
and the judgment of the prescribing physician. Although effective dosage
ranges for
specific biologically active substances of interest are dependent upon a
variety of factors,
and are generally known to one of ordinary skill in the art, some dosage
guidelines can be
generally defined. For most forms of administration, the lipid component will
be
suspended in an aqueous solution and generally not exceed 4.0% (w/v) of the
total
formulation. The drug component of the formulation will most likely be less
than 20%
(w/v) of the formulation and generally greater than 0.01% (w/v).
Methods of administering a composition of an insulin in which the
isoelectric point was altered by binding charged organic molecules to insulin
is
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CA 02864366 2014-09-18
incorporated into a water insoluble target molecule complex are given below.
In an
embodiment, patients with Type I or Type II diabetes are administered an
effective
amount of a hepatocyte targeted composition comprising a mixture of free
recombinant
human insulin isophane plus free recombinant human regular insulin along with
recombinant human insulin isophane and recombinant human regular insulin which
are
both are associated with a water insoluble target molecule complex. In an
embodiment,
recombinant human insulin isophane can be combined with other forms of
insulin, such
as of insulin lispro, insulin aspart, regular insulin, insulin glargine,
insulin zinc, human
insulin zinc extended, isophane insulin, human buffered regular insulin,
insulin glulisine,
to recombinant human regular insulin, recombinant human insulin isophane or
premixed
combinations of any of the aforementioned insulins, a derivative thereof, and
a
combination of any of the aforementioned insulins.
The lipid construct structures of the invention provides a useful agent for
pharmaceutical application for administering insulin to a host. Accordingly,
the
structures of the invention are useful as pharmaceutical compositions in
combination with
pharmaceutically acceptable carriers. Administration of the structures
described herein
can be via any of the accepted modes of administration for insulin that are
desired to be
administered. These methods include oral, parenteral, nasal and other systemic
or aerosol
forms.
After a composition is administered to a patient by subcutaneous injection,
the in situ physiological environment in the injection area, the morphology
and chemical
structures of free recombinant human insulin isophane and the recombinant
human
insulin isophane associated with the water insoluble target molecule complex
begins to
change. As the pH of the environment around the free recombinant human insulin
isophane and the recombinant human insulin isophane associated with the water
insoluble
target molecule complex becomes diluted with physiological media, some
solubilization
occurs for both insulins. As a result of solubilization and equilibrium
conditions
recombinant human insulin isophane can become associated with the target
molecule
complex. The rates at which these equilibrium processes occur differ between
free
recombinant human insulin isophane and recombinant human insulin isophane
associated
with the target molecule complex. The free recombinant human insulin isophane
is
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directly exposed to small changes in pH and physiological dilution. Exposure
of
recombinant human insulin isophane associated with the target molecule complex
to
small changes in pH and dilution at physiological pH is delayed due to the
time required
for diffusion of physiological fluids or media through the lipid bilayer in
the water
insoluble target molecule complex. The delay in the release of insulin from
the lipid
construct as well as the delay of the release of the lipid construct as it
exists within the
precipitated free recombinant human insulin isophane matrix is an essential
discovery of
the invention since it affects and augments the biological and pharmacological
response
in vivo.
Oral administration of a pharmaceutical composition that combines free
recombinant human insulin isophane and recombinant human insulin isophane
associated
with a target molecule complex is followed by intestinal absorption of
recombinant
human insulin isophane associated with the target molecule complex into the
circulatory
system of the body where it is also exposed to the physiological pH of the
blood. All or a
portion of the lipid construct is delivered to the liver.
As the physiological dilution is increased in situ in the subcutaneous space
or upon entering into the circulatory system, free recombinant human insulin
isophane
and recombinant human insulin isophane associated with the target molecule
complex
encounter a normal physiological pH environment of pH 7.4. As a result of
dilution free
recombinant human insulin isophane changes from an insoluble form at
injection, to a
soluble form at physiological pH. In the soluble form, recombinant human
insulin
isophane migrates through the body to sites where it is capable of eliciting a
pharmacological response. Recombinant human insulin isophane associated with
the
water insoluble target molecule complex becomes solubilized and released from
the
complex at a different rate that is slower than that of free recombinant human
insulin
isophane. This is because recombinant human insulin isophane associated with
the water
insoluble target molecule complex has to traverse the core volume and lipid
domains of
the water insoluble target molecule complex before it contacts the bulk phase
media.
The lipid construct structure of the invention provides a useful agent for
pharmaceutical application for administering recombinant human insulin
isophane to a
host. Accordingly, the structures of the invention are useful as
pharmaceutical
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compositions in combination with pharmaceutically acceptable carriers.
Administration
of the structures described herein can be via any of the accepted modes of
administration
for recombinant human insulin isophane that are desired to be administered.
These
methods include oral, parenteral, nasal and other systemic or aerosol forms.
The amount of recombinant human insulin isophane and recombinant
human regular insulin administered will be dependent on the subject being
treated, the
type and severity of the affliction, the manner of administration and the
judgment of the
prescribing physician. Although effective dosage ranges for specific
biologically active
substances of interest are dependent upon a variety of factors, and are
generally known to
one of ordinary skill in the art, some dosage guidelines can be generally
darned. For
most forms of administration, the lipid component will be suspended in an
aqueous
solution and generally not exceed 4.0% (w/v) of the total formulation. The
drug
component of the formulation will most likely be less than 20% (w/v) of the
formulation
and generally greater than 0.01% (w/v).
The amount of insulin administered will be dependent on the subject being
treated, the type and severity of the affliction, the manner of administration
and the
judgment of the prescribing physician. Although effective dosage ranges for
specific
biologically active substances of interest are dependent upon a variety of
factors, and are
generally known to one of ordinary skill in the art, some dosage guidelines
can be
generally defined. For most forms of administration, the lipid component will
be
suspended in an aqueous solution and generally not exceed 4.0% (w/v) of the
total
formulation. The drug component of the formulation will most likely be less
than 20%
(w/v) of the formulation and generally greater than 0.01% (w/v).
Dosage forms or compositions containing active ingredient in the range of
0.005% to 5% with the balance made up from non-toxic carriers may be prepared.
The exact composition of these formulations may vary widely depending
on the particular properties of the drug in question. However, they will
generally
comprise from 0.01% to 5%, and preferably from 0.05% to 1% active ingredient
for
highly potent drugs, and from 2%-4% for moderately active drugs.
The percentage of active ingredient contained in such parenteral
compositions is highly dependent on the specific nature thereof, as well as
the activity of
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the active ingredient and the needs of the subject. However, percentages of
active
ingredient of 0.01% to 5% in solution are employable, and will be higher if
the
composition is a solid which will be subsequently diluted to the above
percentages.
Preferably the composition will comprise 0.2%-2.0% of the active agent in
solution.
