U.S. patent application number 14/977137 was filed with the patent office on 2016-08-25 for liquid insulin formulations and methods relating thereto.
This patent application is currently assigned to Dance BioPharm, Inc.. The applicant listed for this patent is Dance BioPharm, Inc.. Invention is credited to Blaine Bueche, Mei-Chang Kuo, John Patton, Matthew Sander.
Application Number | 20160243199 14/977137 |
Document ID | / |
Family ID | 56689716 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160243199 |
Kind Code |
A1 |
Bueche; Blaine ; et
al. |
August 25, 2016 |
LIQUID INSULIN FORMULATIONS AND METHODS RELATING THERETO
Abstract
Liquid formulations of insulin that contain physically and
chemically stable insulin but do not contain preservatives or
stabilizers are provided. The formulations also lack surfactants.
The formulations are useful for various modes of delivery including
pulmonary delivery.
Inventors: |
Bueche; Blaine; (San Mateo,
CA) ; Kuo; Mei-Chang; (Palo Alto, CA) ;
Patton; John; (San Francisco, CA) ; Sander;
Matthew; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dance BioPharm, Inc. |
Brisbane |
CA |
US |
|
|
Assignee: |
Dance BioPharm, Inc.
Brisbane
CA
|
Family ID: |
56689716 |
Appl. No.: |
14/977137 |
Filed: |
December 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62120573 |
Feb 25, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/0073 20130101;
A61P 3/10 20180101; A61K 38/28 20130101 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61K 9/00 20060101 A61K009/00 |
Claims
1. An insulin formulation, comprising: insulin at a minimal
concentration of 1-13 mM; a salt at a concentration of 50-150 mM; a
pH buffering agent at a concentration of 3-24 mM; zinc at a ratio
of 1.9-2.7 zinc ions per insulin hexamer; and a pH in the range of
7.2 to 8.0, wherein the formulation does not contain preservatives
or stabilizers.
2. The formulation of claim 1, wherein the insulin is human
insulin.
3. The formulation of claim 1, wherein the formulation comprises at
least 30 mg/mL insulin.
4. The formulation of claim 1, wherein the formulation comprises
10-30 mg/mL insulin.
5. The formulation of claim 1, wherein the insulin concentration is
about 30 mg/mL and the pH is about 7.55.
6. The formulation of claim 1, wherein the salt is a chloride
salt.
7. The formulation of claim 1, wherein the salt is NaCl.
8. The formulation of claim 1, wherein the pH buffering agent
comprises citrate.
9. The formulation of claim 1, wherein the tonicity (ionic
strength) of the formulation is 100-300 mOsm.
10. The formulation of claim 1, wherein the formulation is
administrable by inhalation or injection.
11. The formulation of claim 1, wherein the formulation is
administrable by an inhalation device.
12. The formulation of claim 1, wherein the formulation is
aerosolizable.
13. The formulation of claim 1, wherein the formulation does not
contain surfactants.
14. A unit dose of the pharmaceutically acceptable formulation of
claim 1.
15. A method of treating a subject with diabetes mellitus,
comprising administering to a subject having diabetes mellitus a
therapeutically effective amount of the formulation according to
claim 1, wherein the formulation is administered to the subject via
inhalation or injection.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/120,573, filed Feb. 25, 2015, the contents
of which is incorporated by reference in its entirety herein.
BACKGROUND
[0002] Diabetes mellitus is a metabolic disorder in which an
individual's ability to moderate blood glucose levels in response
to insulin is lost. Insulin is a hormone secreted by the pancreas
into the blood that triggers cells to take up glucose. When the
body cannot produce insulin, as occurs in type 1 diabetes, or is no
longer responsive to insulin and/or produces less insulin, as
occurs in type 2 diabetes, blood glucose levels rise. Complications
from diabetes include increased risk of cardiovascular disease,
neuropathy, nephropathy, retinopathy, foot damage, skin conditions,
hearing impairment, and Alzheimer's disease. Treatment for type 1
diabetes involves insulin injections or the use of an insulin pump.
Type 2 diabetes is also often treated with insulin injections or
pumps.
[0003] The human insulin protein is composed of 51 amino acids and
has a molecular weight of 5808 Da. The amino acid sequence is
strongly conserved in invertebrates. While initially synthesized as
a single polypeptide chain, it is cleaved to form an A-chain and a
B-chain that are linked together by disulfide bonds. Insulin is
produced and stored in the body as a zinc-stabilized inactive
hexamer (a unit of six insulin molecules), while the active form is
the monomer. Hexameric insulin is very stable, serving to keep the
highly reactive insulin protected, yet readily available in the
blood. Hexamer formation and disassociation reflect the following
equilibria:
6In3In.sub.2+2Zn.sup.2+In.sub.6(T.sub.6)In.sub.6(R.sub.6).
Insulin monomers above 1 .mu.m concentration can form non-covalent
dimers (In.sub.2). The binding of two zinc molecules facilitates 3
dimers (In.sub.2) assembling into the hexameric form. Hexameric
insulin can transition between two primary states: T.sub.6 and
R.sub.6. The conversion between the T.fwdarw.R forms is related to
the conformational flexibility in the N' terminal portion (B1-B8)
of the B chain. In the T state, the B1-B8 region is in an extended
linear state, while in the R state, it is an alpha helix. (Kim and
Shield (1992) Biochem. Biophys. Res. Comm. 186(2): 1115-120.) The
alpha helix form of the B1-B8 region can stabilize the hexamer. The
interconversion between the T.sub.6 and R.sub.6 conformations of
the insulin hexamer may be modulated by ligand binding to the
T.sub.6 and R.sub.6 forms. Such ligands may include anions,
preservatives, and stabilizers. For example, phenolic preservatives
and stabilizers bind to hydrophobic pockets located near the
surfaces of insulin to promote the R.sub.6 state. (Derewenda et al.
(1989) Nature 338(6216): 594-596; Brzovic et al. (1994)
Biochemistry 33:13057-13069.)
[0004] Insulin degradation during storage is a challenge when
developing commercial liquid insulin formulations. Insulin
formulations degrade or lose potency by a number of physical
processes and chemical reactions, including: a) precipitation
(crystal formation); b) fibril formation (protein denaturation); c)
hydrolysis reactions, especially deamidation of certain amino acid
positions (including A18, A21 and B3); d) covalent dimerizations
via transamidation or Schiff-base formation; and e) disulfide
exchange reactions. (Strickley and Anderson (1997) J. Pharma. Sci.
86(6): 645-53.)
[0005] Fibril formation (aggregation) generally occurs when two or
more partially unfolded insulin monomers are brought together by
shaking or shear force (for example, during shipment). The
hydrophobic surfaces of the insulin molecule bind together,
irreversibly forming an insulin fiber. Insulin monomers continue to
bind, and the fiber elongates until it becomes insoluble in aqueous
solution. The formation of these inactive insulin fibers, results
in visible cloudiness and loss of potency in insulin
formulations.
[0006] Commercial insulin formulations often include the hexameric
configuration of insulin and excipients such as preservatives or
stabilizers (such as phenolic compounds), which contribute to the
rigidity of the protein complex and promote hexamer stability.
Conformational flexibility in the B1-B8 region of the B chain
appears to be a factor in the chemical instability of insulin. An
increase in the rigidity of the N' terminal B chain via increased
interaction (promoting hexamer form), or by adding excipients (such
as phenolic compounds) that induce a helical structure in the B1-B8
region, may decrease the deamidation and covalent dimer reactions
(Berchtold and Hilgenfeld (1999) Biopolymers 51(2): 165-72;
Derewenda et al. (1989) Nature 338(6216): 594-96; Darrington and
Anderson (1994) Pharma. Res. 11(6): 784-793; Brange et al. (1994)
Stability of insulin: studies on the physical and chemical
stability of insulin in pharmaceutical formulation. Dordrecht, The
Netherlands: Kluwer Academic Publishers.)
[0007] Phenolic compounds, in particular phenol and cresol, have
proven so effective at stabilizing the B1 chain and promoting
hexamer formation that they are routine components of commercial
liquid insulin formulations. Many such preservatives and
stabilizers can be mucose irritating and malodorous. However,
formulations lacking such components (see, for example, U.S. Pat.
No. 6,211,144) may have significant protein instability and may be
more susceptible to physical instability during shipment.
BRIEF SUMMARY
[0008] Disclosed herein are liquid insulin formulations suitable
containing insulin at a minimum concentration of 1-13 mM, a salt at
a concentration of 50-150 mM, a pH buffering agent at a
concentration of 3-24 mM, zinc at a ratio of 1.9-2.7 zinc ions per
insulin hexamer (0.35-0.5% w/w zinc to insulin), and a pH in the
range of 7.2 to 8.0. An important feature of these formulations is
that they do not contain preservatives or stabilizers. Also
provided are unit doses of such insulin formulations, and kit
containing such formulations.
[0009] Further disclosed are methods of treating a subject with
diabetes mellitus, the method involving administering to a subject
having diabetes mellitus a therapeutically effective amount of an
insulin formulation containing insulin at a minimum concentration
of 1-13 mM, a salt at a concentration of 50-150 mM, a pH buffering
agent at a concentration of 3-24 mM, zinc at a ratio of 1.9-2.7
zinc ions per insulin hexamer (0.35-0.5% w/w zinc to insulin), and
a pH in the range of 7.2 to 8.0, wherein the formulation does not
contain preservatives or stabilizers. The formulations may be
administered to the subject via inhalation or injection.
[0010] It will be appreciated from a review of the remainder of
this application that further methods and compositions are also
part of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the formation of Iso B3 insulin degradation
product overtime in insulin formulations including different pH
buffering agents (Phosphate, Tris, Citrate, Tris/Citrate) and/or
salt (NaCl) according to some aspects.
[0012] FIG. 2 shows the formation of Insulin Related Substances
(IRS) degradation product overtime in the same insulin formulations
as shown in FIG. 1 according to some aspects.
[0013] FIG. 3 shows the chemical stability, as evidenced by Iso B3
insulin degradation product formation overtime, of insulin
formulations having different pH values according to some
aspects.
[0014] FIG. 4 shows the chemical stability, as evidenced by IRS
degradation product formation overtime, of the same insulin
formulations as shown in FIG. 3 according to some aspects.
[0015] FIG. 5 shows the potency, as evidence by insulin
concentration, of insulin formulations having 300 U/mL or 900 U/mL
insulin, 0.39% zinc, 6 mM sodium, citrate, 70 mM sodium chloride,
at pH 7.5 overtime during cold storage according to some
aspects.
[0016] FIG. 6 shows the accumulation of IRS degradation products
overtime at cold storage for the same formulation as in FIG. 5
according to some aspects.
[0017] FIG. 7 shows the accumulation of Iso B3 degradation products
overtime at cold storage for the same formulation as in FIG. 5
according to some aspects.
[0018] FIG. 8 shows the accumulation of high molecular weight
polymers (HMWP) degradation products overtime at cold storage for
the same formulation as in FIG. 5 according to some aspects.
[0019] FIG. 9 shows the insulin solubility, measured by
concentration change, in formulations containing 0.39% w/w zinc to
insulin over time as a function of pH according to some
aspects.
[0020] FIG. 10 shows the insulin concentration change, measured by
concentration change, in formulations containing 0.45% w/w zinc to
insulin over time as a function of pH according to some
aspects.
[0021] FIG. 11 shows the insulin concentration change, measured by
concentration change, in formulations containing 0.51% w/w to
insulin zinc over time as a function of pH according to some
aspects.
[0022] FIG. 12 shows a comparison of the final concentration of
insulin in formulations containing 0.39% w/w, 0.45% w/w, and 0.51%
w/w zinc to insulin as a function of pH and zinc concentration
according to some aspects.
[0023] FIG. 13 shows a stability assessment following exposure to
shear forces of preservative free formulations containing 280 U/mL
and 840 U/mL insulin in comparison to commercial Humulin R.RTM.
U-500, which contains a phenolic preservative, according to some
aspects. The formulations were shaken over a period of 28 days.
Insulin concentration was measured by HPLC, and potency is plotted
as percent initial insulin concentration over time.
[0024] FIG. 14 shows a stability assessment following exposure to
shear forces of preservative free formulations containing 280 U/mL
and 840 U/mL insulin in comparison to commercial Humulin R.RTM.
