U.S. patent application number 12/376205 was filed with the patent office on 2009-12-31 for compositions for intranasal delivery of human insulin and uses thereof.
This patent application is currently assigned to NASTECH PHARMACEUTICAL COMPANY INC.. Invention is credited to Annemarie Stoudt Cohen, Henry R. Costantino, Anthony P. Sileno.
Application Number | 20090325860 12/376205 |
Document ID | / |
Family ID | 38335606 |
Filed Date | 2009-12-31 |
United States Patent
Application |
20090325860 |
Kind Code |
A1 |
Costantino; Henry R. ; et
al. |
December 31, 2009 |
COMPOSITIONS FOR INTRANASAL DELIVERY OF HUMAN INSULIN AND USES
THEREOF
Abstract
What is described is a pharmaceutical formulation for intranasal
delivery of insulin to a patient, comprising an aqueous mixture of
human insulin, a solubilizing agent, a surface active agent, and a
thickening agent, wherein said formulation provides a ultra-rapid
acting profile to regular human insulin.
Inventors: |
Costantino; Henry R.;
(Woodinville, WA) ; Cohen; Annemarie Stoudt;
(Kirkland, WA) ; Sileno; Anthony P.; (Brookhaven
Hamlet, NY) |
Correspondence
Address: |
Eckman Law Group
1250 Oakmead Parkway, Suite 210
Sunnyvale
CA
94085
US
|
Assignee: |
NASTECH PHARMACEUTICAL COMPANY
INC.
Bothell
WA
|
Family ID: |
38335606 |
Appl. No.: |
12/376205 |
Filed: |
April 19, 2007 |
PCT Filed: |
April 19, 2007 |
PCT NO: |
PCT/US07/67007 |
371 Date: |
February 3, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60825876 |
Sep 15, 2006 |
|
|
|
60868703 |
Dec 5, 2006 |
|
|
|
60894130 |
Mar 9, 2007 |
|
|
|
60821525 |
Aug 4, 2006 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 38/28 20130101;
A61P 3/10 20180101; A61K 9/0043 20130101; A61K 47/40 20130101; A61P
3/08 20180101 |
Class at
Publication: |
514/3 |
International
Class: |
A61K 38/28 20060101
A61K038/28 |
Claims
1.-88. (canceled)
89. An aqueous pharmaceutical formulation comprising an aqueous
mixture of an insulin molecule, a solubilizing agent, a surface
active agent, and a thickening agent, wherein the pharmaceutical
formulation confers an ultra-rapid acting insulin profile to a
non-ultra-rapid acting insulin.
90. The pharmaceutical formulation of claim 89, wherein the
pharmaceutical formulation is able to provide peak serum levels of
the administered human insulin within at least about 40 minutes and
glucose troughs within at least 60 minutes post-administration of
the pharmaceutical formulation to the patient.
91. The pharmaceutical formulation of claim 89, wherein the insulin
molecule is selected from group consisting of a natural human
insulin; LysB3, GluB29-human insulin; LysB3, IleB28 human insulin;
GlyA21, HisB31, HisB32-human insulin; AspB10-human insulin, LysB28,
and ProB29-human insulin.
92. The pharmaceutical formulation of claim 89, wherein the
solubilizing agent is selected from the group consisting of a
cyclodextrin, hydroxypropyl-.beta.-cyclodextrin, sulfobutylether
.beta.-cyclodextrin, methyl-.beta.-cyclodextrin, and mixtures
thereof.
93. The pharmaceutical formulation of claim 89, wherein the
surface-active agent is selected from the group consisting of
nonionic polyoxyethylene ether, fusidic acid and derivatives
thereof, sodium taurodihydrofusidate, L-.alpha.-phosphatidylcholine
didecanoyl, polysorbate 80, polysorbate 20, polyethylene glycol,
cetyl alcohol, polyvinylpyrolidone, polyvinyl alcohol, lanolin
alcohol, sorbitan monooleate, and mixtures thereof.
94. The pharmaceutical formulation of claim 89, wherein the
thickening agent is selected from a group consisting of gelatin,
hydroxypropyl methylcellulose, methylcellulose, a carbomer,
carboxymethylcellulose, and mixtures thereof.
95. An aqueous pharmaceutical formulation comprising an aqueous
mixture of an insulin molecule, a solubilizing agent, a surface
active agent, and a thickening agent, wherein the insulin molecule
is selected from the group consisting of a natural human insulin;
LysB3, GluB29-human insulin; LysB3, IleB28 human insulin; GlyA21,
HisB31, HisB32-human insulin; AspB10-human insulin, LysB28, and
ProB29-human insulin.
96. The pharmaceutical formulation of claim 95, wherein the
solubilizing agent is selected from the group consisting of a
cyclodextrin, hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, methyl-.beta.-cyclodextrin,
and mixtures thereof.
97. The pharmaceutical formulation of claim 95, wherein the
surface-active agent is selected from the group consisting of
nonionic polyoxyethylene ether, fusidic acid and its derivatives,
sodium taurodihydrofusidate, L-.alpha.-phosphatidylcholine
didecanoyl, polysorbate 80, polysorbate 20, polyethylene glycol,
cetyl alcohol, polyvinylpyrolidone, polyvinyl alcohol, lanolin
alcohol, sorbitan monooleate, and mixtures thereof.
98. The pharmaceutical formulation of claim 95, where the
thickening agent is selected from a group consisting of gelatin,
hydroxypropyl methylcellulose, methylcellulose, a carbomer,
carboxymethylcellulose, and mixtures thereof.
99. The pharmaceutical formulation of claim 95, wherein the
formulation has a pH of about 7.
100. The pharmaceutical formulation of claim 95, wherein the
formulation after administration to a subject provides a
bioavailability of insulin in the patient of greater than about
15%.
101. A method of treating a metabolic syndrome in a human by
administration of the pharmaceutical formulation of claim 89 to the
human, wherein the metabolic syndrome is selected from the group
consisting of Type 2 diabetes, Type 1 diabetes, impaired glucose
tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or
insulin resistance syndrome), glucosuria, metabolic acidosis,
arthritis, cataracts, diabetic neuropathy, diabetic nephropathy,
diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions
exacerbated by obesity, hypertension, hyperlipidemia,
atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone
fracture, acute coronary syndrome, short stature due to growth
hormone deficiency, infertility due to polycystic ovary syndrome,
anxiety, depression, insomnia, chronic fatigue, epilepsy, eating
disorders, chronic pain, alcohol addiction, diseases associated
with intestinal motility, ulcers, irritable bowel syndrome,
inflammatory bowel syndrome; short bowel syndrome; and the
prevention of disease progression in Type 2 diabetes.
102. A method of treating a metabolic syndrome in a human by
administration of the pharmaceutical formulation of claim 95 to the
human, wherein the metabolic syndrome is selected from the group
consisting of Type 2 diabetes, Type 1 diabetes, impaired glucose
tolerance, hyperglycemia, metabolic syndrome (syndrome X and/or
insulin resistance syndrome), glucosuria, metabolic acidosis,
arthritis, cataracts, diabetic neuropathy, diabetic nephropathy,
diabetic retinopathy, diabetic cardiomyopathy, obesity, conditions
exacerbated by obesity, hypertension, hyperlipidemia,
atherosclerosis, osteoporosis, osteopenia, frailty, bone loss, bone
fracture, acute coronary syndrome, short stature due to growth
hormone deficiency, infertility due to polycystic ovary syndrome,
anxiety, depression, insomnia, chronic fatigue, epilepsy, eating
disorders, chronic pain, alcohol addiction, diseases associated
with intestinal motility, ulcers, irritable bowel syndrome,
inflammatory bowel syndrome; short bowel syndrome; and the
prevention of disease progression in Type 2 diabetes.