The formulations of the pharmaceutical compositions described herein
may be prepared by any method known or hereafter developed in the art of
pharmacology. In general, such preparatory methods include the step of
bringing the
active ingredient into association with a carrier or one or more other
ingredients, and
then, if necessary or desirable, shaping or packaging the product into a
desired single- or
multi-dose unit.
Although the descriptions of pharmaceutical compositions provided herein
are principally directed to pharmaceutical compositions which are suitable for
ethical
administration to humans, it will be understood by the skilled artisan that
such
compositions are generally suitable for administration to animals of all
sorts.
Modification of pharmaceutical compositions suitable for administration to
humans in
order to render the compositions suitable for administration to various
animals is well
understood, and the ordinarily skilled veterinary pharmacologist can design
and perform
such modification with merely ordinary, if any, experimentation. Subjects to
which
administration of the pharmaceutical compositions of the invention is
contemplated
include, but are not limited to, humans and other primates, mammals including
commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and
dogs.
Pharmaceutical compositions that are useful in the methods of the
invention may be prepared, packaged, or sold in formulations suitable for
oral, parenteral,
pulmonary, intranasal, buccal, or another route of administration.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in bulk, as a single unit dose, or as a plurality of single
unit doses. As
used herein, a "unit dose" is discrete amount of the pharmaceutical
composition
comprising a predetermined amount of the active ingredient. The amount of the
active
ingredient is generally equal to the dosage of the active ingredient which
would be
administered to a subject or a convenient fraction of such a dosage such as,
for example,
one-half or one-third of such a dosage. However, delivery of the active agent
as set forth
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in the invention may be as low as 1/10, 1/100 or 1/1,000 or smaller than the
dose
normally administered because of the targeted nature of the insulin
therapeutic agent.
The relative amounts of the active ingredient, the pharmaceutically
acceptable carrier, and any additional ingredients in a pharmaceutical
composition of the
invention will vary, depending upon the identity, size, and condition of the
subject treated
and further depending upon the route by which the composition is to be
administered. By
way of example, the composition may comprise between 0.1% and 100% (w/w)
active
ingredient.
A formulation of a pharmaceutical composition of the invention suitable
for oral administration may be prepared, packaged, or sold in the form of a
discrete solid
dose unit including, but not limited to, a tablet, a hard or soft capsule, a
cachet, a troche,
or a lozenge, each containing a predetermined amount of the active ingredient.
Other
formulations suitable for oral administration include, but are not limited to,
a powdered
or granular formulation, an aqueous or oily suspension, an aqueous or oily
solution, or an
emulsion.
As used herein, an "oily" liquid is one which comprises a carbon-
containing liquid molecule and which exhibits a less polar character than
water.
A tablet comprising the active ingredient may, for example, be made by
compressing or molding the active ingredient, optionally with one or more
additional
ingredients. Compressed tablets may be prepared by compressing, in a suitable
device,
the active ingredient in a free-flowing form such as a powder or granular
preparation,
optionally mixed with one or more of a binder, a lubricant, an excipient, a
surface active
agent, and a dispersing agent. Molded tablets may be made by molding, in a
suitable
device, a mixture of the active ingredient, a pharmaceutically acceptable
carrier, and at
least sufficient liquid to moisten the mixture. Pharmaceutically acceptable
excipients
used in the manufacture of tablets include, but are not limited to, inert
diluents,
granulating and disintegrating agents, binding agents, and lubricating agents.
Known
dispersing agents include, but are not limited to, potato starch and sodium
starch
glycollate. Known surface active agents include, but are not limited to,
sodium lauryl
sulphate. Known diluents include, but are not limited to, calcium carbonate,
sodium
carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium
hydrogen
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phosphate, and sodium phosphate. Known granulating and disintegrating agents
include,
but are not limited to, corn starch and alginic acid. Known binding agents
include, but
are not limited to, gelatin, acacia, pre-gelatinized maize starch,
polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include, but are not
limited to,
magnesium stearate, stearic acid, silica, and talc.
Tablets may be non-coated or they may be coated using known methods to
achieve delayed disintegration in the gastrointestinal tract of a subject,
thereby providing
sustained release and absorption of the active ingredient. By way of example,
a material
such as glyceryl monostearate or glyceryl distearate may be used to coat
tablets. Further
by way of example, tablets may be coated using methods described in U.S.
Patents
numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled
release
tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a
coloring
agent, a preservative, or some combination of these in order to provide
pharmaceutically
elegant and palatable preparation.
Hard capsules comprising the active ingredient may be made using a
physiologically degradable composition, such as gelatin. Such hard capsules
comprise
the active ingredient, and may further comprise additional ingredients
including, for
example, an inert solid diluent such as calcium carbonate, calcium phosphate,
kaolin or
cellulose acetate hydrogen phthalate.
Soft gelatin capsules comprising the active ingredient may be made using
a physiologically degradable composition, such as gelatin. Such soft capsules
comprise
the active ingredient, which may be mixed with water or an oil medium such as
peanut
oil, liquid paraffin, or olive oil.
Liquid formulations of a pharmaceutical composition of the invention
which are suitable for oral administration may be prepared, packaged, and sold
either in
liquid form or in the form of a dry product intended for reconstitution with
water or
another suitable vehicle prior to use.
Liquid suspensions may be prepared using conventional methods to
achieve suspension of the active ingredient in an aqueous or oily vehicle.
Aqueous
vehicles include, for example, water and isotonic saline. Oily vehicles
include, for
example, almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive,
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sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as
liquid
paraffin. Liquid suspensions may further comprise one or more additional
ingredients
including, but not limited to, suspending agents, dispersing or wetting
agents,
emulsifying agents, demulcents, preservatives, buffers, salts, flavorings,
coloring agents,
and sweetening agents. Oily suspensions may further comprise a thickening
agent.
Known suspending agents include, but are not limited to, sorbitol syrup,
hydrogenated
edible fats, sodium alginate, polyvinylpyrrolid.one, gum tragacanth, gum
acacia, and
cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but
are not
limited to, naturally-occurring phosphatides such as lecithin, condensation
products of an
alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a
partial ester
derived from a fatty acid and a hexitol, or with a partial ester derived from
a fatty acid
and a headtol anhydride (e.g. polyoxyethylene stearate,
heptadecaethyleneoxycetanol,
polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate,
respectively). Known emulsifying agents include, but are not limited to,
lecithin and
acacia. Known preservatives include, but are not limited to, methyl, ethyl, or
n-propyl-
para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents
include, for example, glycerol, propylene glycol, sorbitol, sucrose, and
saccharin. Known
thickening agents for oily suspensions include, for example, beeswax, hard
paraffin, and
cetyl alcohol.