U-500 as in FIG. 13, according to some aspects. Formulations were
shaken over a period of 15 days, and then turbidity was assessed
using a nephelometer. Turbidity is shown plotted against time. The
turbidity reading for the Humulin formulation reached the maximum
threshold for the device by day 8.
[0025] FIG. 15 shows the impact of adding phenol on the chemical
stability of high concentration insulin formulations over time in
cold storage according to some aspects. Formulations containing 70
mg/ml insulin at pH 7.2 and 7.4, including batches containing 0.25%
v/v phenol ("phenol") and not containing phenol ("preservative
free"), were prepared. The formulations were assessed at time 0, 3
months, and 6 months. Insulin concentration (potency) was measured
by HPLC and potency plotted against time.
DETAILED DESCRIPTION
[0026] This detailed description of the aspects and embodiments of
the present disclosure is organized into sections as follows.
Section I provides an introduction to the subject matter of the
disclosure. Section II provides definitions of terms used herein.
Section III describes the formulations. Section IV describes kits
containing the formulations. Section V describes treatment methods.
Section VI provides exemplary formulations and treatment methods.
This detailed description is organized into sections only for the
convenience of the reader, and disclosure found in any section is
applicable to disclosure elsewhere in the specification.
I. Introduction
[0027] Certain embodiments and aspects of the present disclosure
relate to liquid insulin formulations and methods of treating a
subject having diabetes mellitus using the formulations. Relative
to existing commercial formulations, the disclosed formulations are
stable for sufficient periods of time for a viable commercial
product. Importantly, the formulations are stable without
containing preservatives or stabilizers, such as phenolic
compounds. In addition, the described formulations have sufficient
insulin concentration to be useful for delivering a desired dosage
of insulin to a subject in a minimum volume, which can be
particularly useful for pulmonary administration. In some
instances, the formulations may contain relatively high insulin
concentrations relative to commercial formulations. In some
aspects, the formulations may be administered to a subject by
aerosolization in a minimum volume and inhalation time. The
formulations may be particularly suitable for aerosol delivery in
part, by the lack of preservatives and stabilizers, which can be
irritants and can impair dose delivery, by the lack of surfactants,
which can also impair dose delivery, and by the relatively high
insulin concentrations that can be provided. In addition, the
formulations contain soluble insulin that is physically and
chemically stable for extended periods of time even after extended
exposure to shear forces. Long term stability, including chemical
stability during storage and physical stability during shipping,
handling, and patient use scenarios, is a factor in regulatory
approval and commercial viability of an insulin formulation. The
provided formulations may remain stable for an extended period of
time such as, for example, at least 24 months, under appropriate
storage conditions (refrigeration). Developing such insulin
formulations, which are stable and may be relatively concentrated,
required optimizing multiple factors and components to achieve a pH
sufficiently high to maintain insulin solubility but sufficiently
low to minimize chemical degradation (such as deamidation) and to
maintain stability without the aid of preservatives or stabilizers
as additives. The conditions identified for the formulations of
this disclosure include a specific pH range and combination of
components, including salt, zinc ions, and a pH buffering agent. An
aspect of the formulations is that protein stability is controlled
primarily through protein concentration, pH value, type of pH
buffering agent, zinc ion concentration, and salt (ionic strength),
without the need for chemical preservatives or stabilizers.
II. Definitions
[0028] The following definitions are provided to assist the reader.
Unless otherwise defined, all terms of art, notations, and other
scientific or medical terms or terminology used herein are intended
to have the meanings commonly understood by those of skill in the
chemical and medical arts. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
be construed as representing a substantial difference over the
definition of the term as generally understood in the art.
[0029] "Administering" or "administration of" a formulation to a
subject (and grammatical equivalents of this phrase), as used
herein, refers to direct administration, which may be
administration to a subject by a medical professional or may be
self-administration, and/or indirect administration, which may be
the act of prescribing a formulation. For example, a physician who
instructs a subject to self-administer a formulation and/or
provides a subject with a prescription for a formulation is
administering the formulation to the subject.
[0030] "Chemical stability", as used herein, refers to the
reactivity of a pharmaceutical composition in a pharmaceutical
formulation and the propensity of the pharmaceutical composition to
react chemically, or decompose chemically, in the pharmaceutical
formulation. Examples of chemical instability for insulin
formulations include oxidation and hydrolysis of the insulin
protein.
[0031] "Physical stability", as used herein, refers to the ability
of a pharmaceutical composition to retain its normal physical
structure in a pharmaceutical formulation and, as a result, the
propensity of the pharmaceutical composition to aggregate and/or
precipitate out of solution during storage and usage. The physical
stability of insulin formulations is reflected by the ability of
the insulin protein to retain its native configuration in the
formulation. Exemplary physical instability for insulin
formulations includes fibrillation.
[0032] "Potency", as used herein, refers to the activity or amount
of a active agent in a composition, such as the amount of insulin
in a pharmaceutical formulation. Potency may be stated in terms of
the amount of active agent required to produce an effect. For
example, units of insulin per milliliter (U/mL), or milligrams of
insulin per milliliter (mg/mL). As used herein, potency may be used
to refer to the desired insulin concentration of the described
formulations. In some instances, percent (%) potency may be used to
refer to the amount of insulin (concentration) in a formulation as
compared to a starting/initial concentration.
[0033] "Preservative", as used herein, refers to a class of
compounds that prevents or inhibits the growth of microorganisms,
as well as compounds that help control oxidation reactions in
pharmaceuticals. Phenol and EDTA are examples of preservatives.
[0034] "Stabilizer", as used herein, refers to a substance that
acts to stabilize secondary and tertiary structures of proteins in
solution and, as a result, reduces the rate of degradation
(chemical, physical, or both) of the proteins. For the insulin
hexamer, exemplary stabilizers are phenol, meta-cresol, and other
phenolic compounds.
[0035] "Surfactants", as used herein, refers to amphiphilic organic
compounds (having hydrophobic groups and hydrophilic groups) that
aggregate to form micelles in aqueous formulations at critical
concentrations, providing greater solubility for hydrophobic
compounds. Surfactants may be applied to formulations may increase
the physical stability of the formulations, modify their
solubility, or both.
[0036] "Therapeutically effective amount" of an insulin
formulation, as used herein, refers to an amount of an insulin
formulation that, when administered to a subject with diabetes
mellitus, will have the intended therapeutic effect, for example,
increased cellular uptake of blood glucose and reduced blood
glucose levels. A therapeutically effective amount may be
administered in one or more administrations.
[0037] "Treating" or "treatment of" a condition or subject, as used
herein, refers to taking action to obtain beneficial or desired
results, including clinical results, for a subject. For purposes of
this disclosure, beneficial or desired clinical results include,
but are not limited to, increased cellular uptake of blood glucose,
reduced blood glucose levels, or both.
III. Formulations
[0038] Provided are stable insulin formulations that do not contain
preservatives, stabilizers, or surfactants. The formulations may
include 1-13 mM insulin, 50 to 150 mM of a salt, 3 to 24 mM of a
buffering agent; zinc ions at a ratio of 1.9 to 2.7 zinc ions per
insulin hexamer (alternatively referred to as 0.35% to 0.5% wt/wt
of zinc to insulin), and a pH of 7.2 to 8.0. In some instances, the
formulations may include 6 mg/mL to 76 mg/mL insulin.
[0039] Several properties of insulin were taken into account in the
development of the formulations described herein. Development
factored in that the self-association pattern of insulin in
solution is a complex function of protein concentration, metal
ions, pH, ionic strength, and solvent composition. In one aspect,
the formulations include hexameric insulin as the majority insulin
species. The formulations are generally formulated to minimize the
formation of insulin degradation products.
[0040] In one aspect, the formulations contain insulin. In some
instances, the formulations may contain insulin in the range of 1
mM to 13 mM. In some instances, the formulations may contain
insulin in the range of 6 mg/mL to 76 mg/mL. In some instances, the
formulations may contain insulin in the range of 173 U/mL to 2189
U/mL. For example, the formulations may have 1 mM, 2 mM, 3 mM, 4
mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, or 13 mM, or
a concentration within 0.5 mM of any of these concentrations. In
some instances, the formulations may have an insulin concentration
of 6 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 30 mg/mL, 35 mg/mL, 40
mg/mL, 45 mg/mL, 50 mg/mL, 55 mg/mL, 60 mg/mL, 65 mg/mL, 70 mg/mL,
75 mg/mL, or a concentration with 3 m/mL of any of these
concentrations. In some instances, the formulations may have an
insulin concentration of about 170 U/ml, 175 U/ml, 190 U/ml, 225
U/ml, 260 U/ml, 280 U/ml, 300 U/ml, 325 U/ml, 350 U/ml, 375 U/ml,
400 U/ml, 425 U/ml, 450 U/ml, 475 U/ml, 500 U/ml, 525 U/ml, 550
U/ml, 600 U/ml, 625 U/ml, 650 U/ml, 675 U/ml, 700 U/ml, 725 U/ml,
750 U/ml, 775 U/ml, 800 U/ml, 825 U/ml, 850 U/ml, 875 U/ml, 900
U/ml, 925 U/ml, 950 U/ml, 975 U/ml, 1000 U/ml, 1025 U/ml, 1050
U/ml, 1075 U/ml, 1100 U/ml, 1125 U/ml, 1150 U/ml, 1175 U/ml, 1200
U/ml, 1225 U/ml, 1250 U/ml, 1275 U/ml, 1300 U/ml, 1325 U/ml, 1350
U/ml, 1375 U/ml, 1400 U/ml, 1425 U/ml, 1450 U/ml, 1475 U/ml, 1500
U/ml, 1525 U/ml, 1550 U/ml, 1575 U/ml, 1600 U/ml, 1625 U/ml, 1650
U/ml, 1675 U/ml, 1700 U/ml, 1725 U/ml, 1750 U/ml, 1775 U/ml, 1800
U/ml, 1825 U/ml, 1850 U/ml, 1875 U/ml, 1900 U/ml, 1925 U/ml, 1950
U/ml, 1975 U/ml, 2000 U/ml, 2025 U/ml, 2050 U/ml, 2075 U/ml, 2100
U/ml, 1225 U/ml, 1250 U/ml, 1275 U/ml, or 2200 U/ml. The
formulations may contain an insulin concentration of at least 30
mg/mL or 5.17 mM. The formulations may contain an insulin
concentration up to about 76 mg/mL or 13 mM. In some instances, the
formulations may have an insulin concentration of 30 mg/mL to 76
mg/ml or 5.17 mM to 13 mM. In some instances, the formulations may
have an insulin concentration of 10 mg/mL to 30 mg/ml or 1.72 mM to
5.17 mM. In certain aspects, the formulations may contain an
insulin concentration in the range of 6 mg/mL to 35 mg/mL or 1 mM
to 6.03 mM. In some instances, the formulations contain insulin
that is primarily in a hexameric state, which can enhance chemical
stability of insulin in the formulations. In some examples, the
formulations may be 3 to 10 times more concentrated than existing
commercial formulations. In some instances, the formulations may be
up to 20 times more concentrated than existing commercial
formulations such as, for example, Humulin.RTM. R U-100 (100 U/mL,
3.47 mg/ml), pH 7.4, and Humulin.RTM. R U-500 (500 U/mL, 17.35
mg/ml, 2.99 mM), pH 7.4 (Eli Lilly).
[0041] The type of insulin included in the formulations may vary.
In some instances, the insulin may be human, porcine, or bovine
insulin. In some instances, the formulation includes human insulin.
In some instances, the insulin is human insulin. The insulin may be
a recombinant protein derived from human or other mammalian cell
lines. In some instances, the insulin may be a recombinant insulin
protein derived from prokaryotic cells. In some instances, the
insulin may be the full-length, 51 amino acid wild-type sequence of
the insulin protein. In other instances, the insulin may be an
insulin analogue that has a genetically modified sequence. For
example, the insulin may have one or more amino acids deleted
and/or replaced by other amino acids, including non-codeable amino
acids, or may have one or more amino acids added to the protein
sequence. In some instances, as described in Examples 1 to 6, the
formulations may contain recombinant human insulin as commercially
available from various suppliers (for example, Sanofi-Aventis
(Material: 192228, GMID: 341921), EMD Millipore (Cat.