Description
BACKGROUND
[0001] Insulin is an important glucose-regulating protein. Insulin
is a naturally-occurring polypeptide hormone secreted by the
pancreas. Insulin is required by the cells of the body to remove
and use glucose from the blood. Glucose allows the cells to produce
the energy needed to carry out cellular functions. In addition to
being the primary effector in carbohydrate homeostasis, it has
effects on fat metabolism. It can change the liver's ability to
release fat stores. Insulin has various pharmacodynamic effects
throughout the body. In healthy individuals, in response to a
glucose injection, insulin is rapidly secreted reaching an initial
peak within 5-7 minutes and lasting no more than 10-15 minutes
(first-phase), followed by a sustained secretion lasting hours
(second-phase), see FIG. 1. In Type 2 diabetes, patients experience
a loss of first-phase insulin release, despite the enhancement of
second-phase insulin secretion. Human data support a critical role
for first-phase insulin secretion in postprandial glucose
homeostasis (PPG), and evidence supports that increased incidence
of cardiovascular disease is associated with PPG.
[0002] Researchers first gave an active extract of the pancreas
containing insulin to a young diabetic patient in 1922, and the FDA
first approved insulin in 1939. The first recombinant human insulin
was approved by the FDA in 1982. Recombinant human insulin, insulin
lispro, insulin aspart, and insulin glargine are the commonly-used
insulins. Beef and pork insulin are infrequently used.
[0003] Insulin is used medically when treating some forms of
diabetes mellitus. Patients with diabetes mellitus have an
inability to take up and use glucose from the blood, and, as a
result, the glucose level in the blood rises. In type 1 diabetes,
the pancreas cannot produce enough insulin. Therefore, insulin
therapy is needed. In type 2 diabetes, patients produce insulin,
but cells throughout the body do not respond normally to the
insulin. Nevertheless, administration of insulin may also be used
in the treatment of type 2 diabetes in order to overcome cellular
resistance to insulin. By increasing the uptake of glucose by cells
and reducing the concentration of glucose in the blood, insulin
prevents or reduces the long-term complications of diabetes,
including, for example, damage to the blood vessels, eyes, kidneys,
and nerves. Insulin is usually administered by injection under the
skin (subcutaneously). The subcutaneous tissue of the abdomen is
preferred because absorption of the insulin is more consistent from
this location than subcutaneous tissues in other locations.
[0004] Insulin can be injected manually, or can be infused into the
body with the help of a small electronic infusion device called an
insulin pump. Syringes are probably the most common and
cost-effective choice for insulin injection, and are useful for
patients who take two types of insulin mixed together. An
alternative to syringes is an insulin pen, which comes prefilled
with insulin and may either be disposable or reusable (with
disposable insulin cartridges). The device resembles a large pen,
with a fine needle under the cap and a plunger at the other end. A
dial allows the user to regulate the dose. Insulin pens are also
available in the most frequently-prescribed mixtures of insulin
types, such as 70/30 (NPH and regular insulin).
[0005] Another device known as an insulin jet injector works by
using a high-pressure blast of air to send a fine spray of insulin
through the skin. This may be a good option for those patients that
are needle-shy. However, jet injectors require a significant
financial investment and are not always covered by insurance.
[0006] An insulin pump may be a more effective way to control type
1 diabetes for some people because it more closely mimics the
insulin production of a pancreas. An insulin pump is a compact
electronic device with an attached infusion set (or tube) that
administers a small, steady flow of insulin to a patient throughout
the day, known as a "basal rate." Before eating, a pump user
programs the pump to deliver a "bolus " of fast-acting insulin to
cover the corresponding rise in blood glucose levels from the meal.
Pump flow can also be manually adjusted by a user throughout the
day as needed.
[0007] Disadvantages to patient administration of insulin by
injection include discomfort due to multiple daily injections,
reaction and infection at the injection site, variation in
absorption of subcutaneous insulin, and difficulty in simulating
the fast release of endogenous insulin at meal times. Thus, there
is a need to develop modes of administration of insulin other than
by injection.
[0008] When insulin was first discovered and made available for
people with diabetes there was only one kind of short-acting
insulin. This required several injections a day. As time went on,
new insulins were developed that lasted longer, requiring fewer
injections, but requiring strict attention to timing of meals.
Presently, there are different types of insulin available. This
gives more flexibility in the number and timing of administration,
making it easier to maintain target blood glucose levels based on a
patient's lifestyle. Insulin is available in various forms, for
example, rapid-, medium-, and long-acting. Insulin is typically
delivered by SC injections. However, other options such as pump
delivery, and more recently pulmonary delivery are available. A dry
powder formulation of a rapid acting insulin has been described for
lung delivery that comprises a human crystalline zinc insulin
having the amino acid sequence of natural human insulin (U.S. Pat.
No. 6,737,045).
[0009] Regular human insulin (e.g., Novolin R, Humulin R) is
available in vials, cartridges, and prefilled syringes. Regular
human insulin is a molecule known to form molecular complexes via
non-covalent interactions (i.e., dimers and hexamers).
[0010] Several insulin analogs that are prepared with recombinant
DNA technology are available for clinical use. Among these agents
is insulin aspart (NovoLog.TM.; Novo Nordisk Pharmaceuticals),
which is homologous with regular human insulin except for a single
substitution of aspartic acid for proline at position B28. This
single substitution reduces the molecule's tendency to form
hexamers. Therefore, insulin aspart is absorbed more rapidly after
subcutaneous injection and has both a faster onset of action and a
shorter duration of action than short-acting insulins.
[0011] Insulin mixtures are also used, especially for people with
type 2 diabetes. Insulin mixtures allow treatment with different
types of insulins in one combined administration.
[0012] NPH human insulin (Novolin N, Humulin N) is available in
vials, cartridges and prefilled syringes. A mixture of 70% NPH
human insulin and 30% regular human insulin (Novolin 70/30, Humulin
70/30) is available in vials, cartridges and pre-filled
syringes.
[0013] A mixture of 50% NPH human insulin and 50% regular human
insulin (Humulin 50/50) is available in vials. Lente human insulin
(Novolin L, Humulin L) is available in vials. Ultralente human
insulin (Humulin U) is available in vials. Insulin lispro (Humalog)
is available in vials and cartridges. Insulin aspart (NovoLog) is
available in vials and cartridges. Insulin glargine (Lantus) is
available in vials and cartridges.
[0014] Insulin is stabilized in the monomeric state to create a
rapid-acting form of insulin. When the insulin is stabilized in the
hexameric form the time to pharmacodynamic effect (i.e., glucose
reduction) is dramatically increased, as compared to monomeric
insulin, because the insulin molecules must disassociate before
producing the desired biological effect. The injectable insulin
treatments that are characterized as rapid acting are chemically
modified to maintain the monomer, thereby imparting the rapid
pharmacodynamic activity upon injection. Monomeric forms of insulin
include insulin analogs and are known to be rapid acting, e.g.,
insulin glulisine (LysB3, GluB29), HMR-1153 (LysB3, IleB28),
HMR-1423 (GlyA21, HisB31, HisB32), insulin aspart (AspB28) or
(AspB10), and lispro (LysB28, ProB29). In every instance above, the
nomenclature of the analogs is based on a description of the amino
acid substitution at specific positions on the A or B chain of
insulin, numbered from the N-terminus of the chain, in which the
remainder of the sequence is that of natural human insulin.
[0015] There is a need to develop pharmaceutical formulations
comprising ultra-rapid acting insulin, i.e., insulin which are able
to provide peak serum levels in less than 60 minutes and glucose
troughs in less than 90 minutes.
DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: Phases of insulin secretion.
[0017] FIG. 2: Pharmacokinetic data, mean insulin levels, for
groups dosed in rabbits with formulations containing a thickening
agent.
[0018] FIG. 3: Pharmacodynamic data, mean % glucose from initial,
for groups dosed in rabbits with formulations containing a
thickening agent.
[0019] FIG. 4: Pharmacokinetic data, mean insulin levels, for human
subjects dosed with 25 IU, 50 IU, and 100 IU intranasal insulin
formulations, NOVOLOG (aspart insulin), EXUBERA (inhaled insulin)
and placebo.
[0020] FIG. 5: Pharmacokinetic data, mean insulin levels, for human
subjects dosed with 25 IU and 50 IU of intranasal insulin
formulations containing a thickening agent and control formulations
(placebo and NovoLog).
[0021] FIG. 6: Pharmacodynamic data, glucose levels, for human
subjects dosed with 25 IU and 50 IU of intranasal insulin
formulations containing a thickening agent and control formulations
(placebo and NovoLog).