Liquid solutions of the active ingredient in aqueous or oily solvents may
be prepared in substantially the same manner as liquid suspensions, the
primary
difference being that the active ingredient is dissolved, rather than
suspended in the
solvent. Liquid solutions of the pharmaceutical composition of the invention
may
comprise each of the components described with regard to liquid suspensions,
it being
understood that suspending agents will not necessarily aid dissolution of the
active
ingredient in the solvent. Aqueous solvents include, for example, water and
isotonic
saline. Oily solvents include, for example, almond oil, oily esters, ethyl
alcohol,
vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated
vegetable oils,
and mineral oils such as liquid paraffin.
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Powdered and granular formulations of a pharmaceutical preparation of
the invention may be prepared using known methods. Such formulations may be
administered directly to a subject, used, for example, to form tablets, to
fill capsules, or to
prepare an aqueous or oily suspension or solution by addition of an aqueous or
oily
vehicle thereto. Each of these formulations may further comprise one or more
of
dispersing or wetting agent, a suspending agent, and a preservative.
Additional
excipients, such as fillers and sweetening, flavoring, or coloring agents, may
also be
included in these formulations.
A pharmaceutical composition of the invention may also be prepared,
packaged, or sold in the form of oil-in-water emulsion or a water-in-oil
emulsion. The
oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil
such as liquid
paraffin, or a combination of these. Such compositions may further comprise
one or
more emulsifying agents such as naturally occurring gums such as gum acacia or
gum
tragacanth, naturally-occurring phosphatides such as soybean or lecithin
phosphatide,
esters or partial esters derived from combinations of fatty acids and hexitol
anhydrides
such as sorbitan monooleate, and condensation products of such partial esters
with
ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions
may also
contain additional ingredients including, for example, sweetening or flavoring
agents.
As used herein, "parenteral administration" of a pharmaceutical
composition includes any route of administration characterized by physical
breaching of
a tissue of a subject and administration of the pharmaceutical composition
through the
breach in the tissue. Parenteral administration thus includes, but is not
limited to,
administration of a pharmaceutical composition by injection of the
composition, by
application of the composition through a surgical incision, by application of
the
composition through a tissue-penetrating non-surgical wound, and the like. In
particular,
parenteral administration is contemplated to include, but is not limited to,
subcutaneous,
intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic
infusion
techniques.
Formulations of a pharmaceutical composition suitable for parenteral
administration comprise the active ingredient combined with a pharmaceutically
acceptable carrier, such as sterile water or sterile isotonic saline. Such
formulations may
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be prepared, packaged, or sold in a form suitable for bolus administration or
for
continuous administration. Injectable formulations may be prepared, packaged,
or sold in
unit dosage form, such as in ampoules or in multi-dose containers containing a
preservative. Formulations for parenteral administration include, but are not
limited to,
suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and
implantable
sustained-release or biodegradable formulations. Such formulations may further
comprise one or more additional ingredients including, but not limited to,
suspending,
stabilizing, or dispersing agents. In one embodiment of a formulation for
parenteral
administration, the active ingredient is provided in dry (i.e. powder or
granular) foam for
reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior
to parenteral
administration of the reconstituted composition.
The pharmaceutical compositions may be prepared, packaged, or sold in
the form of a sterile injectable aqueous or oily suspension or solution. This
suspension or
solution may be formulated according to the known art, and may comprise, in
addition to
the active ingredient, additional ingredients such as the dispersing agents,
wetting agents,
or suspending agents described herein. Such sterile injectable formulations
may be
prepared using a non-toxic parenterally-acceptable diluent or solvent, such as
water or
1,3-butane diol, for example. Other acceptable diluents and solvents include,
but are not
limited to, Ringer's solution, isotonic sodium chloride solution, and fixed
oils such as
synthetic mono- or di-glycerides. Other parentally-administrable formulations
which are
useful include those which comprise the active ingredient in microcrystalline
form, in a
lipid construct preparation, or as a component of a biodegradable polymer
system.
Compositions for sustained release or implantation may comprise
pharmaceutically
acceptable polymeric or hydrophobic materials such as an emulsion, an ion
exchange
resin, a sparingly soluble polymer, or a sparingly soluble salt.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for pulmonary administration via
the buccal
cavity. Such a formulation may comprise dry particles which comprise the
active
ingredient and which have a diameter in the range from about 0.5 to about 7
microns, and
preferably from about 1 to about 6 microns. Such compositions are conveniently
in the
form of dry powders for administration using a device comprising a dry powder
reservoir
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to which a stream of propellant may be directed to disperse the powder or
using a
self-propelling solvent/powder-dispensing container such as a device
comprising the
active ingredient dissolved or suspended in a low-boiling propellant in a
sealed container.
Preferably, such powders comprise particles wherein at least 98% of the
particles by
weight have a diameter greater than 0.5 microns and at least 95% of the
particles by
number have a diameter less than 7 microns. More preferably, at least 95% of
the
particles by weight have a diameter greater than 1 nanometer and at least 90%
of the
particles by number have a diameter less than 6 microns. Dry powder
compositions
preferably include a solid fine powder diluent such as sugar and are
conveniently
provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a
boiling point of below 65 F at atmospheric pressure. Generally the propellant
may
constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may
constitute
0.1 to 20% (w/w) of the composition. The propellant may further comprise
additional
ingredients such as a liquid non-ionic or solid anionic surfactant or a solid
diluent
(preferably having a particle size of the same order as particles comprising
the active
ingredient).
Pharmaceutical compositions of the invention formulated for pulmonary
delivery may also provide the active ingredient in the form of droplets of a
solution or
suspension. Such formulations may be prepared, packaged, or sold as aqueous or
dilute
alcoholic solutions or suspensions, optionally sterile, comprising the active
ingredient,
and may conveniently be administered using any nebulization or atomization
device.
Such formulations may further comprise one or more additional ingredients
including,
but not limited to, a flavoring agent such as saccharin sodium, a volatile
oil, a buffering
agent, a surface active agent, or a preservative such as
ttiethylhydroxybenzoate. The
droplets provided by this route of administration preferably have an average
diameter in
the range from about 0.1 to about 200 microns.
The formulations described herein as being useful for pulmonary delivery
are also useful for intranasal delivery of a pharmaceutical composition of the
invention.
Another formulation suitable for intranasal administration is a coarse
powder comprising the active ingredient and having an average particle from
about 0.2 to
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500 microns. Such a formulation is administered in the manner in which snuff
is taken
i.e. by rapid inhalation through the nasal passage from a container of the
powder held
close to the nares.