#407709-50MG), Sigma Aldrich (CAS #11061-68-0, Cat. #91077C, EC
#234-279-7, MDL # MFCD00131380)). For reference, 1 U is equivalent
to 0.0347 mg of human insulin (28.8 U/mg). Also, as noted above,
the molecular weight of human insulin is 5808 Daltons (g/mol).
[0042] In one aspect, the formulation may be isotonic. Extremely
hypotonic or hypertonic solutions can cause mucus membrane
irritation. For example, inhalation of low salt solutions (such as
pure water) or high salt solutions can cause irritation of the
throat and lungs, often resulting in coughing and discomfort. In
some instances, the formulations have a tonicity of 100 mOsm-300
mOsm. Various compounds can be used to adjust the tonicity of the
formulations. Tonicity of the formulations may be adjusted by
including a salt or other solute that does not readily cross a cell
membrane. In some instances, the salt concentration may be modified
to optimize ionic strength and tonicity. In some instances, the
concentration of the salt may be varied as the concentration of
insulin in the formulation is varied to achieve the desired
tonicity. In some instances, ionic strength of the formulations may
be optimized to make the formulation comfortable to inhale (such as
for pulmonary administration).
[0043] In some instances, the formulations may include a chloride
salt. In some cases, the salt is a chloride salt. For example, in
some instances, the salt may be sodium chloride. In some cases,
insulin may be particularly stable in a formulation comprising
sodium chloride as compared to other salts. For example, as
described in Examples 1 to 6, the salt may be NaCl. In another
aspect, the formulations may contain a salt, such as a chloride
salt, in the range of about 50 to 150 mM. For example, the salt
concentration may be about 50-70 mM, about 60-80 mM, about 90-100
mM, about 65-75 mM, about 75-85 mM, about 85-95 mM, about 70-140
mM, about 110-120 mM, about 130-150 mM, or any range therein. In
some examples, the salt concentration may be about 50 mM, about 55
mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80
mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, or a
concentration within 2-3 mM of any of these concentrations. In some
instances, as described in Examples 1 to 6, NaCl may be included in
the formulations at a concentration of 70 mM.
[0044] In one aspect, the formulation contains 1.9 to 2.7 zinc ions
per six molecules of insulin (hexamer), which is equivalent to
about 0.35% to 0.5% wt/wt of zinc to insulin. In some instances,
the zinc ions may promote the solubility of the insulin in the
formulation. For example, two zinc ions may promote and/or
stabilize the insulin hexamer. For example, in the absence of zinc
ions, or if the zinc to hexamer ratio is too low (such as less that
1.9:1), the insulin may be physically unstable and unlikely to form
hexamers. Also, for example, if the ratio of zinc ion to insulin
hexamer is too high (such as 3:1 or greater), the insulin may
precipitate out of solution. In some instances, 0.35% to 0.5% wt/wt
of zinc to insulin provides stability to the insulin in
formulation. In some instances, various proportions of the insulin
in the provided formulations may be bound with 0, 1, 2, or 3 zinc
ions, or a mixture thereof, such that the ratio of zinc ions to
insulin hexamer for the formulation is 1.9 to 2.7. The formulations
may contain 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, or 2.7 zinc
ions per insulin hexamer. In some instances, the formulations may
contain 2.0 to 2.3 zinc ions per hexamer, 2.1 to 2.4 zinc ions per
hexamer, 2.2 to 2.5 zinc ions per hexamers, 1.9 to 2.5 zinc ions
per hexamer, 2.2 to 2.7 zinc ions per hexamer, 2.2 to 2.6 zinc ions
per hexamer, or 2.3 to 2.6 zinc ions per hexamer.
[0045] In another aspect, the formulations may have a pH buffering
capacity. For example, the formulations may include a pH buffering
agent. For example, the pH buffering agent can be an ionizable salt
such as phosphate, acetate, citrate, bicarbonate, Tris, and the
like, with a counter-ion. The pH buffering agent acts to reduce
fluctuations in the pH of the formulation. In certain instances,
the pH buffering agent may be citrate, Tris, or Tris-citrate as
described in Example 1 and shown in FIG. 1 and FIG. 2. In another
aspect, the formulations may include a pH buffering agent at a
concentration of about 3 to 24 mM. For example, the pH buffering
agent concentration may be 3-6 mM, 5-10 mM, 8-16 mM, or 12-24 mM.
In some instances, the pH buffering agent concentration may be 3
mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM,
14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23
mM, or 24 mM, or any concentration with 0.5 mM of these
concentrations. In some instances, the pH buffering agent is
citrate. In some instances, the pH buffering agent may be 6 mM
citrate.
[0046] The stability and solubility of insulin may be influenced by
the pH of the formulation. For example, the solubility of insulin
is low at pH values near its isoelectric pH (pH range 4.0-7.0). In
one aspect, the formulations may have a relatively neutral pH such
as, for example, within 0.6 pH units of physiological pH
(approximately 7.4). In some instances, the pH of the formulations
may be slightly alkaline, such as up to pH 8. The pH of the
formulations may be in the range of 7.2 to 8.0. A pH within this
range, in combination with the components of the formulations,
imparts particular stability to the insulin in the formulations.
For example, the pH of the formulation may be about 7.2 to about
8.0. In some instances, the formulation has a pH of 7.2, 7.25, 7.3,
7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8, 7.85, 7.9,
7.95, or 8.0, or a pH within 0.5 of any of these pH values. In some
instances, the chemical and physical stability of formulations may
be reduced at pH less than 7.2. In certain cases, the insulin
protein may start to degrade at pH 8.0. Highly concentrated
solutions of porcine insulin (5000 U/ml, approx. 30 mM) have been
created at acidic pH but the insulin in the formulation is highly
unstable due to deamidation. (Galloway (1981) Diabetes Care 4,
366-376.) Highly concentrated solutions of zinc-free insulin can be
made at slightly alkaline pH (such as pH 8-9) but they are unstable
due to a high rate of polymerization and deamidation. Also, highly
concentrated porcine zinc insulin solutions at neutral pH
comprising phenol are physically stable at elevated temperature but
become supersaturated when the temperature is lowered to 4.degree.
C. (Brange and Havelund, Properties of Insulin in Solution,
Artificial Systems for Insulin Delivery, 1983 (Brunetti et al.
eds., Raven Press, New York), pp. 83-88.) In contrast, the
formulations of this disclosure are stable and may contain
concentrated insulin at a pH of 7.2 to 8.0 without including
preservatives or stabilizers as described in Examples 1 to 6 and
shown in FIGS. 1-15. In one example, as described in Example 3,
formulations may have a pH of about 7.5 and include about 30 mg/mL
insulin. In another example, as described in Example 3,
formulations may have a pH of about 7.5 and include about 10 mg/mL
insulin. In another example, as described in Example 4, the
formulations may contain 35 mg/mL insulin at pH 7.2 and pH 7.3. In
another example, as described in Example 5, the formulations may
contain either 9.7 mg/mL or 29.2 mg/mL at pH 7.55. In another
example, as described in Example 6, the formulations may contain 70
mg/mL insulin at either pH 7.2 and 7.4.
[0047] In some instances, the pH of the formulations may impact
insulin degradation. As mentioned, two primary routes of insulin
degradation are hydrolysis and the formation of high molecular
weight polymers (HMWP). The most common hydrolysis products are B3
deamidated insulin (B3 and Iso B3) and A21 deamidated insulin
(A21), arising from deamidation at the 3.sup.rd amino acid (Asn) in
the B chain and deamidation at the 21.sup.st amino acid (Asn) in
the A chain, respectively. In some instances, hydrolytic
degradation of insulin having an aspargine at amino acid position
21 (A21) may be highest in acidic pH, lowest at a pH of 6.5, and
generally low at neutral pH. In one example, the predominant
hydrolysis product at low pH may be deamidation (loss of an ammonia
group) from Asparagine A21 (A21-Asn). In another example,
hydrolysis may increase at alkaline pH (such as pH 7.5-9.0) with
deamidation generally occurring predominantly at Asparagine B3
(B3-Asn). In some instances, the deamidation of Asn-B3 may proceed
via formation of a cyclic imide intermediate to form aspartic acid
(B3) and iso-aspartic acid (Iso B3), producing two separate
by-products. As A21, B3 and Iso B3 are nearly 100% bioactive
(Brange et al. (1992) Acta Pharm. Nord. 4:223-232), these
degradation products generally do not impair the potency of the
insulin formulations.
[0048] The accumulation of HMWP products can be more problematic
for the production of stable commercial insulin formulations, which
are required to have very low amounts of such degradants for
injectable insulin products (for example, 1.7% limit on HMWP; see
U.S. Pharmacopeia (U.S. Monograph)). In some instances, maximum
stability of aqueous insulin formulations may occur around neutral
pH. In some cases, the rate of HMWP product formation of insulin
may be high at acidic pH in many aqueous formulations but
relatively low at pH in the range of about 6.5 to 8.0. For example,
the rate of formation of HMWP products may be accelerated in
alkaline media above pH 9.0 (for example, because disulfide
reactions may be favored). Limiting HMWP formation is the reason
nearly all commercial injectable insulin formulations are currently
formulated at neutral pH. However, HMWP products may still form at
neutral pH. Formation of these products mainly occurs by
intermolecular aminolysis between N-terminal amines, especially
between the B1-Asn and the amide side chains in the insulin A-chain
of an adjacent insulin molecule, particularly within a hexamer.
[0049] In one aspect, the formulations do not contain preservatives
or stabilizers. In some instances, preservatives and stabilizers
are phenolic compounds. Examples of phenolic compounds include
phenol, cresol, and derivatives thereof. In some instances, the
formulations do not contain organic solvents. In certain aspects,
the formulations do not contain alcohols, including polyols,
sugars, amino acids, or amines. Formulations containing
preservatives and stabilizers are described in U.S. Pat. Nos.
6,489,292 and 6,211,144, which are incorporated herein by
reference. Such preservatives and stabilizers can include phenol
and derivatives thereof such as meta-cresol, chloro-cresol,
methylparaben, ethyl paraben, propyl paraben, thymol, as well as
derivatives thereof and mixtures of such compounds. Some similar
non-phenol preservatives and stabilizers include bi- or tricyclic
aliphatic alcohols and purines, such as a bicyclic aliphatic
alcohol, including a monoterpenol, such as isopinocampheol,
2,3-pinandiol, myrtanol, bomeol, norbomeol or fenchol, a tricyclic
aliphatic alcohol, such as 1-adamantanol, and purines such as
adenine, guanine or hypoxanthine. Other exemplary preservatives and
stabilizers include sodium benzoate, benzalkonium chloride, benzyl
alcohol, and thimerosal. Such preservatives and stabilizers are
generally included to ensure stability of the insulin in
formulations. In contrast, the formulations of the present
disclosure maintain the stability of insulin in a concentrated form
without including preservatives or stabilizers. In some instances,
the formulations do not contain phenol, cresol, or derivatives of
either. In some instances, as described in Table 12 and FIG. 15,
0.25% wt/vol phenol may decrease the stability of an insulin
formulation as compared to a formulation as provided by this
disclosure (preservative free) within a day of formulation and over
a period of 6 months.
[0050] In another aspect, the formulations do not contain
surfactants. For example, the formulations do not contain
amphipathic excipients that modify the surface tension between a
solution and any interface (for example, a liquid/glass vial
interface, an air/liquid interface). Surfactants such as
polysorbate-80 and Triton.TM. X-100 are well-known excipients. As
surfactants may cause foaming and, therefore, loss of physical
stability when a formulation is nebulized or aerosolized, the
formulations provided by this disclosure provide an advantage over
formulations containing surfactants.
[0051] As described in the Examples, the formulations provided in
this disclosure are stable, with low levels of chemical and
physical degradation, for at least 24 months at typical
refrigeration temperatures (2-8.degree. C., approx. 5.degree. C.).
As such, the formulation may be suitable for providing products
with long shelf-stability.