[0022] FIG. 7: Insulin levels and mean glucose levels adjusted to
baseline for human subjects dosed with 25 IU and 50 IU of
intranasal insulin formulations containing a thickening agent and a
control formulation (NovoLog).
DESCRIPTION OF THIS DISCLOSURE
[0023] In order to provide better understanding of the present
disclosure, the following definitions are provided:
[0024] As used herein, any concentration range, percentage range,
ratio range, or integer range is to be understood to include the
value of any integer within the recited range and, when
appropriate, fractions thereof (such as one tenth and one hundredth
of an integer), unless otherwise indicated. Also, any number range
recited herein relating to any physical feature, such as polymer
subunits, size or thickness, are to be understood to include any
integer within the recited range, unless otherwise indicated. As
used herein, "about" or "consisting essentially of" mean .+-.20% of
the indicated range, value, or structure, unless otherwise
indicated. As used herein, the terms "include" and "comprise" are
used synonymously. It should be understood that the terms "a" and
"an" as used herein refer to "one or more" of the enumerated
components. The use of the alternative (e.g., "or") should be
understood to mean either one, both or any combination thereof of
the alternatives.
[0025] In addition, it should be understood that the individual
compounds, or groups of compounds, derived from the various
combinations of the structures and substituents described herein,
are disclosed by the present application to the same extent as if
each compound or group of compounds was set forth individually.
Thus, selection of particular structures or particular substituents
is within the scope of the present disclosure. "Analog" or
"analogue" as used herein refers to a chemical compound that is
structurally similar to a parent compound (e.g., a peptide, protein
or a mucosal delivery enhancing agent), but differs slightly in
composition (e.g., one atom or functional group is different,
added, or removed). The analog may or may not have different
chemical or physical properties than the original compound and may
or may not have improved biological or chemical activity. For
example, the analog may be more hydrophilic or it may have altered
activity as compared to a parent compound. The analog may mimic the
chemical or biological activity of the parent compound (i.e., it
may have similar or identical activity), or, in some cases, may
have increased or decreased activity. The analog may be a naturally
or non-naturally occurring (e.g., chemically-modified, synthetic or
recombinant) variant of the original compound. An example of an
analog is a mutein (i.e., a protein analogue in which at least one
amino acid is deleted, added, or substituted with another amino
acid). Other types of analogs include isomers (enantiomers,
diastereomers, and the like) and other types of chiral variants of
a compound, as well as structural isomers.
[0026] "Derivative" as used herein refers to a chemically or
biologically modified version of a chemical compound (including an
analog) that is structurally similar to a parent compound and
(actually or theoretically) derivable from that parent compound.
Generally, a "derivative" differs from an "analog" in that a parent
compound may be the starting material to generate a "derivative,"
whereas the parent compound may not necessarily be used as the
starting material to generate an "analog."
[0027] As used herein, a thickening agent or thickener includes but
is not limited to a viscosity enhancer, a viscosity enhancing
agent, and a viscosity increasing agent. Within formulations and/or
compositions of the present disclosure, a thickening agent is used
to increase the viscosity of such formulation or composition.
Insulin and Insulin Homologs, Analogs and Derivatives
[0028] As used herein, insulin includes, but is not limited to,
homologs, analogs, and derivatives thereof Insulin, as used herein
encompasses human insulin (e.g., natural, synthetic or
recombinant), insulin glulisine (LysB3, GluB29), HMR-1153 (LysB3,
IleB28), HMR-1423 (GlyA21, HisB31, HisB32), insulin aspart (AspB28)
or (AspB10), and lispro (LysB28, ProB29). Further examples of
insulin according to the present disclosure may be found in Vajo
and Duckworth, Endocrine Reviews 22(5):706-17, 2001; Vajo and
Duckworth Pharmocologic Reviews 52(1):1-9, 2000, and Bhatnagar et
al., Progress in Biophysics and Molecular Biology 91(3):199-228,
2006. The current disclosure focuses primarily on intranasal
administration of insulins, pharmaceutical formulations, and
ultra-rapid acting pharmaceutical formulations comprising insulin
which are able to provide peak insulin serum levels in less than 60
minutes and glucose troughs in less than 90 minutes of
administration. According to the present disclosure insulin also
includes the free bases, acid addition salts or metal salts, such
as potassium or sodium salts of insulin, and peptides or proteins
that have been modified by such processes as amidation,
glycosylation, acylation, sulfation, phosphorylation, acetylation,
cyclization and other well known covalent modification methods.
[0029] Thus, according to the present disclosure, the
above-described peptides are incorporated into pharmaceutical
formulations suitable for transmucosal delivery, especially
intranasal delivery.
Mucosal Delivery Enhancing Agents
[0030] "Mucosal delivery enhancing agents " are defined as
chemicals and other excipients that, when added to a formulation
comprising water, salts and/or common buffers and insulin (the
control formulation) produce a formulation that results in a
significant increase in transport of a insulin across a mucosa as
measured by the maximum blood, serum, or cerebral spinal fluid
concentration (C.sub.max) or by the area under the curve (AUC) in a
plot of concentration versus time. A mucosa includes the nasal,
oral, intestinal, buccal, bronchopulmonary, vaginal, and rectal
mucosal surfaces and includes all mucus-secreting membranes lining
all body cavities or passages that communicate with the exterior.
Mucosal delivery enhancing agents are sometimes called carriers,
excipients, additives, enhancing agents or enhancers (including,
for example, a thickening agent).
[0031] "Endotoxin-free formulation" means a formulation comprising
n insulin and one or more mucosal delivery enhancing agents that is
substantially free of endotoxins and/or related pyrogenic
substances. Endotoxins include toxins that are confined inside a
microorganism and are released only when the microorganisms are
broken down or die. Pyrogenic substances include fever-inducing,
thermostable substances (glycoproteins) from the outer membrane of
bacteria and other microorganisms. These substances can cause
fever, hypotension and shock if administered to humans. Producing
formulations that are endotoxin-free can require special equipment,
expert artisians, and can be significantly more expensive than
making formulations that are not endotoxin-free. Because
intravenous administration of the glucose-regulating peptides,
glucogon-like peptide (GLP) or amylin, simultaneously with infusion
of endotoxin in rodents has been shown to prevent the hypotension
and even death associated with the administration of endotoxin
alone (U.S. Pat. No. 4,839,343), producing endotoxin-free
formulations of insulin would not be expected to be necessary for
non-parental (non-injected) administration.
Non-Infused Administration
[0032] "Non-infused administration" means any method of delivery
that does not involve an injection directly into an artery or vein,
a method which forces or drives (typically a fluid) into something
and especially to introduce into a body part by means of a needle,
syringe or other invasive method. Non-infused administration
includes subcutaneous injection, intramuscular injection,
intraparitoneal injection and the non-injection methods of delivery
to a mucosa.
Methods and Compositions of Delivery
[0033] Improved methods and compositions for mucosal administration
of insulin to mammalian subjects optimize insulin dosing schedules.
The present disclosure describes mucosal delivery of insulin
formulated with one or more mucosal delivery-enhancing agents
wherein insulin dosage release is substantially normalized and/or
sustained for an effective delivery period ranging from about 0.1
to about 2.0 hours; from about 0.4 to about 1.5 hours; from about
0.7 to about 1.5 hours; or from about 0.8 to about 1.0 hours;
following mucosal administration. The sustained release of insulin
achieved may be facilitated by repeated administration of exogenous
insulin utilizing methods and compositions of the present
disclosure.
Compositions and Methods of Sustained Release
[0034] Improved compositions and methods for mucosal administration
of insulin to mammalian subjects allow for the optimization of
insulin dosing schedules. The present disclosure provides improved
mucosal (e.g., nasal) delivery of a formulation comprising insulin
in combination with one or more mucosal delivery-enhancing agents
and an optional sustained release-enhancing agent or agents.