Formulations suitable for nasal administration may, for example, comprise
from about as little as 0.1% (w/w) and as much as 75% (w/w) of the active
ingredient,
and may further comprise one or more of the additional ingredients described
herein.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for buccal administration. Such
formulations
may, for example, be in the form of tablets or lozenges made using
conventional
methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance
comprising an orally dissolvable or degradable composition and, optionally,
one or more
of the additional ingredients described herein. Alternately, formulations
suitable for
buccal administration may comprise a powder or an aerosolized or atomized
solution or
suspension comprising the active ingredient. Such powdered, aerosolized, or
aerosolized
formulations, when dispersed, preferably have an average particle or droplet
size in the
range from about 0.1 to about zoamicrons, and may further comprise one or more
of the
additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared,
packaged, or sold in a formulation suitable for ophthalmic administration.
Such
formulations may, for example, be in the form of eye drops including, for
example, a
0.1%-1.0% (w/w) solution or suspension of the active ingredient in an aqueous
or oily
liquid carrier. Such drops may further comprise buffering agents, salts, or
one or more
other of the additional ingredients described herein. Other opthalmically-
administrable
formulations which are useful include those which comprise the active
ingredient in
microcrystalline form or in a lipid construct preparation.
As used herein, "additional ingredients" include, but are not limited to,
one or more of the following: excipients; surface active agents; dispersing
agents; inert
diluents; granulating and disintegrating agents; binding agents; lubricating
agents;
sweetening agents; flavoring agents; coloring agents; preservatives;
physiologically
degradable compositions such as gelatin; aqueous vehicles and solvents; oily
vehicles and
solvents; suspending agents; dispersing or wetting agents; emulsifying agents,
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demulcents; buffers; salts; thickening agents; fillers; emulsifying agents;
antioxidants;
antibiotics; antifungal agents; stabilizing agents; and pharmaceutically
acceptable
polymeric or hydrophobic materials. Other "additional ingredients" which may
be
included in the pharmaceutical compositions of the invention are known in the
art and
described, for example in Genera, ed., 1985, Remington's Pharmaceutical
Sciences,
Mack Publishing Co., Easton, PA.
Typically dosages of the active ingredient in the composition of the
invention which may be administered to an animal, preferably a human, range in
amount
from 1 micrograms to about 100 g per kilogram of body weight of the animal.
While the
precise dosage administered will vary depending upon any number of factors,
including
but not limited to, the type of animal and type of disease state being
treated, the age of the
animal and the route of administration. Preferably, the dosage of the active
ingredient
will vary from about 1 mg to about 10 g per kilogram of body weight of the
animal.
More preferably, the dosage will vary from about 10 mg to about 1 g per
kilogram of
body weight of the animal.
The composition may be administered to an animal as frequently as
several times daily, or it may be administered leas frequently, such as once a
day, once a
week, once every two weeks, once a month, or even lees frequently, such as
once every
several months or even once a year or less. The frequency of the dose will be
readily
apparent to the skilled physician and will depend upon any number of factors,
such as,
but not limited to, the type and severity of the disease being treated, the
type and age of
the animal, etc.
The invention also includes a kit comprising the composition of the
invention and an instructional material which describes administering the
composition to
a tissue of a mammal. In another embodiment, this kit comprises a (preferably
sterile)
solvent suitable for dissolving or suspending the composition of the invention
prior to
administering the composition to the mammal.
As used herein, an "instructional material" includes a publication, a
recording, a diagram, or any other medium of expression which can be used to
communicate the usefulness of the protein of the invention in the kit for
effecting
alleviation of the various diseases or disorders recited herein. Optionally,
or alternately,
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the instructional material may describe one or more methods of alleviation the
diseases or
disorders in a cell or a tissue of a mammal. The instructional material of the
kit of the
invention may, for example, be affixed to a container which contains the
components of
the invention or be shipped together with a container which contains the
components of
the invention. Alternatively, the instructional material may be shipped
separately from
the container with the intention thet the instructional material and the
composition be
used cooperatively by the recipient.
The pharmaceutical compositions useful for practicing the invention may
be administered to deliver a dose equivalent to standard doses of insulin.
Although the descriptions of pharmaceutical compositions provided herein
are principally directed to pharmaceutical compositions which are suitable for
ethical
administration to humans, it will be understood by the skilled artisan that
such
compositions are generally suitable for administration to animals of all
sorts.
Modification of pharmaceutical compositions suitable for administration to
humans in
order to render the compositions suitable for administration to various
animals is well
understood, and the ordinarily skilled veterinary pharmacologist can design
and perform
such modification with merely ordinary, if any, experimentation. Subjects to
which
administration of the pharmaceutical compositions of the invention is
contemplated
include, but are not limited to, humans and other primates, companion animals
and other
mammals.
Pharmaceutical compositions that are useful in the methods of the
invention may be prepared, packaged, or sold in formulations suitable for oral
or
injectable routes of administration.
The relative amounts of the active ingredient, the pharmaceutically
acceptable carrier, and any additional ingredients in a pharmaceutical
composition of the
invention will vary, depending upon the identity, size, and condition of the
subject treated
and further depending upon the route by which the composition is to be
administered.
EXPERIMENTAL EXAMPLES
The invention is now described with reference to the following Examples.
These Examples are provided for the purpose of illustration only and the
invention should
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in no way be construed as being limited to these Examples, but rather should
be
construed to encompass any and all variations which become evident as a result
of the
teaching provided herein.
The materials and methods used in the experiments presented in this
Experimental Example are now described.
Experimental Example 1. Pharmaceutical Composition 1
A lipid construct comprises a mixture of the lipids 1,2-distearoyl-sn-
glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-distearoyl-sn-
glycero-3-
phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(succinyl),
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt), the
receptor
binding molecule 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolarnine-N-(Cap
Biotinyl)
and insulin.
Experimental Example 2. Pharmaceutical Composition 2
A lipid construct comprises a mixture of the lipids 1,2-distearoyl-sn-
glycero-3-phosphocholine, cholesterol, dicetyl phosphate, 1,2-distearoyl-sn-
glycero-3-
phosphoethanolamine, 1,2-dipalmitoyl-sn-glyeero-3-phosphoethanolamine-N-
(succinyl),
1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycero)] (sodium salt), insulin,
the
receptor binding molecule 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-
(Cap
Biotinyl), and/or polychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)
carbamoyhnethyl)
imino]diacetic acid). The lipid anchoring-hepatocyte receptor binding molecule
1,2-
dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap Biotinyl) and polychromium-
poly(bis)-[N-(2,6-(diisopropylphenyl)earbamoyl methyl)imino diacetic acid] had
been
added to the lipid construct at a level of 1.68% 0.5% by weight and 1.2%
0.5% by
weight, respectively.
Experimental Example 3. Pharmaceutical Composition 3
A lipid construct comprises a mixture of the amphipathic lipids 1,2-
distearoyl-sn-glycero-3-phosphocholine (12.09 g), cholesterol (1.60 g),
dicetyl phosphate
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(3.10 g), polychromium-poly(bis)-[N-(2,6-(diisopropylphenyl)
carbamoylmethypimino]
diacetic acid] (0.20 g) and insulin. The mixture was added to a aqueous medium
and the
total mass was 1200 g.