[0052] Long term stability can be assessed by analysis of the
physical and chemical properties of the formulations. For example,
the physical appearance of the formulation may reveal the presence
of precipitates, other insoluble components, and discoloring
contaminants. Another example is that a change in the pH of the
formulation may occur if stability decreases. Another example is
that the potency of the formulation (the amount of soluble insulin)
may decrease if stability decreases. Another example is the purity
of the insulin in the formulation may decrease such as, for
example, by an increase in the amount of desamido content (such as
Iso B3 and A21 impurities), HMWP products (such as insulin dimers
and polymers), and other insulin related substances (IRS). The term
Insulin related substances (IRS) generally encompasses all insulin
degradation products except A21, B3 and Iso B3 deamidated insulin.
Specifications for commercial formulations are often established by
pharmacopeial monographs, such as the USP Monograph: Insulin Human
(USP29--NF 24, Pharmacopeial Forum: Vol. 31(5): 1135) and European
Pharmacopeia: Insulin Human (01/2005:0838, pg. 1800-1801, Acta.
Pharm. Nord. 223-232). These specifications are useful as
guidelines for development of insulin inhalation solutions. For
example, specifications for the stability of injectable insulin may
be found in the European Pharmacopeia as set forth below and may be
used as guidelines for a stable, commercially viable insulin
inhalation formulation:
TABLE-US-00001 Property Guideline Potency 90-110% of target High
Molecular Weight Polymers (% HMWP) .ltoreq.1.7% Insulin Related
Substances (% IRS) .ltoreq.6%
[0053] In some instances, the formulations may meet the
requirements set forth in the table above for one or more of
potency, % HWMP, or % IRS for at least 6 months, at least 12
months, at least 18 months, at least 24 months, or at least the
duration of the shelf life of a commercial product comprising the
formulations. In certain cases, the formulations may maintain
relatively low levels of contaminants in the form of desamido
products, HMWP products, or other IRS over time during storage. In
some instances, the formulations provided may maintain relatively
low levels of one or more of A21, Iso B3, and B3 insulin products
over time during refrigerated storage. For example, as described in
Examples 1-3, the amount of Iso B3 remained below 5% for the
formulations provided by this disclosure for up to 23-24 months.
These formulations also maintained IRS levels below 6% for up to
23-24 months. In another example, as described in Example 3, the
provided formulations maintain an amount of A21 of less than 5% and
an amount of HMWP of less than 1.7%.
[0054] In some instances, the insulin formulations provided are
chemically and physical stable. In some instances, stability is
related to the pH buffering agent of the formulation. For example,
as described in Table 2, the rate of Iso B3 formation may range
from about 0.14-0.25% per month depending on the pH buffering
agent. In some instances, as shown in FIG. 1, a pH buffering agent
like citrate, or a Tris-citrate mixture, may result in only about
1% Iso B3 formation after 6 months and only about 2.5% after 18
months at typical refrigeration temperatures (2-8.degree. C.,
approx. 5.degree. C.). In some instances, other pH buffering agents
like Tris or phosphate buffer alone, may result in only about
4.0-4.5% Iso B3 formation after 18 months and only about 4.5-5.9%
after 24 months at typical refrigeration temperatures (2-8.degree.
C., approx. 5.degree. C.). In some examples, the rate of IRS
formation may range from about 0.11-0.12% per month for pH
buffering agents such as citrate, Tris, and phosphate and may be
about 0.16% per month for citrate-Tris mixture buffer, as described
in Table 3. For example, as shown in FIG. 2, the a pH buffering
agent like citrate, or a Tris-citrate mixture, may result in only
about 1% IRS formation after 6 months, only about 1.8% after 18
months for a citrate buffer, or only about 2.5% after 18 months for
a citrate-Tris buffer, at typical refrigeration temperatures
(2-8.degree. C., approx. 5.degree. C.). In some instances, other pH
buffering agents like Tris or phosphate buffer alone, may result in
IRS formation at rates intermediate to those observed for citrate
and Tris-citrate at typical refrigeration temperatures (2-8.degree.
C., approx. 5.degree. C.). In some instances, the provided
formulations may contain 31.2 mg/mL (900 U/mL) insulin and 70 mM
NaCl with 0.42% w/w zinc to insulin (about 2.2 zinc per insulin
hexamer) at pH 7.5 as described in Table 2, FIG. 1, and FIG. 2.
[0055] In some instances, chemical and physical stability of the
formulations is related to pH. For example, as described in Example
2, the formation of Iso B3 may decrease, and the formation of IRS
may increase, as the pH of the formulation increases and vice
versa. In some instances, as shown in Table 4 and shown in FIG. 3,
the rate of formation of Iso B3 at typical refrigeration
temperatures may increase with increasing pH from 0.13% per month
at pH 7.0 to 0.21% per month at pH 7.8. In contrast, as described
in Table 5 and shown in FIG. 4, in some instances the rate of
formation of IRS at typical refrigeration temperatures decreases
with increasing pH from 0.21% per month at pH 7.0 to 0.09% per
month at pH 7.8. In another example, the stability of the
formulations decreases substantially under very basic conditions
such as pH 8.7 when stored at typical room temperature (about 22 to
27.degree. C.) as described in Table 6. In some instances, the
formulations described in Tables 4 and 5 and FIGS. 3-4, may contain
31.2 mg/mL (900 U/mL) insulin, 6 mM citrate, and 70 mM NaCl, with
0.42% w/w zinc to insulin (about 2.2 zinc per insulin hexamer).
[0056] In some instances, as described in Example 3, formulations
having 300 U/mL and 900 U/mL generally have similar chemical and
physical stability over time. For example, as shown in FIG. 5, the
formulations may retain at least about 100-101% of the initial
insulin after 12 months in storage at typical refrigeration
temperatures (2-8.degree. C., approx. 5.degree. C.). In some
instances, also as shown in FIG. 5, the formulations may retain at
least about 98% of the initial concentration of insulin in solution
after 18 months in storage at typical refrigeration temperatures
(2-8.degree. C., approx. 5.degree. C.). In certain instances, also
as shown in FIG. 5, the formulations may retain at least about 100%
of the initial concentration of insulin in solution after 24 months
in storage at typical refrigeration temperatures (2-8.degree. C.,
approx. 5.degree. C.). In some instances, as described in Table 7
and Table 8, the formulations may show a potency of about 99% after
18 months of storage at typical refrigeration conditions. In some
instances, as described in Table 7 and Table 8, the formulations
may show a potency of about 101% after 24 months of storage at
typical refrigeration conditions. For example, the formulations may
not exhibit a significant drop in potency over time. In some
instances, such as shown in FIG. 6, the formulations may accumulate
only about 1-2% IRS after 6 month at typical refrigeration
temperatures. Also, in some instances, such as shown in FIG. 6, the
formulations may accumulate only about 2-3% IRS after 18 month at
typical refrigeration temperatures. Also, in some instances, such
as shown in FIG. 6, the formulations may accumulate only about 2-4%
IRS after 24 month at typical refrigeration temperatures. For
example, the formulations may show % IRS accumulation of about
2.5-2.7% after 18 months of storage at typical refrigeration
conditions, as described in Table 7 and Table 8. In some instances,
the formulations may show % IRS accumulation of about 2.9-3.1%
after 24 months of storage at typical refrigeration conditions, as
described in Table 7 and Table 8. In certain instances, as shown in
FIG. 7, the formulations may accumulate only about 0.5-0.7% Iso B3
after 6 month at typical refrigeration temperatures. In some
instances, as shown in FIG. 7, the formulations may accumulate only
about 2.0-2.8% Iso B3 after 18 month at typical refrigeration
temperatures. In certain instances, as shown in FIG. 7, the
formulations may accumulate only about 2.6-3.3% Iso B3 after 24
month at typical refrigeration temperatures. For example, the
formulations may show % Iso B3 accumulation of about 2.29-2.45%
after 18 months of storage at typical refrigeration conditions, as
described in Table 7 and Table 8. In some instances, the
formulations may show % Iso B3 accumulation of about 3.26-3.23%
after 24 months of storage at typical refrigeration conditions, as
described in Table 7 and Table 8. In some instances, as shown in
FIG. 8, the formulations may accumulate only about 0.4-0.9% HMWP
products after 9 month at typical refrigeration temperatures. In
some cases, as shown in FIG. 8, the formulations may accumulate
only about 1.0-1.6% HMWP after 18 month at typical refrigeration
temperatures. In some instances, as shown in FIG. 8, the
formulations may accumulate only about 0.8-1.9% HMWP after 24 month
at typical refrigeration temperatures. For example, the
formulations may show % HMWP product accumulation of about
1.29-1.33% after 18 months of storage at typical refrigeration
conditions, as described in Table 7 and 8. In some instances, the
formulations may show % HMWP product accumulation of about
1.40-1.43% after 24 months of storage at typical refrigeration
conditions, as described in Table 7 and 8. As described in Example
3, pH of the formulations may stay stable over 24 months at typical
refrigeration temperatures. In some instances, the formulations may
contain 31.23 mg/mL (900 U/mL) or 10.41 mg/mL (300 U/mL) insulin, 6
mM citrate, and 70 mM NaCl, with 0.39% w/w zinc to insulin (2.0
zinc per insulin hexamer) at pH 7.5, as described in Table 7 and
Table 8 and FIGS. 5-8.
[0057] In certain instances, as described in Example 4, the
stability of insulin in the formulations may be impacted by pH and
zinc concentration. For example, as shown in FIGS. 9-12, the
solubility of insulin may be greater (improved) in formulations
having higher pH (such as at least pH 7.2) and lower zinc
concentration (such as less than or equal to about 0.5% wt/wt zinc
to insulin, 2.7 zinc per insulin hexamer). In one example, as shown
in Example 4 and FIG. 12, the solubility of insulin decreases below
pH 7.2, particularly when the zinc concentration increases. In some
instances, the formulations may contain 35 mg/mL (1009 U/mL)
insulin, 6 mM citrate, and 70 mM NaCl as described in FIGS. 9-12.
In some instances, the formulations are stable for at least 25
weeks under such conditions.
[0058] In some instances, as described in Example 5, the disclosed
insulin formulations may be physically stable when exposed to shear
forces. Formulations experience shear forces during shipment,
handling, and typical use of the product by a subject. A factor
that influences shear force includes the movement of formulation
within the container. As such, experiments in which a formulation
is shaken under controlled conditions are often used as a
laboratory model for assessing the impact of shear forces on a
formulation. As discussed above, insulin formulations may reflect
instability by the formation of insulin fibrils, which may
aggregate and come out of solution. Fibril formation decreases the
potency of a formulation and can promote further destabilization
via additional fibrillation. In addition, the fibril sediment is
undesirable for therapeutic formulations, particularly inhaled
formulations delivered into the respiratory system. In some
instances, as shown in Table 10 and FIG. 13, the provided insulin
formulations may retain greater than 90% potency (original insulin
concentration) when exposed to constant agitation shear forces over
a period of 26 days (refrigerated conditions). In some cases, the
formulations are substantially more stable than commercial Humulin
R.RTM. U-500 formulation (containing 0.25% wt/wt phenol) that
retains less than 50% potency over the same period of time. In some
instances, turbidity of a formulation may be used to assess insulin
aggregation. For example, as shown in Table 11 and FIG. 14, the
agitated formulations described in Example 5 and discussed with
respect to FIG. 13, may have increased turibity after 5 days of
constant agitation. In some instances, increased insulin
concentration may facilitate fibrilation in a formulation, as shown
by the increased rate of at which the 840 U/mL formulation becomes
increasing turbid as compared to the 280 U/mL formulation. In some
instances, the preservative free formulations of the disclosure
show substantially less fibrillation as detected by turbidity as
compared to the commercial Humulin R.RTM. U-500 formulation over a
period of 8 days, as shown in FIG. 14. In some instances, the
preservative free formulations of the disclosure may experience a
cumulative loss of potency of about 0.5-1.7 mg/mL over a period of
28 days when exposed to shear forces. This is in contrast to an 8.9
mg/mL loss of potency observed under the same conditions for the
commercial Humulin R.RTM. U-500 formulation. In some instances, the
disclosed formulations may experience about 4-18 fold less loss in
potency as compared to the commercial Humulin R.RTM. U-500
formulation. Said another way, the commercial Humulin R.RTM. U-500
formulation may experience about 4-18 fold greater loss in potency
as compared to the disclosed formulations. In some instances, the
formulations may contain containing 280 U/mL (29.2 mg/mL) and 840
U/mL (9.7 mg/mL) insulin, 6 mM citrate, 70 mM NaCl, 2.6 zinc
molecules per hexamer, at pH 7.55 as described in FIGS. 9-12.