Mucosal delivery-enhancing agents of the present disclosure yield
an effective increase in delivery, e.g., an increase in the maximal
plasma concentration (C.sub.max) to enhance the therapeutic
activity of mucosally-administered insulin. Another factor
affecting therapeutic activity of insulin in the blood plasma and
CNS is residence time (RT). Sustained release-enhancing agents, in
combination with intranasal delivery-enhancing agents, increase
C.sub.max and increase residence time (RT) of insulin. An increase
in residence time at the mucosal delivery site (e.g., nasal mucosa)
and/or systemic circulation are contemplated herein. Polymeric
delivery vehicles and other agents and methods of the present
disclosure that yield sustained release-enhancing formulations, for
example, polyethylene glycol (PEG), are disclosed herein. The
present disclosure describes an improved insulin delivery method
and dosage form for treatment of symptoms related to metabolic
disease in mammalian subjects.
[0035] Within the mucosal delivery formulations and methods of this
disclosure, the insulin is frequently combined or coordinately
administered with a suitable carrier or vehicle for mucosal
delivery. As used herein, the term "carrier" includes
pharmaceutically acceptable solid or liquid filler, diluent or
encapsulating material. As used herein, a carrier may be a mucosal
delivery enhancing agent. A water-containing liquid carrier can
contain pharmaceutically acceptable additives such as acidifying
agents, alkalizing agents, antimicrobial preservatives,
antioxidants, buffering agents, chelating agents, complexing
agents, solubilizing agents, humectants, solvents, suspending
and/or viscosity-increasing agents (e.g., a thickener), tonicity
agents, wetting agents or other biocompatible materials. As
disclosed herein, humectants include, but are not limited to,
propylene glycol, glycerine, glyceryl triacetate, a polyol, a
polymeric polyol, lactic acid, and urea. Within this disclosure,
pharmaceutical formulations may contain one humectant or any
combination or mixture of more than one humectant. A tabulation of
ingredients listed by the above categories can be found in the U.S.
Pharmacopeia National Formulary, 1990, 1857-1859. Solubilizing
agents as disclosed herein include, but are not limited to,
cyclodextrin, hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin and methyl-.beta.-cyclodextrin.
Such solubilizing agents may be used in a pharmaceutical
formulation alone or in any mixture or combination of more than one
solubilizing agent. Some examples of the materials which can serve
as pharmaceutically acceptable carriers are sugars, such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters such as ethyl
oleate and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen free water;
isotonic saline, acetate, glycine, histidine, arginine, glutamate,
lysine, methionine, lactate, formate, and glycolate; Ringer's
solution, ethyl alcohol and phosphate buffer solutions, as well as
other non toxic compatible substances used in pharmaceutical
formulations. Pharmaceutical formulations set forth herein may
include any one buffering agent or any combination or mixture of
more than one buffering agent. A buffering agent may have a
pK.sub.a ranging from about 5 to about 9, or from about 6 to about
8. Wetting agents, emulsifiers and lubricants such as sodium lauryl
sulfate and magnesium stearate, as well as coloring agents, release
agents, coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the
compositions, according to the desires of the formulator. Examples
of pharmaceutically acceptable antioxidants include water soluble
antioxidants such as ascorbic acid, cysteine hydrochloride, sodium
bisulfite, sodium metabisulfite, sodium sulfite and the like;
oil-soluble antioxidants such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol and the like; and metal-chelating
agents such as citric acid, ethylenediamine tetraacetic acid
(EDTA), ethylene glycol tetraacetic acid, (EGTA), sorbitol,
tartaric acid, phosphoric acid and the like. In accordance with the
present disclosure, any one or any mixture or combination of
chelating agents may be contained in a pharmaceutical formulation.
The amount of active ingredient that can be combined with the
carrier materials to produce a single dosage form will vary
depending upon the particular mode of administration.
[0036] Within the mucosal delivery compositions and methods of this
disclosure, various mucosal delivery-enhancing agents are employed
which enhance delivery of insulin into or across a mucosal surface.
In this regard, delivery of insulin across the mucosal epithelium
can occur "transcellularly" or "paracellularly." The extent to
which these pathways contribute to the overall flux and
bioavailability of the insulin depends upon the environment of the
mucosa, the physico-chemical properties the active agent, and the
properties of the mucosal epithelium. Paracellular transport
involves only passive diffusion, whereas transcellular transport
can occur by passive, facilitated or active processes. Generally,
hydrophilic, passively transported, polar solutes diffuse across a
mucosal surface through the paracellular route, while more
lipophilic solutes use the transcellular route. Absorption and
bioavailability (e.g., as reflected by a permeability coefficient
or physiological assay), for diverse, passively and actively
absorbed solutes, can be readily evaluated, in terms of both
paracellular and transcellular delivery components, for any
selected insulin within this disclosure. For passively absorbed
drugs, the relative contribution of paracellular and transcellular
pathways to drug transport depends upon the pKa, partition
coefficient, molecular radius and charge of the drug, the pH of the
luminal environment in which the drug is delivered, and the area of
the absorbing surface. The paracellular route represents a
relatively small fraction of accessible surface area of the nasal
mucosal epithelium. In general terms, it has been reported that
cell membranes occupy a mucosal surface area that is a thousand
times greater than the area occupied by the paracellular spaces.
Thus, the smaller accessible area, and the size- and charge-based
discrimination against macromolecular permeation would suggest that
the paracellular route would be a generally less favorable route
than transcellular delivery for drug transport. Surprisingly, the
methods and compositions of this disclosure provide for
significantly enhanced transport of biotherapeutics into and across
mucosal epithelia via the paracellular route. Therefore, the
methods and compositions of this disclosure successfully target
both paracellular and transcellular routes, alternatively or within
a single method or composition.
[0037] As used herein, mucosal delivery-enhancing agents include
agents which enhance or otherwise modulate the release or
solubility (e.g., from a formulation delivery vehicle), diffusion
rate, penetration capacity and timing, uptake, residence time,
stability, effective half-life, peak or sustained concentration
levels, clearance and other desired mucosal delivery
characteristics (e.g., as measured at the site of delivery, or at a
selected target site of activity such as the bloodstream or central
nervous system) of insulin or other biologically active
compound(s). Enhancement of mucosal delivery can thus occur by any
one or more of a variety of mechanisms, for example by increasing
the diffusion, transport, persistence or stability of insulin,
increasing membrane fluidity, modulating the availability or action
of calcium and other ions that regulate intracellular or
paracellular permeation, solubilizing mucosal membrane components
(e.g., lipids), changing non-protein and protein sulfhydryl levels
in mucosal tissues, increasing water flux across the mucosal
surface, modulating epithelial junctional physiology, reducing the
viscosity of mucus overlying the mucosal epithelium, reducing
mucociliary clearance rates, and other mechanisms.
[0038] As used herein, a "mucosally effective amount of insulin"
contemplates effective mucosal delivery of insulin to a target site
for drug activity in a subject in need thereof that may involve a
variety of delivery or transfer routes. For example, a given active
agent may find its way through clearances (e.g., spaces) between
cells of the mucosa and reach an adjacent vascular wall, while by
another route the agent may, either passively or actively, be taken
up (i.e., internalized) into mucosal cells to act within the cells
or be discharged (e.g., released) or transported out of the cells
to reach a secondary target site, such as the systemic circulation.
The methods and compositions of this disclosure may promote the
translocation of active agents along one or more such alternate
(transcellular or paracellular) routes, or may act directly on the
mucosal tissue or proximal vascular tissue to promote absorption or
penetration of the active agent(s). The promotion of absorption or
penetration in this context is not limited to these mechanisms.
[0039] As used herein "peak concentration (C.sub.max) of insulin in
a blood plasma", "area under concentration vs. time curve (AUC) of
insulin in a blood plasma", "time to maximal plasma concentration
(t.sub.max) of insulin in a blood plasma" are pharmacokinetic
parameters known to one skilled in the art. Laursen et al., Eur. J.
Endocrinology 135:309-315, 1996. The "concentration vs. time curve"
measures the concentration of insulin in a blood serum of a subject
vs. time after administration of a dosage of insulin to the subject
either by intranasal, intramuscular, subcutaneous, or other
parenteral route of administration. "C.sub.max" is the maximum
concentration of insulin in the blood serum of a subject following
a single dosage of insulin to the subject. "t.sub.max" is the time
to reach maximum concentration of insulin in a blood serum of a
subject following administration of a single dosage of insulin to
the subject.