Experimental Exam_ple 4. Preparation of a Lipid construct Containing Insulin
The lipid construct was formed by preparing a mixture of amphipathic
lipid molecules and an extended amphipathic lipid, preparing a lipid construct
from the
mixture of amphipathic lipid molecules and an extended amphipathic lipid, and
combining insulin into the lipid construct.
A mixture of amphipathic lipid molecules and an extended amphipathic
lipid was produced using the following procedure. A mixture of the lipid
components
[total mass of 8.5316 g] of the lipid construct was prepared by combining
aliquots of the
lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (5.6881 g), cholesterol
crystalline
(0.7980 g), dicetyl phosphate (1.5444 g), 1,2-dipalmitoyl-sn-glycero-3-
phosphoethanolamine-N-(Cap Biotinyl) (0.1436 g), 1,2-distearoyl-sn-glyc,ero-3-
phosphoethanolamine (01144 g), 1,2-dipahnitoyl-sn-glycero-3-
phosphoethanolarnine-N-
(succinyl) (0.1245 g) and 1,2-dipahnitoyl-sn-g,lycero-3-[phospho-rac-(1-
glycerol)]
(sodium salt) (0.1186 g).
A 100 ml solution of chloroform:methnnol (2:1 v:v) was dehydrated over
5.0 grams of molecular sieves. The mixture of the lipid components of lipid
construct
was placed in a 3 liter flask and 45 rats of the chloroform/methanol solution
was added to
the lipid mixture. The solution was placed in flask on a rotoevaporator with a
water bath
at 60 C 12 C and turned slowly. The chloroform/methanol solution was removed
under
vacuum on a rotary evaporator using an aspirator for approximately 45 minutes,
followed
by a vacuum pump for approximately two hours to remove residual solvent, and
the solid
mixture of the lipids formed. The dried mixture of lipids can be stored in a
freezer at
approximately -20 C-0 C for an indefinite time period.
The lipid construct was prepared from the mixture of amphipathic lipid
molecules
and an extended amphipathic lipid using the following procedure. The lipid
mixture was
mixed with approximately 600 ml of 28.4 mM sodium phosphate (monobasic-
dibasic)
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buffer at pH 7Ø The lipid mixture was swirled, then placed in a heated water
bath at 80
C 4 C for 30 minutes while slowly turning to hydrate the lipids.
A M-110 Em microfluidizer was preheated to 70 C 10 C using SWI
with a pH between 6.5 - 7.5. The suspension of the hydrated target complex was
transferred to the microfluidizer and microfluidized at approximately 9000
psig using one
pass of the suspension of the hydrated target molecule complex through the
fluidizer.
After passing through the microfluidizer, an unfiltered sample (2.0 - 5.0 ml)
of the
fluidized suspension was collected for particle size analysis using unimodal
distribution
data from a Coulter N-4 plus particle size analyzer. Prior to all particle
size
determinations, the sample was diluted with 0.2 micron filtered SWI that has
been pH
adjusted to between 6.5 - 7.5. The particle size was required to range from
0.020 - 0.40
microns. If the particle size was not within this range, the suspension was
passed through
the microfluidizer again at approximately 9000 psig, and the particle size was
analyzed
again until the particle size requirements are reached. The microfluidized
target molecule
complex was collected in a sterile container.
The microfluidized target molecule complex was maintained at 60 C
2 C while filtered twice through a sterile 0.8 micron + 0.2 micron gang filter
attached to a
5.0 ml syringe. An aliquot of the filtered suspension was analyzed to
determine the
particle size range of particles in the suspension. The particle size range of
the final 0.2
micron filtered sample should be in the range from 0.0200 - 0.2000 microns as
determined from the unimodal distribution printout Row the particle size
analyzer.
Insulin is loaded into the construct by reverse loading of the construct
using the methods described in U.S. 5,104,661.
Experimental Example 5. Method of Use
The efficacy of hepatic directed vesicle (HDV) insulin on hepatic
glycogen was evaluated in a rat model. A total of 60 Male Sprague-Dawley rats
(8 weeks of
age; 250g) were divided into five treatment groups as described below.
For the first day of the study, all rats were fasted for 24 hours with ab
libitum water. On the second day, the rats were injected intraperitoneally
with a mixture
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of alloxan and streptozotocin (AS). The mixture of alloxan and streptozotocin
was
prepared in pH 70.01 M phosphate buffer by weighing 5 mg per mL of each
material so
that the final concentration is 5 mg alloxan per mL and 5 mg streptozotocin
per mL. The
AS mixture was administered 0.5 mL of the mixture of alloxan and
streptozotocin via
intraperitoneal injection at 20 mg/kg body weight (10 mg/kg alloxan and 10
mg/kg
streptozotocin). AS will cause a massive release of insulin resulting in a
profound and
transient hypoglycemia a few hours after injecting AS. A 10% glucose in water
solution
was injected subcutaneously as needed to prevent hypoglycemia and keep the
rats
adequately hydrated during the second day. A normal chow diet and water were
available ad libitum.
On the third day, a baseline tail-vein blood glucose sample is taken at 0
Minutes, followed immediately by a subcutaneous injection of one of the
following
solutions at 0.32 U insulin/rat, corresponding to the group to which the rat
was assigned.
(1) HDV-insulin with a Cr-disofenin [polychromium-poly(bis)-[N-(2,6-
(diisopropylphenyl) carbamoyl methypimino diacetic acid]]
hepatocyte target molecule (HTM) (Positive) control. There was no
extended amphipathic lipid present. The amount of amphipathic lipids
present provided a dose of about 14.5 micrograms of amphipathic
lipids per kilogram of rat.
(2) Regular insulin (negative) control;
(3) HDV-insulin test material 1, where the extended amphipathic lipid was
Biotin-X DHPE [triethylammonium 2,3-diacetoxypropyl 24645-
((3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-cl]imidazol-4-y1)
pentanamido)hexanamido)ethyl phosphate]. The amount of
amphipathic lipids present provided a dose of about 14.5 micrograms
of amphipathic lipids per kilogram of rat. The amount of extended
amphipathic lipid present provided a dose of about 191 nanograms of
extended amphipathic lipid per kilogram of rat.
(4) HDV-insulin test material 2, where the extended amphipathic lipid was
Biotin DHPE [triethylammonium 2,3-diacetoxyproPY1245-
((3a8,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-y1)
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pentanamido)ethyl phosphate). The amount of amphipathic lipids
present provided a dose of about 7.25 micrograms of amphipathic
lipids per kilogram of rat. The amount of extended amphipathic lipid
present provided a dose of about 95.5 nanograms of extended
amphipathic lipid per kilogram of rat.