[0059] In some instances, the disclosed preservative-free
formulations may be more stable than similar formulations
containing phenol as a preservative/stabilizer as described in
Example 6. For example, as shown in Table 12 and FIG. 15, insulin
formulations containing saturating amounts of insulin at either pH
7.2 or pH 7.4 may retain at least 90% potency (original insulin
concentration) over 6 months in refrigerated storage. In another
example, similar formulations that also included 0.25% wt/vol
phenol retained only between 20% and 35% potency over 6 months. In
some instances, approximately 6% more insulin may be retained in
the preservative free formulations at pH 7.4 as compared to pH 7.2
after 6 months in storage. In other instances, approximately 15%
more insulin may be retained in the phenol containing formulations
at pH 7.4 as compared to pH 7.2 after 6 months in storage. In some
instances, the phenol containing formulations may become instable
quickly, losing potency within a day, as shown in Table 12 and FIG.
15, with a greater loss at pH 7.2 than at pH 7.4. In some
instances, the provided formulations may contain containing 2017
U/mL (70 mg/mL) in 6 mM citrate and 70 mM NaCl, with 2.6 zinc
molecules per insulin hexamer at pH 7.2 or 7.4 as described in
Table 12 and FIG. 15.
[0060] A further aspect is that the formulation may be packaged as
a single use "unit dose" container or as a multi-dose container. In
some instances, a unit dose of the insulin formulations described
in this disclosure is provided. Examples of single use containers
are blister packs or capsules. Examples of multi-dose containers
are drop dispensers, or vials.
[0061] In some instances, provided is an insulin formulation that
includes insulin at a minimum concentration of 1-13 mM, a salt at a
concentration of 50-150 mM, a pH buffering agent at a concentration
of 3-24 mM, zinc at a ratio of 1.9-2.7 zinc ions per insulin
hexamer, and a pH in the range of 7.2 to 8.0, but does not contain
preservatives or stabilizers. In some instances, the formulations
may have an insulin concentration of 6-76 mg/mL. In some instances,
the formulations may have an insulin concentration of 173 U/mL to
2189 U/mL. In some instances, the insulin may be human insulin. In
some cases, the formulation may include at least 30 mg/mL insulin
or at least 5.17 mM insulin or at least 864 U/mL insulin. In some
instances, the formulation may contain 10-30 mg/mL insulin or
1.72-5.17 mM. In some instances, the insulin formulation may have
an insulin concentration of about 30 mg/mL or 5.17 mM or 864 U/mL
and a pH of about 7.55. In some cases, the insulin concentration
may be 30 mg/mL or 5.17 mM or 864 U/mL and the pH may be 7.55. In
some instances, the pH of the formulation may be in the range of
7.4 to 7.6. In some instances, the salt may be a chloride salt. In
some cases, the salt may be sodium chloride (NaCl). In some
instances, the pH buffering agent may be citrate. In one example,
the pH buffering agent may be sodium citrate. In some instances,
the tonicity (ionic strength) of the formulation may be 100-300
mOsm. In some cases, the formulation may contain 70 mM NaCl or a
salt having similar ionic strength. In some instances, the
formulation does not contain surfactants. In some cases, the
formulation may be administrable by inhalation or injection. In
some instances, the formulation may be administrable by an
inhalation device. In some instances, the formulation may be
aerosolizable. In some instances, provided is a unit dose of an
insulin formulation as described in this paragraph or elsewhere in
this disclosure.
IV. Kits
[0062] Another aspect of this disclosure is kits containing the
insulin formulations described in Section III and elsewhere in this
disclosure. In some instances, the kits may include one or more
unit doses of a described insulin formulation and a device for
administering the formulation. Kits may include a single use "unit
dose" container or a multi-dose container. Examples of single use
containers are blister packs or capsules. Examples of multi-dose
containers are drop dispensers, or vials. In some instances, the
device for administering the formulation may be an aerosolization
device. For example, in some instances, the device may be an
aerosolizer, an inhaler, or a nebulizer. Exemplary aerosolization
devices that may be included in the kit are described in U.S.
Patent Application Publication Nos. 20110168172; 20110017431;
20130269684; 20130269694; and 20130269684; U.S. application Ser.
Nos. 14/743,763; 14/743,711; 14/732,247; and 14/732,446; and
International PCT Publication Nos. WO 2013/158352 and WO
2013/158353, each of which is incorporated herein by reference in
their entirety. Other devices for aerosolization of liquid
formulations are well-known in the art. In some instances, the kits
may include a device for administrating the formulation via
injection. For example, the kits may include one or more syringes.
In another example, the kits may include one or more needles. In
another example, the kits may include one or more syringes and one
or more needles. The kits may also include a pump or a pen device
for administering the formulation via injection. In some instances,
the kit may include instructions describing use of the device to
administer the formulation.
V. Methods of Treatment
[0063] As noted above, certain embodiments and aspects of the
present disclosure relate to a method of treating a subject having
diabetes mellitus using the formulations described in Section III
and elsewhere in this disclosure. In one aspect, the method can
include administering to a subject having diabetes mellitus a
therapeutically effective amount of the formulation. The
formulation can be administered to the subject via inhalation or
injection. For example, the formulation can be administered using
an inhalation device such as an aerosolizer, an inhaler, or a
nebulizer, or by injection (intravenous, intramuscular,
intraperitoneal), including by pump or pen.
[0064] Exemplary aerosolization devices for administering the
provided preservative free formulations are described in U.S.
Patent Application Publication Nos. 20110168172; 20110017431;
20130269684; 20130269694; and 20130269684; U.S. application Ser.
Nos. 14/743,763; 14/743,711; 14/732,247; and 14/732,446; and
International PCT Publication Nos. WO 2013/158352 and WO
2013/158353, each of which is incorporated herein by reference in
their entirety. Other devices for aerosolization of liquid
formulations such as those described herein are well-known in the
art.
[0065] In one aspect, a therapeutically effective amount of the
formulations may be 20 U to 500 U delivered via the pulmonary
route, with a typical dosage being 150 U. In some instances, a
therapeutically effective amount of the formulations delivered via
the pulmonary route may be 150 U. In one aspect, a therapeutically
effective amount of the formulations may be 2 U to 28 U delivered
via subcutaneous injection, with a typical dosage being 21 U. In
some instances, a therapeutically effective amount of the
formulations delivered via subcutaneous injection may be 21 U.
Typical dosing varies with route of administration because
pulmonary efficiency is approximately 5%-30% of the efficiency of
the subcutaneous route. For reference, 1 U is equivalent to 0.0347
mg of human insulin (28.8 U/mg).
[0066] In some cases, suitable dosing with an insulin formulation
by pulmonary delivery, such as through inhalation of an aerosolized
formulation, is performed using formulations with at least 6 mg/mL
(1 mM; 168 U/mL) to achieve the desired dosage with minimal
administrations. For example, if the insulin formulation has a low
concentration, repeated administrations may be required to achieve
the desired dosage. In one aspect, the efficiency of delivery of
insulin into the blood via the inhalation pathway is approximately
8-20% relative to the injectable route. In one aspect, the relative
efficiency is 14%. In some aspects, the insulin formulation can
administer a dose of 200 U/mL insulin to a subject via inhalation
of the aerosolized formulation in one or two administrations, in
particular, via one or two inhalations by a subject. In some
instances, the insulin concentration of the formulation may be in
the range of 1-13 mM (or 6-76 mg/mL or 173-2189 U/mL) to provide a
sufficient dosage with minimal administrations. In some instances,
the insulin concentration of the formulation may contain 10-30
mg/ml insulin (1.72-5.17 mM) to provide a sufficient dosage with
minimal administrations. In some instances, the insulin
concentration of the formulation may contain at least 30 mg/ml
insulin to provide a sufficient dosage with minimal
administrations.
[0067] In another aspect, the formulation is administered prior to
the subject eating a meal. For example, the formulation may be
administered just prior to the subject eating a meal. In another
example, the formulation may be administered at least 15 minutes
prior to the subject eating a meal. In some examples, the
formulation may be administered at least once a day. In certain
instances, the formulation may be administered up to 1, 2, 3, or 4
times per day.
VI. Examples
[0068] The following examples are offered to illustrate, but not to
limit, the claimed invention.
Example 1
Purpose
[0069] To establish the chemical stability of buffered insulin
formulations containing 900 U/mL (31.2 mg/mL, 5.37 mM) insulin in
70 mM sodium chloride, with a variety of pH buffering agents to
identify a pH buffering agent to maintain stable insulin in
formulation. This study looks specifically at the buffer type as a
variable. The insulin and sodium chloride levels were held
constant. Insulin concentration was fixed at 900 U/mL (31.2
mg/mL).
Formulation Components
[0070] The formulations were made using USP recombinant human
insulin (Sanofi-Aventis, Material: 192228, GMID: 341921), which
contained 0.42% w/w zinc to insulin (about 2.2 zinc per six insulin
monomers). The following materials were obtained from JT Baker: USP
sodium citrate dihydrate; USP Trizma.RTM. Base; USP sodium
phosphate, monobasic; HPLC grade water; hydrochloric acid (37%),
zinc chloride, and sodium hydroxide pellets.
Method Description
[0071] Six separate formulations were prepared. For each,
recombinant human insulin was weighed and dissolved to 80% of final
volume in 87.5 mM hydrochloric acid. Buffer (citrate, Tris,
phosphate, or no buffer) was weighed into each formulation to
concentrations listed in Table 1, relative to final dilution
volume. For the +1 Zn condition, zinc chloride was added to a final
zinc concentration of 0.61% w/w zinc to insulin. Sodium hydroxide
(1M) was added to adjust the pH to 7.5. The formulation was
adjusted to final volume with water and the pH was rechecked, and,
if necessary, re-adjusted to pH 7.5.
TABLE-US-00002 TABLE 1 Buffers/Concentrations Buffer Concentration
pH Citrate 15 mM 7.5 Citrate/Tris 15 mM/20 mM Tris 20 mM Tris + 1
Zn 20 mM Phosphate 5 mM Reference (no buffer) --
Procedure
[0072] Each formulation was placed into refrigerated storage
(2-8.degree. C.; approx. 5.degree. C.). Refrigerated formulations
were stored for up to 23 months and were sampled at two or more of
time points 0, 6, 12, 18 and 23 months. Samples were analyzed by
High Performance Liquid Chromatography at time points from 0 to 23
months. Each sample was analyzed by Reverse Phase High Performance
Liquid Chromatography (RP-HPLC) for insulin related substances
(IRS) and Iso B3 at 5.degree. C. at the start of the experiment and
then at 6 months, 12 months, 18, months, and 23 months.
HPLC Assays
[0073] The content of Iso B3 and Insulin Related Substances (IRS)
was measured by an Reversed Phase (RP)-HPLC method using a C-18,
3.times.250 mm column on a Waters (Milford, Mass.) HPLC module with
diode array detector. The detector was set at 214 nm. Flow rate was
0.55 mL/minute. Samples were diluted in 100 mM phosphate buffer, pH
7.8 and tested at 2 mg/ml concentration. For Iso B3 assessment,
mobile phase A was 7% acetonitrile and 93% 140 mM sodium
perchlorate (pH adjusted to 2.3 with phosphoric acid) and mobile
phase B was 57% acetonitrile, 65 mM sodium perchlorate (pH adjusted
to 2.3 with phosphoric acid). The assay conditions were isocratic
at 55% B for the first 30 minutes, from 30 to 56 minutes there was
a gradient to 80% B, and after 56 minutes, the mobile phase
percentages were returned to initial conditions. For IRS
assessment, the mobile phase A was 18% acetonitrile, 82% 200 mM
sodium sulfate (pH adjusted to 2.8 with phosphoric acid) and mobile
phase B was 50% acetonitrile, 50% 200 mM sodium sulfate (pH was
adjusted to 2.8 with phosphoric acid). The initial starting
condition were isocratic at 22-30% B for 32 minutes, the % B was
adjusted for insulin retention time between 17-24 minutes, from
32-57 minutes the % B was increased in a linear gradient to 52% B,
and at 57 minutes mobile phases return to starting conditions.