[0040] As used herein, "area under concentration vs. time curve
(AUC) of insulin in a blood plasma" is calculated according to the
linear trapezoidal rule and with addition of the residual areas. A
decrease of 23% or an increase of 30% between two dosages would be
detected with a probability of 90% (type II error .beta.=10%). The
"delivery rate" or "rate of absorption" is estimated by comparison
of the time (t.sub.max) to reach the maximum concentration
(C.sub.max). Both C.sub.max and t.sub.max are analyzed using
non-parametric methods. Comparisons of the pharmacokinetics of
intramuscular, subcutaneous, intravenous and intranasal insulin
administrations were performed by analysis of variance (ANOVA). For
pair wise comparisons a Bonferroni-Holmes sequential procedure is
used to evaluate significance. The dose-response relationship
between the three nasal doses is estimated by regression analysis.
P<0.05 is considered significant. Results are given as mean
values .+-.SEM.
[0041] While the mechanism of absorption promotion may vary with
different mucosal delivery-enhancing agents of this disclosure,
useful reagents in this context will not substantially adversely
affect the mucosal tissue and will be selected according to the
physicochemical characteristics of the particular insulin or other
active or delivery-enhancing agent. In this context,
delivery-enhancing agents that increase penetration or permeability
of mucosal tissues will often result in some alteration of the
protective permeability barrier of the mucosa. For such
delivery-enhancing agents to be of value within this disclosure, it
is generally desired that any significant changes in permeability
of the mucosa may be reversible within a time frame appropriate to
the desired duration of drug delivery. Furthermore, there should be
no substantial, cumulative toxicity, nor any permanent deleterious
changes induced in the barrier properties of the mucosa with
long-term use.
[0042] Within certain aspects of this disclosure,
absorption-promoting agents for coordinate administration or
combinatorial formulation with insulin are selected from small
hydrophilic molecules, including but not limited to, dimethyl
sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and
the 2-pyrrolidones. Alternatively, long-chain amphipathic
molecules, for example, deacylmethyl sulfoxide, azone, sodium
laurylsulfate, oleic acid, and the bile salts, may be employed to
enhance mucosal penetration of the insulin. In additional aspects,
surfactants (e.g., polysorbates) are employed as adjunct compounds,
processing agents, or formulation additives to enhance intranasal
delivery of the insulin. Agents such as DMSO, polyethylene glycol,
and ethanol can, if present in sufficiently high concentrations in
delivery environment (e.g., by pre-administration or incorporation
in a therapeutic formulation), enter the aqueous phase of the
mucosa and alter its solubilizing properties, thereby enhancing the
partitioning of the insulin from the vehicle (e.g., the therapeutic
or pharmaceutical formulation) into the mucosa.
[0043] Additional mucosal delivery-enhancing agents that are useful
within the coordinate administration and processing methods and
combinatorial formulations include, but are not limited to, mixed
micelles; enamines; nitric oxide donors (e.g.,
S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4--which are
preferably co-administered with an NO scavenger such as
carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol
esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetate or
1,2-isopropylideneglycerine-3-acetoacetate); and other
release-diffusion or intra- or trans-epithelial
penetration-promoting agents that are physiologically compatible
for mucosal delivery.
[0044] Other absorption-promoting agents are selected from a
variety of carriers, bases and excipients that enhance mucosal
delivery, stability, activity or trans-epithelial penetration of
the insulin. These include, inter alia, cyclodextrins (e.g.,
cyclodextrin) and .beta.-cyclodextrin derivatives (e.g.,
hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, methyl-.beta.-cyclodextrin and
heptakis(2,6-di-O-methyl-.beta.-cyclodextrin)). These compounds,
optionally conjugated with one or more of the active ingredients
and further optionally formulated in an oleaginous base, enhance
bioavailability of a glucose regulating peptide contained in the
mucosal formulations of this disclosure. Yet additional
absorption-enhancing agents adapted for mucosal delivery include
medium-chain fatty acids, including mono- and diglycerides (e.g.,
sodium caprate--extracts of coconut oil, Capmul), and triglycerides
(e.g., amylodextrin, Estaram 299, Miglyol 810).
[0045] The mucosal therapeutic and prophylactic compositions of the
present disclosure may be supplemented with any suitable
penetration-promoting agent that facilitates absorption, diffusion,
or penetration of insulin across mucosal barriers. The penetration
promoting agent may be any such agent that is pharmaceutically
acceptable. Thus, in more detailed aspects of this disclosure
compositions are provided that incorporate one or more of the
penetration-promoting agents selected from sodium salicylate and
salicylic acid derivatives (acetyl salicylate, choline salicylate,
salicylamide, etc.); amino acids and salts thereof (e.g.,
monoaminocarboxlic acids such as glycine, alanine, phenylalanine,
proline, hydroxyproline, etc.; hydroxyamino acids such as serine;
acidic amino acids such as aspartic acid, glutamic acid, etc.; and
basic amino acids such as lysine etc.--inclusive of their alkali
metal or alkaline earth metal salts); and N-acetylamino acids
(N-acetylalanine, N-acetylphenylalanine, N-acetylserine,
N-acetylglycine, N-acetyllysine, N-acetylglutamic acid,
N-acetylproline, N-acetylhydroxyproline, etc.) and their salts
(alkali metal salts and alkaline earth metal salts). Also provided
as penetration-promoting agents within the methods and compositions
of this disclosure are substances which are generally used as
emulsifiers (e.g., sodium oleyl phosphate, sodium lauryl phosphate,
sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene
alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid,
lactic acid, malic acid and citric acid and alkali metal salts
thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic
acid esters, N-alkylpyrrolidones, proline acyl esters, and the
like.
[0046] Within various aspects of this disclosure, improved nasal
mucosal delivery formulations and methods are provided that allow
for the delivery of an insulin and/or other therapeutic agents
across a mucosal barriers (e.g., mucosal surface) between
administration and one or more selected target sites. Certain
formulations may be specifically adapted for a selected target
cell, tissue or organ, or even a particular disease state. In other
aspects of the instant disclosure, improved nasal delivery
formulations and methods provide for efficient, selective endo- or
transcytosis of insulin specifically routed along a defined
intracellular or intercellular pathway. As appreciated herein, the
insulin may be efficiently loaded at an effective concentration in
a carrier or other delivery vehicle, which is then administered and
maintained in a stabilized format when, for example, administered
to the nasal mucosa and/or during passage through one or more
intracellular compartments and/or membranes to a target site for
drug action (e.g., the blood stream or a defined tissue, organ, or
extracellular compartment). The insulin may be provided in a
delivery vehicle or otherwise modified (e.g., in the form of a
prodrug), wherein release or activation of the insulin is triggered
by a physiological stimulus (e.g., pH change, lysosomal enzymes,
etc.) In certain aspects, the insulin may be pharmacologically
inactive until it reaches its target site for activity. The insulin
and other formulation components are non-toxic (or reduce toxicity
to an acceptable amount) and non-immunogenic. In this context,
carriers and other formulation components are generally selected
for their ability to be rapidly degraded and/or excreted under
physiological conditions. At the same time, formulations are
chemically and physically stable in dosage form for effective
storage.
Peptide and Protein Derivatives, Analogs and Mimetics
[0047] Included within the definition of biologically active
peptides and proteins for use within the context of this disclosure
are natural or synthetic, therapeutically or prophylactically
active, peptides (comprised of two or more covalently linked amino
acids), proteins, peptide or protein fragments, peptide or protein
analogs, and chemically modified derivatives or salts of active
peptides or proteins. A wide variety of useful analogs, derivatives
and/or mimetics of insulin are therefore contemplated for use
within this disclosure and can be produced and tested for
biological activity according to known methods. Often, the peptides
or proteins of an insulin or other biologically active peptides or
proteins for use within this disclosure are muteins that are
readily obtainable by partial substitution, addition, or deletion
of amino acids within a naturally occurring or native (e.g.,
wild-type, naturally occurring mutant, or allelic variant) peptide
or protein sequence. Additionally, biologically active fragments of
native or non-native peptides or proteins are included within the
context of a biologically active peptide and/or protein described
herein. Such mutant derivatives and fragments substantially retain
the desired biological activity of the native peptide or proteins.