(5) HDV-insulin test material 3, where the extended amphipathic lipid was
Biotin DHPE jtriethylammonium 2,3-diacetoxypropyl 245-
((3aS,6aR)-2-oxohexahydm-1H-thieno[3,4-d]imidazol-4-y1)
pentanamido)ethyl phosphate]. The amount of amphipathic lipids
present provided a dose of about 14.5 micrograms of amphipathic
lipids per kilogram of rat. The amount of extended amphipathic lipid
present provided a dose of about 191 nanograms of extended
amphipathic lipid per kilogram of rat.
For treatment ?pups 1 and 3-5, the amphipathic lipids were a mixture
of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and dicetyl
phosphate.
At "0" minutes, each rat was also gavaged with 375 mg glucose in 3.75 ml
water (10% glucose).
Half of the animals of each group were anesthetized and euthanized using
ketamine (150mg/kg)/xylazine (15mg/kg) at one hour minutes and the remaining
rats at 2
hours via LP. Previous studies with Cr-disofenin HTM have shown the
statistically
significant effect over 2 hours. The entire liver was removed and stored in
liquid
nitrogen at -80 C until analyzed for hepatic glycogen.
Hepatic glycogen was determined by the following procedure which is
described by Ong KC and Kho HE, Life Sciences 67 (2000) 1695-1705. Weighed
amounts (0.3-0.5g) of frozen liver tissue were homogenized in 10 volumes of
ice-cold
30% KOH and then boiled at 100 C for 30 minutes. Glycogen was precipitated
with
ethanol, pelleted, washed, and resolubilized in distilled water. Glycogen
content was
determined by treating the aqueous solution with anthrone reagent (1 g
anthrone
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dissolved in 500 ml conc. %SO4). The absorbance of the solution at 625 am was
measured in a spectrometer and the amount of glycogen present was calculated.
The results are shown in Figure 17, which compares the concentration of
glycogen present in the liver for the five treatment groups. The values are
the average of
the one and two hour values, which were similar to each other. Regular
insulin, which
has been shown to be ineffective as a stimulant for hepatic glucose and
glycogen storage,
was used as a negative control. HDV-Insulin with the Cr-disofenin HTM was the
positive control and it had a significantly higher glycogen content (p<0.05)
than did the
regular insulin negative control. Thus the expected statistical and
biologically significant
differences between the negative and positive controls post dosing were
observed.
Test materials 1 and 3, which had the extended amphipathic lipids biotin
DHPE [triethylanunonium 2,3-diacetoxypropyl 2-(54(36,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-y1) pentanamido)ethyl phosphate] and biotin-X DHPE
[triethylammonium 2,3-diacetoxypropyl 2-(6-(5-((3aS,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-y1) pentanamido)hexanamido) ethyl phosphate] had
statistically
higher (p-A.05) glycogen levels than did the regular insulin. Test material 2,
which also
had biotin-X DHPE, but with lipid concentrations one-half of those in test
material 3, had
glycogen levels that were higher, but the within group variability was great
enough to
give a p=0.08.
Experimental Example 6. Pharmaceutical Composition of HDV-Glargine Insulin
A hepatocyte targeted composition comprises a mixture of free glargine
insulin and glargine insulin associated with a water insoluble target molecule
complex.
The complex comprises multiple linked individual units and a lipid construct
matrix,
comprising a mixture of 1,2-distearoyl-sn-glycero-3-phosphocholine,
cholesterol, dicetyl
phosphate. The bridging agent polychromium poly(bis) [N-(2,6-
dfisopropylphenylcarbamoylmethyl) iminodiacetic acid] is present within the
complex.
Experimental Example 7. Proaration of HDV-Glarzine Insulin
An intermediate mixture of the components of a target molecule complex
was produced by the following procedure. A mixture of the components [total
mass of
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2.830 g] of a target molecule complex was prepared by adding aliquots of the
lipids 1,2-
distearoyl-sn-glycero-3-phosphocholine (2.015 g), crystalline cholesterol
(0.266 g), and
dicetyl phosphate (0.515 g) to the bridging agent, polychromium poly(bis) [N-
(2,6-
diisopropylphenylcarbamoylmethyl) iminodiacetic acid] (0.034 g). A solution of
chloroform (50 ml) and methanol (25 ml) had been dehydrated over molecular
sieves.
The mixture of the components of the target molecule complex was added to the
chloroform/methanol solution, which was then placed in a water bath at 60 C 12
C to
form a solution. The chloroform/methanol solution was removed under vacuum on
a
rotary evaporator using an aspirator, followed by a vacuum pump, and the solid
intermediate mixture formed.
A target molecule complex was produced by the following process. The
pH of 530 ml of Sterile Water for Injection, USP (SWI) was adjusted to between
pH 6.5 -
7.5 by the addition of a 105 1 of 0.1 N NaOH solution. Sufficient water was
added to
make 200 g of product. The pH adjusted SWI was added to the intermediate
mixture
(2.830 g) and the intermediate mixture was hydrated by placing the mixture in
a water
bath at 80 C 2 C while rotating the mixture for approximately 30 minutes
15 minutes,
or until the mixture was a uniform appearing suspension. During the previous
process,
the pH of the suspension decreased. The pH of the suspension was then adjusted
to pH
5.44 0.5 pH units by the addition of approximately 1.0 ml 0.1 N NaOH.
The suspension of the hydrated target complex was transferred to a model
M-110 Em microfluidizer that was preheated to 70 C 10 C with 28 mM sodium
phosphate buffer at pH 7Ø The suspension was microfluidized at 9,000 psig
using one
pass of the suspension of the hydrated target molecule complex through the
fluidizer.
After passing through the tnicrofluidizer, an unfiltered sample (2.0¨ 5.0 ml)
of the
fluidized suspension was collected for particle size analysis using unimodal
distribution
data from a Coulter N-4 plus particle size analyzer. Prior to all particle
size
determinations, the sample was diluted with 0.2 micron filtered SWI that has
been pH
adjusted to between 6.5 ¨ 7.5. The particle size was required to range from
0.020 ¨ 0.40
microns. If the particle size was not within this range, the suspension was
passed through
the microfluiclizer again, and the particle size was analyzed again until the
particle size
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requirements was reached. The microfluidized target molecule complex was
collected in
a sterile container.
The suspension of the microfluidized target molecule complex was
maintained at 60 C 2 C while filtered twice through a sterile 0.8 micron +
0.2 micron
= 5 gang filter attached to a 5.0 ml syringe. An aliquot of the filtered
suspension was
analyzed to determine the particle size range of particles in the suspension.
The particle
size of the final 0.2 micron filtered sample was in the range from 0.0200 -
0.2000
microns, as determined from the unimodal distribution printout from the
particle size
analyzer. The pH of the filtered suspension of the target molecule complex was
3.74
0.2 pH units before pH adjustment. Samples were stored in a refrigerator
between 2 -8 C
until further use.