[0074] The HPLC chromatograms were evaluated to determine the area
of insulin degradation product peaks relative to intact insulin.
The total percent of degradation products are referred to as
Insulin Related Substances (% IRS). These included all insulin
related degradation peaks except the A21 and Iso B3 desamido peaks.
These two substances are addressed separately by the USP and EP
Monographs and are not typically included in % IRS. The area
represented by the IRS peaks as a percent of total insulin (all
peaks) was determined, and the data points were analyzed by linear
regression to establish trend lines. % Iso B3 and % IRS change per
month were calculated from the slope of trend lines.
Results
[0075] The analytical results for this experiment are shown in
Table 2 and Table 3 and graphically in FIG. 1 and FIG. 2 and are
described below. The 20 mM Tris+1 Zn (0.61% wt/wt zinc to insulin)
physically precipitated, resulting in a small potency drop (data
not shown). The Reference (no buffer) solution is identified in
FIGS. 1-2 as "NaCl".
TABLE-US-00003 TABLE 2 Chemical Stability of Insulin vs. Buffer
Type - Area % Iso B3 (5.degree. C.) % Iso B3/ Buffer 0 mo 6 mo 18
mo 23 mo mo Reference (no buffer) 0.05 0.97 NS 4.74 0.21
NaCl/Phosphate 0.05 1.32 NS 5.75 0.25 20 mM Tris 0.05 NS 4.09 NS
0.23 20 mM Tris + 1 Zn 0.05 NS 4.46 NS 0.25 15 mM Citrate 0.05 NS
2.46 NS 0.14 15 mM Citrate/10 mM Tris 0.05 NS 2.54 NS 0.14 NS = Not
Sampled
TABLE-US-00004 TABLE 3 Chemical Stability of Insulin vs. Buffer
Type - Area % IRS (5.degree. C.) Buffer 0 mo 6 mo 12 mo 18 mo 23 mo
% IRS/mo Reference (no 0.52 1.02 NS 1.83 2.45 0.11 buffer)
NaCl/Phosphate 0.52 1.01 NS 2.23 2.76 0.12 20 mM Tris 0.52 NS 1.72
1.78 NS 0.10 20 mM Tris + 0.52 NS 1.62 1.64 NS 0.09 1 Zn 15 mM
Citrate 0.52 NS 2.00 1.86 NS 0.10 15 mM Citrate/ 0.52 NS 2.41 2.60
NS 0.14 10 mM Tris NS = Not Sampled
[0076] Iso B3 forms more quickly as pH increases (see Example 1).
Buffer solutions limit pH changes by absorbing environmental
protons and electrons. The results in Table 2 show that the rate of
formation of Iso B3 at 5.degree. C. ranges from 0.14-0.25% per
month depending on the selected buffer type. This data suggests
reduced rates of Iso B3 formation when the buffer used to control
pH includes citrate. Tris, phosphate, and unbuffered solutions show
significantly higher rates of Iso B3 formation relative to
citrate-containing formulas, despite each solution maintaining a pH
of 7.5.
[0077] Formation of IRS range from 0.11-0.12% per month, with the
exception of the citrate/Tris combination, which formed IRS at
0.14% per month. These results suggest that the formation of
insulin dimers and hydrolysis products other than Iso B3 are
generally similar regardless of buffer. The combination of Tris and
citrate shows slightly higher % IRS, which may be the result of a
number of factors including the presence of two buffer types or the
increased osmolality of the solution.
[0078] This study suggests that citrate buffer buffers pH
sufficiently to both limit insulin hydrolysis (Iso B3) and covalent
dimer formation (IRS).
Example 2
Purpose
[0079] To establish the chemical stability of buffered insulin
formulations containing 900 U/mL (31.2 mg/mL, 5.37 mM) insulin in 6
mM sodium citrate and 70 mM sodium chloride over a range of pH
values to identify a pH range to maintain stable insulin in
formulation. This study looks specifically at the pH as a variable.
The insulin, sodium chloride, and sodium citrate levels were held
constant.
Formulation Components
[0080] The formulations were made using USP recombinant human
insulin (Sanofi-Aventis, Material: 192228, GMID: 341921), which
contained 0.42% wt/wt zinc to insulin (about 2.2 zinc per six
insulin monomers). The following materials were obtained from JT
Baker: USP sodium citrate dihydrate; HPLC grade water; hydrochloric
acid (37%), and sodium hydroxide pellets.
Method Description
[0081] Recombinant human insulin was weighed and dissolved to 80%
of final volume in 87.5 mM hydrochloric acid. Sodium citrate
dihydrate was added to the formulation to a concentration of 6 mM
(relative to the final dilution volume). Sodium hydroxide (1M) was
added to adjust the pH to 7.0. The formulation was adjusted to
final volume with water and the pH was rechecked. This bulk
formulation was aliquoted into 6 separate glass containers with
Teflon.TM. seals and screw-cap closures. The pH of each separate
container was carefully adjusted to a different pH value: 7.0, 7.2,
7.4, 7.6, 7.8, and 8.7 with small volumes of sodium hydroxide.
Procedure
[0082] An aliquot of each formulation was placed into refrigerated
storage (2-8.degree. C., approx. 5.degree. C.). A aliquot of each
formulation was also stored at 25.degree. C. (incubator).
Refrigerated formulations were stored for up to 24 months and were
sampled at time 0, 5, 9, 15, 18, and 24 months. Samples stored at
25.degree. C. were held for 2 weeks and then sampled. Samples were
analyzed by HPLC to determine % Iso B3 content and % IRS content as
described in Example 1.
Results
[0083] The analytical results for this experiment are shown in
Tables 4-6 and are described below. FIG. 3 and FIG. 4 show the
growth rate of Iso B3 and IRS, respectively, across 15-18 months at
5.degree. C. for each pH condition. The data points were analyzed
by linear regression to establish trend lines. % Iso B3 and % IRS
change per month were calculated from the slope of trend lines.
TABLE-US-00005 TABLE 4 Chemical Stability of Insulin by pH - Area %
Iso B3 (5.degree. C.) Time (mo): 0 5 15 18 24 % Iso B3/mo pH 7.0
0.05 0.65 1.93 2.31 2.91 0.12 pH 7.2 0.05 0.74 2.03 2.53 3.31 0.14
pH 7.4 0.05 0.88 2.37 2.95 3.83 0.16 pH 7.6 0.05 1.01 2.73 3.37
4.37 0.18 pH 7.8 0.05 1.16 3.10 3.88 4.89 0.20
TABLE-US-00006 TABLE 5 Chemical Stability of Insulin vs. pH - Area
% IRS (5.degree. C.) Time (mo): 0 5 9 15 18 24 % IRS/mo pH 7.0 0.50
1.39 2.60 3.15 3.38 4.26 0.15 pH 7.2 0.50 1.21 1.99 2.39 2.46 3.20
0.11 pH 7.4 0.50 1.03 1.55 1.80 2.70 2.89 0.10 pH 7.6 0.50 0.94
1.36 1.59 1.92 2.42 0.08 pH 7.8 0.50 0.81 1.20 1.42 1.67 1.97
0.06
[0084] The relationship between pH and insulin degradation is
complex. In order to maximize the stability of an insulin
formulation it is very important to control pH. The results in
Table 5 show that the rate of formation of IRS at 5.degree. C.
decreases with increasing pH from 0.15% per month at pH 7.0 to
0.06% per month at pH 7.8. The increase of pH has the opposite
effect on the generation of Iso B3, as shown in Table 4, where the
rate of formation increases with increasing pH from 0.12% per month
at pH 7.0 to 0.20% per month at pH 7.8. As pH increases, Iso B3
formation increases and IRS formation declines (pH range: 7.4-8.0).
At pH greater than 8.0, hydrolysis of insulin appears to occur at a
more rapid rate.
[0085] The increased levels of Iso B3 observed are less significant
to the stability of the formulation than the decreased levels of
IRS, even at the highest pH because Iso B3 remains largely
bioactive (it retains >90% of the insulin activity). As such,
insulin formulations at pH 7.6 and pH 7.8 retain potency, and this
level of Iso B3 formation is acceptable for commercial
formulations.
[0086] Table 6 shows the formation of Iso B3 at 25.degree. C.
storage for 2 weeks. Iso B3 formation occurred rapidly with
increased temperature (25.degree. C.) and pH (pH 8.7).
TABLE-US-00007 TABLE 6 Chemical Stability of Insulin by pH - Area %
Iso B3 (25.degree. C.) 2 Week Time Point Iso B3 (% Area) pH 7.4
1.08 pH 7.6 1.19 pH 7.8 1.33 pH 8.7 1.96
[0087] This study suggests that maintaining the pH range of the
formulation between 7.4 and 7.8 limits insulin hydrolysis (Iso B3)
and covalent dimer formation (insulin related substances)
sufficiently in long term storage conditions.
Example 3
Purpose
[0088] This study assessed formulations having different insulin
concentrations in glass containers outfitted with drop dispensers
simulating commercial packaging in order to determine the
anticipated shelf life (stability) of the insulin formulations. The
chemical stability of buffered insulin formulations containing
10.41 mg/mL (300 U/mL, 1.79 mM) and 31.23 mg/mL (900 U/mL, 5.38 mM)
insulin (0.39% zinc w/w zinc to insulin) at pH 7.5 in 6 mM sodium
citrate and 70 mM sodium chloride was assessed over 24 months.
Formulation Components
[0089] The formulations were made using USP recombinant human
insulin (Sanofi-Aventis, Material: 192228, GMID: 341921), which
contained 0.39% w/w zinc to insulin (about 2.2 zinc per six insulin
monomers). The following materials were obtained from JT Baker: USP
sodium citrate dehydrate, HPLC grade water, hydrochloric acid
(37%), and sodium hydroxide pellets.
Method Description
[0090] Recombinant human insulin was dissolved to 80% of final
volume in 87.5 mM hydrochloric acid. Sodium citrate was weighed
into the formulation at 6 mM concentration (relative to the final
dilution volume). Sodium hydroxide (1 M) was added at 7% of final
volume. Formulations were brought to final volume with water. The
pH was checked, and adjusted to final pH of 7.5 with sodium
hydroxide. Final batch volumes of 1 L; final insulin
concentrations: 10.41 mg/mL (300 U/mL, 1.79 mM) and 31.23 mg/mL
(900 U/mL, 5.38 mM).
Procedure
[0091] Each formulation was aliquoted into 4 glass containers and
outfitted with drop dispensers. The fill volume was 8 mL. Vials
were purchased from Gerresheimer, 10 mL volume, part #
F008X1P20-0150. Drop dispensers were purchased from Aero Pump,
PN#70019500. These drop dispensers represented an example of final
packaging. Samples were assessed at time points from 0 to 24
months.
[0092] Potency was determined by RP-HPLC in comparison to a USP
Human Insulin reference standard by following the USP monograph
method. Iso B3 desamido and A21 content were determined by RP-HPLC
as described in Example 1. The amount of high molecular weight
polymers (HMWP) was determined by Size Exclusion-High Performance
Liquid Chromatography (SEC-HPLC). Solubility was determined by
visual inspection (observation of any crystal formation) and by
potency measurement. pH was measured using a typical pH meter
equipped with probe (Orion Star A211 with a Ag/AgCl combination pH
semi micro expoxy electrode).
[0093] Stability parameters were measured against acceptance
criteria that were set to establish a minimally stable formulation.
Acceptance criteria were set based on US Pharmacopeia and European
Pharmacopeia recommendations for insulin formulations and based on
known criteria for the overall physical and chemical stability of
insulin. The pH acceptance criteria was set based on the reduced
solubility of insulin below pH 7.2 observed in Example 2 and
increased hydrolytic reactions above pH 7.8. The acceptance
criteria were set as follows: 90-110% potency, .ltoreq.6.0% IRS,
.ltoreq.5.0% Iso B3, .ltoreq.5.0% A21, .ltoreq.2.0% HMWP, and pH at
7.2-7.8. Formulations meeting these acceptance criteria over a 24
month period will be considered sufficiently stable for
commercialization.