In the case of peptides or proteins having a carbohydrate
modification (native or non-native), biologically active variants
identified by alterations to such carbohydrate species are also
included within this disclosure.
[0048] As used herein, "derivative" refers to a chemically or
biologically modified version of a chemical compound (e.g., a
peptide or protein) that is structurally similar to a parent
compound and is (actually or theoretically) derivable from that
parent compound. Generally, a "derivative" differs from an
"analogue" in that a parent compound may be the starting material
to generate a "derivative," whereas the parent compound may not
necessarily be used as the starting material to generate an
"analogue." An analogue (or a derivative) may have different
chemical or physical properties of the parent compound. For
example, a derivative may be more hydrophilic or it may have
altered reactivity as compared to the parent compound.
[0049] As used herein and as appreciated by one of skill in the
art, the term "conservative amino acid substitution" refers to the
general interchangeability of amino acid residues having similar
side chains. For example, a commonly interchangeable group of amino
acids having an aliphatic side chain is alanine, valine, leucine,
and isoleucine; a group of amino acids having an aliphatic-hydroxyl
side chain is serine and threonine; a group of amino acids having
an amide-containing side chain is asparagine and glutamine; a group
of amino acids having an aromatic side chain is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having a basic
side chain is lysine, arginine, and histidine; and a group of amino
acids having a sulfur-containing side chain is cysteine and
methionine. Examples of conservative substitutions include the
substitution of a non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another. Likewise,
the present disclosure contemplates the substitution of a polar
(hydrophilic) residue such as between arginine and lysine, or
between glutamine and asparagine, or between threonine and serine.
Additionally, the substitution of a basic residue such as lysine,
arginine or histidine for another or the substitution of an acidic
residue such as aspartic acid or glutamic acid for another is also
contemplated. Exemplary conservative amino acids substitution
groups are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine. By
aligning a peptide or protein analog optimally with a corresponding
native peptide or protein, and by using appropriate assays, e.g.,
adhesion protein or receptor binding assays, to determine a
selected biological activity, one can readily identify operable
peptide and protein analogs for use within the methods and
compositions of this disclosure. Operable peptide and protein
analogs as set forth in this disclosure may be specifically
cross-reactive (e.g., immunoreactive) with antibodies raised to the
corresponding native peptide or protein.
[0050] An approach for stabilizing certain peptide and/or protein
formulations of this disclosure is to increase the physical
stability by using a solid, e.g., lyophilized peptide or protein
formulation. Such stabilization will inhibit peptide or protein
aggregation via hydrophobic interactions as well as via covalent
modification(s) that may increase as a peptide or protein unfolds
or is otherwise denatured. Stabilizing formulations in this context
may include a polymer-based formulation, for example a
biodegradable hydrogel formulation/delivery system. As noted
herein, the critical role of water in protein structure, function,
and stability is well known to one of skill in the relevant art.
Typically, proteins are relatively stable in the solid state with
bulk water removed. However, solid therapeutic protein formulations
may become hydrated upon storage at elevated humidities or during
delivery from a sustained release composition or device. The
stability of proteins may drop with increasing hydration. Water can
also play a significant role in solid protein aggregation, for
example, by increasing protein flexibility resulting in enhanced
accessibility of reactive groups, by providing a mobile phase for
reactants, and by serving as a reactant in several deleterious
processes such as beta-elimination and hydrolysis.
[0051] Solid (e.g., lyophilized) protein preparations containing
from about 6% to about 28% water (hydration) are the most unstable.
Below this level, the mobility of bound water and the internal
motion of a protein are low. Above this level, water mobility and
protein motion approach those of full hydration. Increased
susceptibility toward solid-phase aggregation with increasing
hydration has been observed in several systems. However, at higher
water content, less aggregation may be less obvious because of the
dilution effect.
[0052] In accordance with these principles, an effective method for
stabilizing peptides and proteins against solid-state aggregation
for mucosal delivery is to control the water content in a solid
formulation and maintain the water activity in the formulation at
optimal levels. This level depends on the nature of the protein,
but in general, proteins maintained below their "monolayer" water
coverage will exhibit superior solid-state stability.
[0053] Within this disclosure, a variety of additives, diluents,
bases and delivery vehicles are provided which effectively control
water content and consequently improve protein stability. Such
reagents and carrier materials effective as anti-aggregation agents
include, for example, polymers of various functionalities, such as
polyethylene glycol, dextran, diethylaminoethyl dextran, and
carboxymethyl cellulose, which significantly increase the stability
and reduce the solid-phase aggregation of peptides and proteins
admixed therewith or linked thereto. In some instances, the
activity or physical stability of a peptide or protein can also be
enhanced by various additives to aqueous solutions comprising the
peptide or protein drugs. For example, additives, such as polyols
(including sugars), amino acids, proteins such as collagen and
gelatin, and various salts may be used.
[0054] Certain additives, in particular sugars and other polyols,
also impart significant physical stability to dry (e.g.,
lyophilized) peptides or proteins. These additives can also be used
within the context of this disclosure in order to protect a peptide
or protein against aggregation not only during the lyophilization
process but also during storage of the lyophilized product in the
dry state. For example, sucrose and Ficoll 70 (a polymer with
sucrose units) exhibit significant protection against peptide or
protein aggregation during solid-phase incubation under various
conditions. These additives may also enhance the stability of a
solid protein embedded within polymer matrices.
[0055] Yet additional additives, for example sucrose, stabilize
peptides or proteins against solid-state aggregation in humid
atmospheres at elevated temperatures, as may occur in certain
sustained-release formulations of this disclosure. Proteins such as
gelatin and collagen also serve as stabilizing or bulking agents to
reduce denaturation and/or aggregation of unstable proteins in this
context. These additives can be incorporated into polymeric melt
processes and compositions within the disclosure. For example,
polypeptide microparticles can be prepared by simply lyophilizing
or spray drying a solution containing various stabilizing additives
described herein. Sustained release of unaggregated peptides and
proteins can thereby be obtained over an extended period of
time.
[0056] Various additional preparative components and methods, as
well as specific formulation additives, are provided herein which
yield formulations for mucosal delivery of a therapeutically
effective amount of aggregation-prone peptides and proteins,
wherein the peptide or protein is stabilized in a substantially
pure, unaggregated form as a consequence of using a solubilization
agent. As used herein, a "therapeutically effective amount" means
an amount of an active pharmaceutical ingredient or agent (e.g., a
mucosal delivery-enhancing agent) that is sufficient, in the
subject (e.g., mammal) in need thereof and to which it is
administered, to treat or prevent or otherwise modulate the stated
disease, disorder or condition. A range of components and additives
are contemplated for use within the methods and formulations of the
present disclosure. Exemplary of such solubilization agents are
cyclodextrins (CDs) and derivatives thereof, which selectively bind
hydrophobic side chains of polypeptides. These CDs have been found
to bind to hydrophobic patches of proteins in a manner that
significantly inhibits aggregation. This inhibition is selective
with respect to both the CD and the protein involved. Such
selective inhibition of protein aggregation provides additional
advantages within the intranasal delivery methods and compositions
of the disclosure. Additional agents for use in this context
include CD dimers, trimers and tetramers with varying geometries
controlled by linkers that specifically block aggregation of
peptides and/or protein. Yet solubilization agents and methods for
incorporation within this disclosure may involve the use of
peptides, peptide derivatives, analogues, and peptide mimetics to
selectively block protein-protein interactions. In one aspect, the
specific binding of hydrophobic side chains reported for CD
multimers is extended to proteins via the use of peptides and
peptide mimetics that similarly block protein aggregation. A wide
range of suitable methods and anti-aggregation agents are available
for incorporation within the compositions and procedures
contemplated herein.