The pharmaceutical composition comprising a mixture of free glargine
insulin and glargine insulin associated with a water insoluble target molecule
complex,
also referred to as HDV-glargine insulin, was produced was produced by the
following
process. The pH of a 5.0 ml aliquot of the twice filtered suspension of the
target
molecule complex was adjusted from an initial pH of pH 3.74 + 0.2 to pH 5.2 +
pH 0.5
by the sequential addition of sterile 0.1 NaOH according to the following
procedure:
pH 3.74 + 10 1 0.1 N NaOH pH 3.96
pH 3.96 + 20 1 0.1 N NaOH pH 4.52
pH 4.52 -I- 10 1 0.1 N NaOH -q:tfl 4.69
pH 4.69 + 101t1 0.1 N NaOH -+ pH 5.01
pH 5.01 + 10 1,110.1 N NaOH -+ pH 5.20
A 1.6 ml aliquot of the target molecule complex suspension at pH 5.20
+0.5 was combined with 18.4 ml of SW!, which had been adjusted to pH 3.95
0.2. The
pH of the resulting suspension was adjusted from pH 4.89 to pH 5.27 0.5 by
the
addition of 10 1 1.0 pl of 0.1 N NaOH.
The pH of 5.0 ml aliquot of Lantuse Glargine - U-100 Insulin was
increased from pH 3.88 + 0.2 to pH 4.78 0.5 by the addition of 60 I 2 I
of sterile
0.1 N NaOH with mixing. A 2.5 ml 0.1 ml aliquot of the target molecule
complex
suspension at pH 5.27 + 0.5 was added to 5.0 ml 0.1 ml of the solution of
Glargine
insulin at pH 4.78+ 0.5 to produce the pharmaceutical composition containing a
mixture
of free glargine insulin and glargine insulin associated with the water
insoluble target
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molecule complex. The product contained 66.1 IU of glargine insulin/ml
suspension. In
an embodiment, the mixture of free glargine insulin and glargine insulin
associated with
the complex can be produced in a vial of glargine insulin in situ in order to
manufacture
individual dosage forms.
Example 8. Method of Use of HDV-Glargine Insulin for the Control of Blood
Glucose in
Type I Diabetes Mellitus Patients
HDV-glargine insulin was administered to patients to determine the ability
of HDV-glargine insulin to control post prandial blood glucose levels. Seven
Type I
diabetes mellitus patients were selected. The patients were carefully screened
and
selected according to criteria listed in the study protocol. The patients were
treated with
basal glargine insulin and a short-acting insulin at meal times prior to
entering the HDV-
glargine insulin treatment period. Patients were monitored (via diary cards
and site
contact) for four days prior to administering HDV-glargine insulin to assure
that they
were in acceptable control of their blood glucose levels. Morning fasting
glucose levels
were established to be in the range of 100-150 mg/d1.
During the study, the dose of HDV-glargine insulin for each patient was
1.2X their usual daily dose of basal glargine insulin to compensate for the
amount of
short-acting insulin that they would not receive on the test days. Blood
samples were
taken according to a set schedule over 13 hours. HDV was added to glargine
insulin
using the method previously described to produce a suspension with a final
concentration
of 66.1 IU glargine/m1 and 0.37 mg HDV/ml. The patients were injected with HDV-
glargine insulin one hour prior to the morning breakfast. At each of the three
daily meals,
breakfast, lunch and dinner, a 60 gam carbohydrate meal was prescribed by a
dietitian.
The results of the experiments presented in this Experimental Example are
now described. HDV-glargine insulin was well tolerated by the patients and no
adverse
reactions were observed at the injection sites. Hypoglycemic reactions were
not observed
in patients receiving this treatment. The blood glucose values of patients
treated with
HDV-glargine insulin are graphically presented in Figure 18. Figure 18 shows
that blood
glucose concentrations increased, as anticipated, following meals and glucose
concentrations decreased over time until the next meal was eaten. This pattern
was
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observed for all four patients. Figure 19 shows the effect of a single dose of
HDV-
glargine insulin on average blood glucose concentrations in patients consuming
three
meals during the day. As with the individual patients, blood glucose
concentrations
increased following meals and glucose concentrations decreased over time until
the next
meal was eaten. Average blood glucose concentrations were above the baseline
value at
all time points. The curve suggests that the efficacy of HDV-glargine insulin
improved
throughout the day because there was less variation between the high and low
concentrations after the lunch and dinner meals than the breakfast meal. The
effect of
HDV-glargine insulin on blood glucose concentrations over time relative to
blood
glucose concentrations during fasting are shown in Figure 20. Blood glucose
concentrations increased following meals then decreased over time towards the
glucose
concentration during fasting until the next meal was eaten. Blood glucose
concentrations
were above fasting concentrations throughout the study. Treatment of patients
with
HDV-glargine insulin resulted in some degree of post-prandial blood glucose
level
control, indicating that HDV was able to carry sufficient quantifies of
glargine-insulin to
the liver at mealtimes to provide this control. Blood glucose levels were
typical of Type I
patients that usually receive basal insulin therapy plus short-acting insulins
at meal times.
Experimental Example 9. Pharmaceutical Composition of HDV-Humulin NPH insulin
#1
A hepatocyte targeted composition comprises a mixture of free
recombinant human insulin isophane and recombinant human insulin isophane
associated
with a water insoluble target molecule complex. The complex comprises multiple
linked
individual units and a lipid construct matrix comprising a mixture of 1,2-
distearoyl-sn-
glycero-3-phosphocholine, cholesterol, dicetyl phosphate. The bridging agent
polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic
acid]
is present within the complex.
Experimental Example 10. Pharmaceutical Composition of HDV-Humulin NPH insulin
#2
A hepatocyte targeted composition comprises a mixture of free
recombinant human insulin isophane, free recombinant human regular insulin,
and
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recombinant human insulin isophane and recombinant human regular insulin
associated
with a water insoluble target molecule complex. The complex comprises multiple
linked
individual units and a lipid construct matrix comprising a mixture of 1,2-
distearoyl-sn-
glycero-3-phosphocholine, cholesterol, dicetyl phosphate. The bridging agent
polychromium poly(bis)[N-(2,6-diisopropylphenylcarbamoylmethyl) irninodiacetic
acid]
is present within the complex.
Experimental Example 11. Preparation of HDV-Humulin NPH insulin
An intermediate mixture of the components of a target molecule complex
was produced by the following procedure. A mixture of the components [total
mass of
2.830 g] of a target molecule complex was prepared by adding aliquots of the
lipids 1,2-
distearoyl-sn-glycero-3-phosphocholine (2.015 g), crystalline cholesterol
(0.266 g), and
dic,etyl phosphate (0.515 g) to the bridging agent, polychromium poly(bis)[N-
(2,6-
diisopropylphenylcarbamoylmethyl) iminodiacetic acid] (0.034 g). A solution of
chloroform (50 ml) and methanol (25 ml) had been dehydrated over molecular
sieves.