[0094] The data points were analyzed by linear regression to
establish trend lines for potency, % Iso B3, % IRS, and % HMWP. The
trend lines were used to estimate the rate of change of purity and
potency over the shelf life of the product and for prediction of
the anticipated shelf life based on the set acceptance
criteria.
Results
[0095] The analytical results for this experiment are shown in
Tables 7 and 8 and FIGS. 5-8 and are described below. Data is n=4
replicates, presented as average.+-.standard deviation.
TABLE-US-00008 TABLE 7 300 U/mL Insulin Formulation Stability
Analysis Time (months) Acceptance Criteria 0 6 9 12 18 24 Potency
90-110% 102.9 .+-. 0.7 NS NS 100.4 .+-. 0.5 99.4 .+-. 0.5 101.5
.+-. 1.1 % IRS .ltoreq.6.0% 0.52 .+-. 0.1 1.33 .+-. 0.3 NS NS 2.69
.+-. 0.4 2.90 .+-. 0.7 % Iso B3 .ltoreq.5.0% 0.03 .+-. 0.0 0.60
.+-. 0.0 NS NS 2.29 .+-. 0.1 3.26 .+-. 0.2 % A21 .ltoreq.5.0% 0.50
.+-. 0.0 0.48 .+-. 0.0 NS NS 0.47 .+-. 0.0 0.47 .+-. 0.0 % HMWP
.ltoreq.2.0% 0.16 .+-. 0.0 NS 0.71 .+-. 0.1 NS 1.33 .+-. 0.2 1.40
.+-. 0.4 pH 7.2-7.8 7.39 .+-. 0.0 7.25 .+-. 0.0 NS 7.26 .+-. 0.0
7.27 .+-. 0.0 7.31 .+-. 0.0 NS: Not sampled.
TABLE-US-00009 TABLE 8 900 U/mL Insulin Formulation Stability
Analysis Time (months) Acceptance Criteria 0 6 9 12 18 24 Potency
90-110% 101.9 .+-. 0.7 NS NS 101.0 .+-. 0.3 99.6 .+-. 0.3 101.1
.+-. 0.7 % IRS .ltoreq.6.0% 0.52 .+-. 0.1 1.08 .+-. 0.1 NS NS 2.58
.+-. 0.1 3.10 .+-. 0.3 % Iso B3 .ltoreq.5.0% 0.03 .+-. 0.0 0.67
.+-. 0.0 NS NS 2.45 .+-. 0.1 3.23 .+-. 0.1 % A21 .ltoreq.5.0% 0.50
.+-. 0.0 0.46 .+-. 0.0 NS NS 0.47 .+-. 0.0 0.47 .+-. 0.0 % HMWP
.ltoreq.2.0% 0.16 .+-. 0.0 NS 0.65 .+-. 0.1 NS 1.29 .+-. 0.1 1.43
.+-. 0.1 pH 7.2-7.8 7.40 .+-. 0.0 7.29 .+-. 0.0 NS 7.31 .+-. 0.0
7.29 .+-. 0.0 7.30 .+-. 0.0 NS: Not sampled.
[0096] The results in Tables 7 and 8 show a potency of 101.1-101.5%
of target insulin concentration after 24 months of storage at
5.degree. C. This result equates to a potency drop of 0.8-1.4%. The
results are shown graphically in FIG. 5 (results are graphed as a
percent of their initial value). The results are linear and
indicate that potency remains within the established acceptance
criteria of 90-110% of target for significantly longer than an
anticipated 2 year product shelf life. Potency is predicted to
reach 90% (minimum acceptance criteria) after 132 months based on
the trend line slope.
[0097] The results in Tables 7 and 8 show insulin related
substances (% IRS) of between 2.9%-3.1% after 24 months of storage
at 5.degree. C. This percentage is significantly lower than
acceptance criteria of 6%. The results are shown graphically in
FIG. 6. Based on the trend line, % IRS is predicted to reach the
acceptance criteria of 6% after 50 months. This result indicates
that IRS will be within acceptable limits during an anticipated 2
year product shelf life.
[0098] The results in Tables 7 and 8 show Iso B3 values of
approximately 3.2% after 24 months of storage at 5.degree. C. The
results are shown graphically in FIG. 7. Trend data predicts that %
Iso B3 will reach the acceptance criteria of 5% after 38 months.
This result indicates that Iso B3 will be within acceptable limits
during an anticipated 2 year product shelf life.
[0099] The results in Tables 7 and 8 show % HMWP values of
approximately 1.4% after 24 months of storage at 5.degree. C. The
results are shown graphically in FIG. 8. Trend data predicts that %
HMWP will reach the acceptance criteria of 2% after 33 months. This
result indicates that % HMWP will be within acceptable limits
during an anticipated 2 year product shelf life.
[0100] pH dropped slightly over the initial study period, followed
by no significant trend. This result is consistent with typical
aqueous solutions, which draw carbon dioxide from the environment,
causing slight acidification. pH is predicted to be stable during
an anticipated 2 year shelf life.
[0101] Student T-test analysis of the data indicated no significant
difference (p>0.05) between the results observed for the 300
U/mL and 900 U/mL formulations. These results confirm the null
hypothesis that the 300 U/mL and 900 U/mL formulations were
acceptably similar.
[0102] Important criteria for determining shelf-life for an insulin
formulation are potency (% of asserted/desired formulation
concentration), IRS levels, and HMWP levels. Stability of pH is
also important, particularly for very concentrated insulin
formulations (for example, as saturation limit is neared) to avoid
isoelectric precipitation or recrystallization of the insulin. The
provided formulations remained very clear over the course of the
study (indicating little, if any, protein precipitation) and within
the set acceptance criteria. The formulations also showed
remarkable stability at 5.degree. C. for at least 2 years of
refrigerated storage. The results of this study suggest that the
described formulations provide acceptable long term stability at
both 300 U/mL and 900 U/mL insulin by limiting the formation of Iso
B3, IRS, and HMWP.
Example 4
Purpose
[0103] To assess the relationship between pH and zinc content on
the physical stability of the insulin formulations and to identify
the optimal ranges for these parameters to maintain physical
stability of the insulin formulations. In this experiment, insulin,
sodium chloride, and citrate ion were held constant. Zinc content
and pH were varied.
Formulation Components
[0104] The formulations were made using USP recombinant human
insulin (Sanofi-Aventis, Material: 192228, GMID: 341921), which
contained 0.39% w/w zinc to insulin. The following materials were
obtained from JT Baker: USP sodium citrate dihydrate; HPLC grade
water; hydrochloric acid (37%), zinc chloride, and sodium hydroxide
pellets.
Method Description
[0105] Four 35 mg/ml insulin (6.03 mM, 1008 U/mL) formulations were
prepared. Formulations were prepared by adding sodium citrate
dihydrate and sodium chloride to final concentrations of 6 mM and
70 mM, respectively. Sodium hydroxide was used to adjust the pH of
the formulations to 7.0, 7.1, 7.2, and 7.3, respectively. These
four formulations were each then separated into three equal parts.
Additional zinc (in the form of zinc chloride) was spiked into one
part at each pH in very small relative volumes (0.1-0.2% of final
formulation volume). Final zinc concentrations were 0.39% w/w,
0.45% w/w, and 0.51% w/w zinc to insulin, equivalent to 2.08, 2.40,
and 2.73 zinc per hexamer, respectively. The pH of the formulations
was re-checked, and adjusted to 7.0, 7.1, 7.2, and 7.3, if
necessary, using small volumes of sodium hydroxide. The formulation
matrix is shown in Table 9.
TABLE-US-00010 TABLE 9 Formulations with Varying Zinc and pH pH
Zinc Content 7.3 0.39% 0.45% 0.51% 7.2 0.39% 0.45% 0.51% 7.1 0.39%
0.45% 0.51% 7.0 0.39% 0.45% 0.51%
Procedure
[0106] Formulations were placed into refrigerated storage
(2-8.degree. C.; approx. 5.degree. C.) and were stored for up to 25
weeks. Samples were taken time 0, 2 weeks, 5 weeks, 10 weeks, and
25 weeks in storage. The insulin concentration of the formulations
(insulin solubility) was determined via ultra violet spectroscopy
at 276 nm wavelength.
Results
[0107] The analytical results for this experiment are shown in
FIGS. 9-12 and are described below. Specifically, FIGS. 9-11 show
the insulin concentration change in the formulations over time as a
function of pH, and FIG. 12 shows a comparison of the final
concentration of insulin in each formulation as a function of pH
and zinc concentration.
[0108] As shown in FIG. 9, the insulin concentration of the
formulations remain the same within experimental error at pH 7.2
and 7.3 and 0.39% zinc content, but drop over time at pH 7.0 and
7.1 with 0.39% zinc content. Loss occurs gradually and cannot be
observed at pH 7.1 until after the 8 week time point. The drop at
pH 7.0 is 14.4 mg/mL, equating to an 41.9% loss. The drop at pH 7.1
is 8.3 mg/mL equating to a 23.9% loss.
[0109] As shown in FIG. 10, the insulin concentration of the
formulations remain the same within experimental error at pH 7.3
and 0.45% zinc content, but drop over time at pH 7.0, 7.1 and 7.2
with 0.45% zinc content. Loss occurs gradually and cannot be
observed at pH 7.2 until after the 8 week time point. The drop at
pH 7.0 is 17.7 mg/mL, equating to an 51.2% loss. The drop at pH 7.1
is 12.3 mg/mL equating to a 35.6% loss. The drop at pH 7.2 is 2.5
mg/mL equating to a 7.6% loss.
[0110] As shown in FIG. 11, the insulin concentration of the
formulations remain the same within experimental error at pH 7.3
and 0.51% zinc content, but drop over time at pH 7.0, 7.1 and 7.2
with 0.51% zinc content. Loss occurs gradually and cannot be
observed at pH 7.2 until after the 8 week time point. The drop at
pH 7.0 is 18.6 mg/mL, equating to an 54.1% loss. The drop at pH 7.1
is 14.0 mg/mL equating to a 41.2% loss. The drop at pH 7.2 is 7.8
mg/mL equating to a 22.5% loss.
[0111] FIG. 12 illustrates the insulin concentration of the
formulations at the 25 week time point as an overall function of
zinc content and pH. The results show a trend towards greater
solubility at higher pH and lower zinc concentration. All solutions
were fully soluble at pH 7.3. In general, above pH 7.3 (for
example, at pH 7.3-7.8), insulin easily remained soluble at 35
mg/mL at zinc concentrations of 0.51% w/w relative to insulin.
However, as noted in Example 1, zinc concentrations of 0.61% w/w
relative to insulin showed precipitation at pH 7.5.
[0112] The results of this study indicate a complex relationship
between pH, zinc concentration, and the physical stability of
insulin formulations over time. Because of the time dependent
relationship between pH, zinc concentration and solubility, it is
important to assess insulin formulations for long term stability
comparable to anticipated shelf life expectations or requirements.
The results of this study indicate that, in addition to pH, the
concentration of zinc in insulin formulations is a critical
component for long term physical stability.
Example 5
Purpose
[0113] To establish the physical stability of provided insulin
formulations exposed to shaking. Shaking simulates conditions
during shipment and handling of insulin formulations, which can
lead to fibrillation. Insulin formulations having different buffer
were compared to a commercially available insulin formulation
containing metacresol as a stabilizer/preservative and
glycerin.
Formulation Components
[0114] Formulations were made using USP recombinant human insulin
(Sanofi-Aventis, Material: 192228, GMID: 341921), which contained
0.50% w/w zinc to insulin (about 2.6 zinc per six insulin
monomers). The following materials were obtained from JT Baker: USP
sodium citrate dihydrate; HPLC grade water; hydrochloric acid
(37%), and sodium hydroxide pellets.
Method Description
[0115] The formulations were prepared containing 29.2 mg/mL (840
U/mL, 5.03 mM) and 9.7 mg/mL (280 U/mL, 1.67 mM) insulin in 6 mM
sodium citrate and 70 mM sodium chloride. Recombinant human insulin
was weighed and dissolved to 80% of final volume in 87.5 mM
hydrochloric acid. Sodium citrate dihydrate was added to the
formulation to a concentration of 0, 3, 6, 12, and 24 mM
concentration (relative to the final dilution volume). Sodium
hydroxide (1M) was added to adjust the pH to 7.55. The formulations
were adjusted to final volume with water and the pH was rechecked.