Charge Modifying and pH Control Agents and Methods
[0057] To improve the transport characteristics of biologically
active agents (including a insulin, or other active peptides and
proteins, and macromolecular and small molecule drugs) for enhanced
delivery across hydrophobic mucosal membrane barriers, this
disclosure also provides techniques and reagents for the "charge
modification" of selected biologically active agents or
delivery-enhancing agents described herein. In this regard, the
relative permeability of a macromolecule is generally related to
its partition coefficient. A molecules degree of ionization, which
is dependent on the pK.sub.a of the molecule and the pH at the
mucosal membrane surface, also affects its permeability. As set
forth herein, the permeation and partitioning of biologically
active agents, including insulin and analogs thereof, for mucosal
delivery may be facilitated by charge alteration or charge
spreading of the active agent or permeabilizing agent, which may be
achieved, for example, by alteration of charged functional groups,
by modifying the pH of the delivery vehicle or solution in which
the active agent (or precursor thereto) is delivered, or by
coordinate administration of a charge- or pH-altering reagent with
the active agent (or precursor).
[0058] Consistent with these general teachings, mucosal delivery of
charged macromolecular species, including insulin and other
biologically active peptides and proteins, within the methods and
compositions of this disclosure is substantially improved when the
active agent is delivered to the mucosal surface in a substantially
un-ionized, or neutral, electrical charge state.
[0059] Certain insulin(s) and other biologically active peptide and
protein components of one or more mucosal formulations within this
disclosure may be charge modified in order to provide an increase
in the positive charge density of the peptide or protein. These
modifications extend also to cationization of peptide and protein
conjugates, carriers and other delivery forms disclosed herein.
Cationization offers a convenient means of altering the
biodistribution and transport properties of proteins and
macromolecules within this disclosure. Cationization is undertaken
in a manner that substantially preserves the biological activity of
the active agent and limits potentially adverse side effects,
including tissue damage and toxicity.
[0060] A "buffer" is generally used to maintain the pH of a
solution at a nearly constant value. A buffer maintains the pH of a
solution, even when small amounts of strong acid or strong base are
added to the solution, by preventing or neutralizing large changes
in concentrations of hydrogen and hydroxide ions. A buffer
generally consists of a weak acid and its appropriate salt (or a
weak base and its appropriate salt). The appropriate salt for a
weak acid contains the same negative ion as present in the weak
acid (see Lagowski, Macmillan Encyclopedia of Chemistry 1:273-4,
1997, Simon & Schuster, New York. The Henderson-Hasselbach
Equation, pH=pKa+log10 [A-]/[HA], is used to describe a buffer, and
is based on the standard equation for weak acid dissociation,
HA=[H+]+[A-]. Examples of commonly used buffer salts include the
following: glutamate, acetate, citrate, glycine, histidine,
arginine, lysine, methionine, lactate, formate, glycolate,
tartrate, phosphate and mixtures thereof.
[0061] The "buffer capacity" means the amount of acid or base that
can be added to a buffer solution before a significant pH change
will occur. If the pH lies within the range of pK-1 and pK+1 of the
weak acid the buffer capacity is appreciable, but outside this
range it falls off to such an extent as to be of little value.
Therefore, a given system only has a useful buffer action in a
range of one pH unit on either side of the pK of the weak acid (or
weak base) (see Dawson, Data for Biochemical Research, Third
Edition, Oxford Science Publications, 1986, p. 419). Generally,
suitable concentrations are chosen so that the pH of the solution
is close to the pKa of the weak acid (or weak base) (see Lide, CRC
Handbook of Chemistry and Physics, 86th Edition, Taylor &
Francis Group, 2005-2006, p. 2-41). Further, solutions of strong
acids and bases are not normally classified as buffer solutions,
and they do not display buffer capacity between pH values 2.4 to
11.6.
Degradative Enzyme Inhibitory Agents and Methods
[0062] Another excipient that may be included in a trans-mucosal
delivery formulation is a degradative enzyme inhibitor. Exemplary
mucoadhesive polymer-enzyme inhibitor complexes that are useful
within the mucosal delivery formulations and methods of this
disclosure include, but are not limited to:
Carboxymethylcellulose-pepstatin (with anti-pepsin activity);
Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);
Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylic
acid)-elastatinal (anti-elastase);
Carboxymethylcellulose-elastatinal (anti-elastase);
Polycarbophil--elastatinal (anti-elastase); Chitosan--antipain
(anti-trypsin); Poly(acrylic acid)--bacitracin (anti-aminopeptidase
N); Chitosan--EDTA (anti-aminopeptidase N, anti-carboxypeptidase
A); Chitosan--EDTA--antipain (anti-trypsin, anti-chymotrypsin,
anti-elastase). As described in further detail below, certain
embodiments of this disclosure incorporate a novel chitosan
derivative or chemically modified form of chitosan. One such novel
derivative within this disclosure is denoted as a
.beta.-[1.fwdarw.4]-2-guanidino-2-deoxy-D-glucose polymer
(poly-GuD).
[0063] Any inhibitor that inhibits the activity of an enzyme (e.g.,
a protease) to protect the biologically active agent(s) may be
usefully employed in the compositions and methods of this
disclosure. Useful enzyme inhibitors for the protection of
biologically active proteins and peptides include, for example,
soybean trypsin inhibitor, exendin trypsin inhibitor, chymotrypsin
inhibitor and trypsin and chrymotrypsin inhibitor isolated from
potato (solanum tuberosum L.) tubers. A combination or mixtures of
inhibitors may be employed. Additional inhibitors of proteolytic
enzymes within this disclosure include ovomucoid-enzyme, gabaxate
mesylate, alpha1-antitrypsin, aprotinin, amastatin, bestatin,
puromycin, bacitracin, leupepsin, alpha2-macroglobulin, pepstatin
and egg white or soybean trypsin inhibitor. These and other
inhibitors can be used alone or in any combination. The
inhibitor(s) may be incorporated in or bound to a carrier, e.g., a
hydrophilic polymer, coated on the surface of the dosage form which
is to contact the nasal mucosa, or incorporated in the superficial
phase of the surface, in combination with the biologically active
agent or in a separately administered (e.g., pre-administered)
formulation.
[0064] The amount of the inhibitor, e.g., of a proteolytic enzyme
inhibitor that is optionally incorporated in the compositions of
this disclosure will vary depending on (a) the properties of the
specific inhibitor, (b) the number of functional groups present in
the molecule (which may be reacted to introduce ethylenic
unsaturation necessary for copolymerization with hydrogel forming
monomers), and (c) the number of lectin groups, such as glycosides,
which are present in the inhibitor molecule. It may also depend on
the specific therapeutic agent that is intended to be administered.
A useful amount of an enzyme inhibitor may be from about 0.1 mg/ml
to about 50 mg/ml, often from about 0.2 mg/ml to about 25 mg/ml,
and more commonly from about 0.5 mg/ml to about 5 mg/ml of the of
the formulation (i.e., a separate protease inhibitor formulation or
combined formulation with the inhibitor and biologically active
agent).
[0065] In the case of trypsin inhibition, suitable inhibitors may
be selected from, e.g., aprotinin, BBI, soybean trypsin inhibitor,
chicken ovomucoid, chicken ovoinhibitor, human exendin trypsin
inhibitor, camostat mesilate, flavonoid inhibitors, antipain,
leupeptin, p-aminobenzamidine, AEBSF, TLCK (tosyllysine
chloromethylketone), APMSF, DFP, PMSF, and poly(acrylate)
derivatives. In the case of chymotrypsin inhibition, suitable
inhibitors may be selected from, e.g., aprotinin, BBI, soybean
trypsin inhibitor, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO,
FK-448, chicken ovoinhibitor, sugar biphenylboronic acids
complexes, DFP, PMSF, .beta.-phenylpropionate, and poly(acrylate)
derivatives. In the case of elastase inhibition, suitable
inhibitors may be selected from, e.g., elastatinal,
methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK), BBI,
soybean trypsin inhibitor, chicken ovoinhibitor, DFP, and PMSF.