The mixture of the components of the target molecule complex was added to 25.0
mls the
chloroform/methanol solution, which was then placed in a water bath at 60 C
0.2 C to
form a solution. The chloroform/methanol solution was removed under vacuum on
a
rotary evaporator using an aspirator, followed by a vacuum pump, and the solid
intermediate mixture formed.
A target molecule complex was produced by the following process.
Approximately 200 ml of 28 mM sodium phosphate buffer at pH 7.0 was added to
the
intermediate mixture to form a aqueous suspension. The aqueous suspension was
hydrated in a water bath at 80 C E 2 C while rotating the mixture for
approximately 30
minutes 15 minutes or until the mixture was a uniform appearing suspension.
The suspension of the hydrated target complex was transferred to a model
M-110 EHI microfluidizer that was preheated to 70 C 10 C with 28 mM sodium
phosphate buffer at pH 7Ø The suspension was microfluidized at 9,000 psig
using one
pass of the suspension of the hydrated target molecule complex through the
fluidizer.
After passing through the microfluidizer, an unfiltered sample (2.0¨ 5.0 ml)
of the
fluidized suspension was collected for particle size analysis using unimodal
distribution
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data from a Coulter N-4 plus particle size analyzer. Prior to all particle
size
determinations, the sample was diluted with 28 naM sodium phosphate buffer pH
7Ø If
the particle size was not within the range of 0.020 ¨ 0.40 microns, the
suspension was
passed through the microfluidizer again, and the particle size was analyzed
again. This is
repeated until the particle size is within the range of 0.020 ¨ 0.40 microns.
The
suspension of the microfluidized target molecule complex was collected in a
sterile
container.
The suspension of the microfluidized target molecule complex was
maintained at 60 C 2 C while filtered through a sterile 0.8 micron + 0.2
micron gang
filter attached to a 5.0 ml syringe. An aliquot of the filtered suspension was
analyzed to
determine the particle size range of particles in the suspension. The particle
size of the
final 0.2 micron filtered sample was in the range from 0.0200 ¨ 0.2000
microns, as
determined from the unimodal distribution printout from the particle size
analyzer. The
pH of the filtered suspension of the target molecule complex was 7.0 d 0.5 pH
units.
Samples were stored in a refrigerator between 2 -8 C until further use.
The filtered HDV-lipid suspension contained 14.15 mg of HDV lipid/ml.
A 0.8 ml aliquot of this suspension was added to a 10.0 ml vial of Humulin R.
insulin and
allowed to incubate for several days at 2 -8 C. Then 5.0 ml of the 10.0 ml
Humulin R
insulin HDV suspension was removed with a sterile syringe. To the remaining
5.0 ml of
Humulin R insulin in the vial, 5.0 ml of Humulin NPH insulin was added to form
the
final HDV product. The final HDV composition contained 93.6 units of combined
HDV
Humulin R and HDV Humulin NPH insulin/ml of suspension and 0.52 mg of HDV
lipid/ml. This composition, which can be produced in situ to manufacture
individual
dosage forms, comprised a mixture of free Humulin R insulin, free Humulin NPH
insulin
and both Humulin R insulin and Humulin NPH insulin associated with a lipid
construct.
Example 12. Method of Use of combined HDV Humulin R insulin and HDV-Humulin
NPH insulin for the Control of Blood Glucose in Tyne I Diabetes Mellitus
Patients
HDV-Humulin NPH insulin was administered to patients to determine the
ability of HDV-Humulin NPH insulin to control post prandial blood glucose
levels.
Seven Type I diabetes mellitus patients were selected. The patients were
carefully
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CA 02864366 2014-09-18
screened and selected according to criteria listed in the study protocol. The
patients were
treated with basal Humulin NPH insulin and a short-acting insulin at meal
times prior to
entering the HDV-Humulin NPH insulin treatment period. Patients were monitored
(via
diary cards and site contact) for four days prior to administering HDV-Humulin
NPH
insulin to assure that they were in acceptable control of their blood glucose
levels.
Morning fasting glucose levels were established to be in the range of 100-150
mg/d1.
During the study, the dose of HDV-Humulin NPH insulin for each patient
was 1.2X their usual daily dose of basal Humulin NPH insulin to compensate for
the
amount of short-acting insulin that they would not receive on the test days.
Blood
samples were taken according to a set schedule over 13 hours. HDV was added to
Humulin NPH insulin using the method previously described to produce a
suspension
with a final concentration of 93.6 units of combined HDV Humulin R. insulin
and HDV
Humulin NPH insulin/ml. The final suspension contained 0.52 mg of HDV
lipid/ml.
The patients were injected with the combined HDV-insulins one hour prior to
the
morning breakfast. At each of the three daily meals, breakfast, lunch and
dinner, a 60
gram carbohydrate meal was prescribed by a dietitian.
The results of the experiments presented in this Experimental Example are
now described. HDV-Humulin NPH insulin was well tolerated by the patients and
no
adverse reactions were observed at the injection sites. Hypoglycemic reactions
were not
observed in patients receiving this treatment. The blood glucose values of
patients
treated with HDV-Humulin NPH insulin are graphically presented in Figure 21.
Figure
21 shows that blood glucose concentrations increased, as anticipated,
following meals
and glucose concentrations decreased over time until the next meal was eaten.
This
pattern was observed for all four patients. Figure 22 shows the effect of a
single dose of
HDV-Humulin NPH insulin on average blood glucose concentrations in patients
consuming three meals during the day. As with the individual patients, blood
glucose
concentrations increased following meals and glucose concentrations decreased
over time
until the next meal was eaten. Average blood glucose concentrations were above
the
baseline value at all time points. The curve suggests that the efficacy of HDV-
Humulin
NPH insulin improved throughout the day because there was less variation
between the
high and low concentrations after the lunch and dinner meals than the
breakfast meal.
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The effect of HDV-Humulin NPII insulin on blood glucose concentrations over
time
relative to blood glucose concentrations during fasting are shown in Figure
23. Blood
glucose concentrations increased following meals then decreased over time
towards the
glucose concentration during fasting until the next meal was eaten. Blood
glucose
concentrations were above fasting concentrations throughout the study.
Treatment of
patients with HDV-Humulin NPR insulin resulted in some degree of post-prandial
blood
glucose level control, indicating that HDV was able to carry sufficient
quantities of
Humulin NPR insulin to the liver at mealtimes to provide this control. Blood
glucose
levels were typical of Type I patients that usually receive basal insulin
therapy plus short-
acting insulins at meal times.
While the invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations of the
invention may
be devised by others skilled in the art. The scope of the claims should not be
limited by the
preferred embodiments or the examples but should be given the broadest
interpretation
consistent with the description as a whole.
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