For ease of reference, these formulations will be referred to as
preservative free formulations.
[0116] Preservative free formulations were compared to commercial
insulin formulation Humulin R.RTM. U-500 (HI-500) (NDC
0002-8501-01) containing 500 U/mL (17.4 mg/mL) insulin, 0.017 mg
zinc/100 insulin units (equivalent to 0.49% zinc, or about 2.6 zinc
per insulin hexamer), 16 mg/mL (1.6%) glycerin, and 2.5 mg/mL
(0.25%) metacresol, with HCl and/or NaOH added for pH adjustment.
Humulin, Lot No. C398930A, EXP: October 2016 was purchased from Eli
Lilly and Company. This formulation is referred to as "Humulin"
below.
Procedure
[0117] 10 mL of each formulation was aliquotted into 20 mL Trace
Clean (VWR Part #159000-020) scintillation vials. Vials were
secured to an orbital shaker and shaken at 120 RPM (120 revolutions
per minute with an orbital diameter of 18 mm). Samples were
analyzed at time points from 0-28 days or 0-15 days using two
orthogonal methods designed to assess the concentration of
insoluble insulin fibrils (aggregated insulin).
[0118] 1. Potency by HPLC [0119] Potency is a measurement of
insulin concentration. As insulin fibrillates and precipitates, the
potency of insulin in solution decreases. Lower potency correlates
to greater aggregation (physical instability). Assessments were
made at time 0, day 6, day 18, and day 28.
[0120] 2. Turbidity by Nephelometer [0121] Turbidity is a
measurement of the cloudiness or haziness of a solution. For the
purpose of this analysis, turbidity describes the presence of
insoluble particulate (insulin fibrils) having precipitated from
sample solutions. Higher turbidity correlates to greater
aggregation (physical instability). Assessments were made a time 0,
day 4, day 6, day 8, day 11, and day 15.
Results
[0122] The analytical results for this experiment are shown in
Tables 10-11 and FIG. 13 and FIG. 14 and are described below.
TABLE-US-00011 TABLE 10 Potency of Shaken Insulin Formulations
Insulin Concentration (mg/mL) Sample 0 6 days 18 days 28 days
Humulin (500 U/mL) 17.1 17.1 13.2 8.2 9.7 mg/mL 0 mM Citrate
NS.sup.1 9.7 9.3 8.7 Insulin 3 mM Citrate NS.sup.1 NS.sup.2 9.7 9.1
6 mM Citrate 10.0 9.6 9.4 9.0 12 mM Citrate NS.sup.1 9.8 9.7 9.5 24
mM Citrate NS.sup.1 9.8 9.4 8.7 29.2 mg/mL 0 mM Citrate NS.sup.1
29.0 28.3 28.3 Insulin 3 mM Citrate NS.sup.1 28.7 28.1 28.2 6 mM
Citrate 29.6 28.9 28.3 28.1 12 mM Citrate NS.sup.1 28.8 27.7 28.2
24 mM Citrate NS.sup.1 28.6 28.1 27.9 .sup.1Not Sampled. Assumed to
be equivalent for formulations at each concentration at T = 0.
.sup.2Not Sampled. Data was not available for this sample at this
time point.
TABLE-US-00012 TABLE 11 Turbidity of Shaken Insulin Formulations
Turbidity (NTU) Sample 0 4 days 6 days 8 days 11 days 15 days
Humulin (500 U/mL) 2 42 129 >200.sup.2.sup. >200.sup.2.sup.
>200.sup.2.sup. 9.7 0 mM Citrate N/A.sup.1 2 7 26 52 86 mg/mL 3
mM Citrate N/A.sup.1 2 2 3 23 30 Insulin 6 mM Citrate 2 2 9 53 74
109 12 mM Citrate N/A.sup.1 2 2 2 2 3 24 mM Citrate N/A.sup.1 2 2
62 86 124 29.2 0 mM Citrate N/A.sup.1 12 46 82 101 172 mg/mL 3 mM
Citrate N/A.sup.1 18 79 122 151 173 Insulin 6 mM Citrate 5 13 89
129 168 188 12 mM Citrate N/A.sup.1 4 51 103 145 166 24 mM Citrate
N/A.sup.1 6 84 129 141 174 .sup.1Not Applicable. Assumed to be
equivalent for samples at each concentration at T = 0.
.sup.2Turbidity was >200; above threshold for accurate
measurement.
[0123] The preservative free insulin formulations were found to
have increased resistance to fibrillation (shear force) compared to
the commercial Humulin formulation, as described further below.
[0124] Formulations described herein showed potencies of 8.7-9.5
mg/mL and 27.9-28.3 mg/mL after 28 days for 280 U/mL and 840 U/mL
concentrations, respectively, compared to starting concentrations
of 10.0 and 29.6 mg/mL. This amounts to a cumulative loss due to
fibrillation of approximately 0.5-1.7 mg/mL.
[0125] Humulin showed a potency of 8.2 mg/mL after 28 days,
compared to a starting potency of 17.1 mg/mL. This amounted to a
cumulative loss due to fibrillation of 8.9 mg/mL.
[0126] This data demonstrates that the commercial Humulin
formulation experienced a cumulative loss in potency of 4-18 fold
greater than that of the preservative free formulations described
herein.
[0127] The Humulin formulation showed an increase in turbidity
(presence of insoluble fibrils) as early as day 4, with readings
above the measurable range of the device by day 8. In contrast, the
preservative free formulations described herein did not show
significant increases in turbidity until days 6 or 8, with
measurements still within range at day 15.
[0128] This study indicates that formulations described herein have
substantial resistance to fibrillation and, further, are more
resistant than commercial formulations having the stabilizing
effect of a phenolic preservative.
Example 6
[0129] The purpose of this study was to evaluate whether phenol
affects the solubility of insulin in insulin formulations having a
very high concentration of insulin. The study evaluated the
physical stability of preservative free insulin formulations
according to this disclosure relative to insulin formulations
containing a phenolic preservative/stabilizer, both formulations
containing saturating levels of insulin (70 mg/mL, 12 mM, 2016
U/mL). The phenol-containing formulation included 0.25% wt/vol
phenol, which is the concentration found in common commercial
formulations (such as, for example, Humulin.RTM. R 500; see Example
5). While phenol is added to Humulin.RTM. on a wt/wt basis, the
phenol-containing formulations assessed in the following
experiments included phenol on a wt/vol basis. Wt/wt and wt/vol are
essentially interchangeable in this context because the density of
Humulin.RTM. and the phenol-containing formulations is very close
to 1 g/mL.
Formulation Components
[0130] The formulations were made using USP recombinant human
insulin (Sanofi-Aventis, Material: 192228, GMID: 341921), which
contained 0.50% wt/wt zinc to insulin (about 2.6 zinc per six
insulin monomers). The following materials were obtained from JT
Baker: USP sodium citrate dihydrate; USP grade phenol; HPLC grade
water; hydrochloric acid (37%), and sodium hydroxide pellets.
[0131] 1. Bulk Formulation: [0132] A bulk formulation was prepared
and was later separated into four aliquots. Recombinant human
insulin was weighed and dissolved to 80% of final volume in 87.5 mM
hydrochloric acid. Sodium citrate dihydrate was added to the
formulation to a final concentration 6 mM. Aliquots of the bulk
were removed to prepare preservative free and preserved
formulations, as described below. After these final additions, the
insulin concentration was 70 mg/mL. This formulation was intended
to be a saturated solution such that an insulin concentration of 70
mg/mL is near the limit of insulin solubility at the pH values
evaluated. Final concentration of NaCl of 70 mM.
[0133] 2. Preservative Free Formulations: [0134] Two aliquots were
removed from the bulk formulation. Sodium hydroxide (1M) was added
to adjust the pH to 7.20 and 7.40, respectively. The formulations
were adjusted to final volume with water and the pH was rechecked.
Final concentration of NaCl of 70 mM.
[0135] 3. Phenolic Formulations: [0136] Two aliquots were removed
from the bulk formulation. Phenol was added at a concentration of
0.25% wt of final volume. Sodium hydroxide (1M) was added to adjust
the pH to 7.20 and 7.40, respectively. The formulations were
adjusted to final volume with water and the pH was rechecked.
Procedure
[0137] 20 mL of each formulation was placed at 5.degree. C. and
analyzed for insulin potency at time points of 0, 3 months, and 6
months. Samples were prepared by centrifuging an aliquot of
formulation (approximately 300 .mu.L) to remove insoluble protein
precipitates. The supernatant was evaluated by HPLC for potency
(reported as concentration in mg/mL) as described in Example 3.
Results
[0138] The analytical results for this experiment are shown in
Table 12 and FIG. 15 and are described below.
TABLE-US-00013 TABLE 12 Potency (Concentration) of Preservative
Free and Preserved Formulations Concentration (mg/mL) (% Original)
Sample 0 mo* 3 mo 6 mo Phenol, 0.25%, pH 7.2 59.21 (84.6%) 23.1
(33.0%) 23.6 (33.7%) Phenol, 0.25%, pH 7.4 66.41 (94.9%) 33.91
(48.4%) 33.7 (48.1%) Preservative Free, pH 7.2 69.31 (99.0%) 68.11
(97.3%) 65.2 (93.1%) Preservative Free, pH 7.4 68.41 (97.7%) 67.51
(96.4%) 69.4 (99.1%)
[0139] Formulations containing phenol showed immediate
precipitation, bringing their concentrations below 70 mg/mL at
initial analysis and lost over half of their potency by 3 months.
After 6 months, the phenol containing formulations showed potencies
of 33.7% and 48.1% of their theoretical potency at pH 7.2 and 7.4,
respectively. In contrast, the preservative free formulations
experienced a small potency loss between 3 and 6 months of storage
for the pH 7.2 formulation, while the pH 7.4 formulation maintained
its potency over the course of the study. At 6 months, the potency
of the preservative free formulations was 93.1% and 99.1% for the
preservative-free formulations at pH 7.2 and 7.4, respectively.
[0140] The high concentration preservative-free insulin
formulations thus showed increased physical stability (solubility)
compared to phenol containing formulations. This result is
surprising because, as discussed above, preservatives are generally
added to enhance the chemical stability of injectable insulin
formulations. Importantly, the decrease in potency was observed
with extended analysis. Saturated solubility studies are sometimes
performed using short time courses (often less than one day). If
this study had not extended to longer time points, the extent of
the loss of potency would not have been observed for the phenol
containing formulations.
[0141] Achieving high insulin concentration with acceptable
physical stability is important for inhalable insulin inhalation
solutions and, as such, the preservative free formulations of this
disclosure appear to present an advantage towards this end.
[0142] It is understood that the numerical values set forth in this
disclosure may include a degree of variability within reasonable
experimental error. For example, the recited values may encompass
values 10% above or below the recited value. In some instances, the
variability accounts for transitioning between the units used to
refer to the feature (such as, for example, converting between
mg/mL and U/mL). In some instances, this variability is indicated
by use of the term "about" but it is implied in all values relating
to the features of the formulations and other aspects of this
disclosure regardless of whether the value is modified by the term
"about".
[0143] The foregoing description of certain embodiments, including
illustrated embodiments, has been presented only for the purpose of
illustration and description and is not intended to be exhaustive
or to limit the disclosure to the precise forms disclosed. Numerous
modifications, adaptations, and uses thereof will be apparent to
those skilled in the art without departing from the scope of the
disclosure. Certain features that are described in this
specification in the context of separate embodiments can also be
implemented in combination in a single implementation. Conversely,
various features that are described in the context of a single
implementation can also be implemented in multiple ways separately
or in any suitable subcombination. Moreover, although features may
be described above as acting in certain combinations, one or more
features from a combination can in some cases be excised from the
combination, and the combination may be directed to a
subcombination or variation of a subcombination. Thus, particular
embodiments have been described. Other embodiments are within the
scope of the disclosure.
[0144] All printed patents and publications referred to in this
application are hereby incorporated herein in their entirety by
this reference.
* * * * *