[0066] Additional enzyme inhibitors within this disclosure are
selected from a wide range of non-protein inhibitors that vary in
their degree of potency and toxicity. As described in further
detail below, immobilization of these adjunct agents to matrices or
other delivery vehicles, or development of chemically modified
analogues, may be readily implemented to reduce or even eliminate
toxic effects, when they are encountered. Among this broad group of
candidate enzyme inhibitors within this disclosure are
organophosphorous inhibitors, such as diisopropylfluorophosphate
(DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent,
irreversible inhibitors of serine proteases (e.g., trypsin and
chymotrypsin). The additional inhibition of acetylcholinesterase by
these compounds makes them highly toxic in uncontrolled delivery
settings. Another candidate inhibitor,
4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF), has an
inhibitory activity comparable to DFP and PMSF, but it is markedly
less toxic. (4-Aminophenyl)-methanesulfonyl fluoride hydrochloride
(APMSF) is another potent inhibitor of trypsin, but is toxic in
uncontrolled settings. In contrast to these inhibitors,
4-(4-isopropylpiperadinocarbonyl)phenyl 1,
2,3,4,-tetrahydro-1-naphthoate methanesulphonate (FK-448) is a low
toxic substance, representing a potent and specific inhibitor of
chymotrypsin. Further representatives of this non-protein group of
inhibitor candidates, and also exhibiting low toxic risk, are
camostat mesilate (N,N'-dimethyl
carbamoylmethyl-p-(p'-guanidino-benzoyloxy)phenylacetate
methane-sulphonate).
[0067] Yet another type of enzyme inhibitory agent within the
methods and compositions of this disclosure are amino acids and
modified amino acids that interfere with enzymatic degradation of
specific therapeutic compounds. For use in this context, amino
acids and modified amino acids are substantially non-toxic and can
be produced at a low cost. However, due to their low molecular size
and good solubility, they are readily diluted and absorbed in
mucosal environments. Nevertheless, under proper conditions, amino
acids can act as reversible, competitive inhibitors of protease
enzymes. Certain modified amino acids can display a much stronger
inhibitory activity. A desired modified amino acid in this context
is known as a `transition-state` inhibitor. The strong inhibitory
activity of these compounds is based on their structural similarity
to a substrate in its transition-state geometry, while they are
generally selected to have a much higher affinity for the active
site of an enzyme than the substrate itself. Transition-state
inhibitors are reversible, competitive inhibitors. Examples of this
type of inhibitor are .alpha.-aminoboronic acid derivatives, such
as boro-leucine, boro-valine and boro-alanine. The boron atom in
these derivatives can form a tetrahedral boronate ion that is
believed to resemble the transition state of peptides during their
hydrolysis by aminopeptidases. Such amino acid derivatives are
potent and reversible inhibitors of aminopeptidases and it is
reported that boro-leucine is more than 100-times more effective in
enzyme inhibition than bestatin and more than 1000-times more
effective than puromycin. Another modified amino acid for which a
strong protease inhibitory activity has been reported is
N-acetylcysteine, which inhibits enzymatic activity of
aminopeptidase N. This adjunct agent also displays mucolytic
properties that can be employed within the methods and compositions
of this disclosure to reduce the effects of the mucus diffusion
barrier.
[0068] Still other useful enzyme inhibitors for use within the
coordinate administration methods and combinatorial formulations of
this disclosure may be selected from peptides and modified peptide
enzyme inhibitors. An important representative of this class of
inhibitors is the cyclic dodecapeptide, bacitracin, obtained from
Bacillus licheniformis. In addition to these types of peptides,
certain dipeptides and tripeptides display weak, non-specific
inhibitory activity towards some protease. By analogy with amino
acids, their inhibitory activity can be improved by chemical
modifications. For example, phosphinic acid dipeptide analogues are
also `transition-state` inhibitors with a strong inhibitory
activity towards aminopeptidases. They have reportedly been used to
stabilize nasally administered leucine enkephalin. Another example
of a transition-state analogue is the modified pentapeptide
pepstatin, which is a very potent inhibitor of pepsin. Structural
analysis of pepstatin, by testing the inhibitory activity of
several synthetic analogues, demonstrated the major
structure-function characteristics of the molecule responsible for
the inhibitory activity. Another special type of modified peptide
includes inhibitors with a terminally located aldehyde function in
their structure. For example, the sequence
benzyloxycarbonyl-Pro-Phe-CHO, which fulfills the known primary and
secondary specificity requirements of chymotrypsin, has been found
to be a potent reversible inhibitor of this target proteinase. The
chemical structures of further inhibitors with a terminally located
aldehyde function, e.g., antipain, leupeptin, chymostatin and
elastatinal, are also known in the art, as are the structures of
other known, reversible, modified peptide inhibitors, such as
phosphoramidon, bestatin, puromycin and amastatin.
[0069] Due to their comparably high molecular mass, polypeptide
protease inhibitors are more amenable than smaller compounds to
concentrated delivery in a drug-carrier matrix. Additional agents
for protease inhibition within the formulations and methods of this
disclosure involve the use of complexing agents. These agents
mediate enzyme inhibition by depriving the intranasal environment
(or preparative or therapeutic composition) of divalent cations,
which are co-factors for many proteases. For instance, the
complexing agents EDTA and DTPA as coordinately administered or
combinatorially formulated adjunct agents, in suitable
concentration, will be sufficient to inhibit selected proteases to
thereby enhance intranasal delivery of biologically active agents
according to this disclosure. Further representatives of this class
of inhibitory agents are EGTA, 1,10-phenanthroline and
hydroxychinoline. In addition, due to their propensity to chelate
divalent cations, these and other complexing agents are useful
within this disclosure as direct absorption-promoting agents.
[0070] As noted in more detail elsewhere herein, it is also
contemplated to use various polymers, particularly mucoadhesive
polymers, as enzyme inhibiting agents within the coordinate
administration, multi-processing and/or combinatorial formulation
methods and compositions of this disclosure. For example,
poly(acrylate) derivatives, such as poly(acrylic acid) and
polycarbophil, can affect the activity of various proteases,
including trypsin, chymotrypsin. The inhibitory effect of these
polymers may also be based on the complexation of divalent cations
such as Ca.sup.2+ and Zn.sup.2+. It is further contemplated that
these polymers may serve as conjugate partners or carriers for
additional enzyme inhibitory agents, as described above. For
example, a chitosan-EDTA conjugate has been developed and is useful
within this disclosure that exhibits a strong inhibitory effect
towards the enzymatic activity of zinc-dependent proteases. The
mucoadhesive properties of polymers following covalent attachment
of other enzyme inhibitors in this context are not expected to be
substantially compromised, nor is the general utility of such
polymers as a delivery vehicle for biologically active agents
within this disclosure expected to be diminished. On the contrary,
the reduced distance between the delivery vehicle and mucosal
surface afforded by the mucoadhesive mechanism will minimize
presystemic metabolism of the active agent, while the covalently
bound enzyme inhibitors remain concentrated at the site of drug
delivery, minimizing undesired dilution effects of inhibitors as
well as toxic and other side effects caused thereby. In this
manner, the effective amount of a coordinately administered enzyme
inhibitor can be reduced due to the exclusion of dilution
effects.
[0071] Exemplary mucoadhesive polymer-enzyme inhibitor complexes
that are useful within the mucosal formulations and methods of this
disclosure include, but are not limited to:
Carboxymethylcellulose-pepstatin (with anti-pepsin activity);
Poly(acrylic acid)-Bowman-Birk inhibitor (anti-chymotrypsin);
Poly(acrylic acid)-chymostatin (anti-chymotrypsin); Poly(acrylic
acid)-elastatinal (anti-elastase);
Carboxymethylcellulose-elastatinal (anti-elastase);
Polycarbophil--elastatinal (anti-elastase); Chitosan--antipain
(anti-trypsin); Poly(acrylic acid)--bacitracin (anti-aminopeptidase
N); Chitosan--EDTA (anti-aminopeptidase N, anti-carboxypeptidase
A); Chitosan--EDTA--antipain (anti-trypsin, anti-chymotrypsin,
anti-elastase).
Mucolytic and Mucus-Clearing Agents and Methods
[0072] Effective delivery of biotherapeutic agents via intranasal
administration must take into account the decreased drug transport
rate across the protective mucus lining of the nasal mucosa, in
addition to drug loss due to binding to glycoproteins of the mucus
layer. Normal mucus is a viscoelastic, gel-like substance
consisting of water, electrolytes, mucins, macromolecules, and
sloughed epithelial cells. It serves primarily as a cytoprotective
and lubricative covering for the underlying mucosal tissues. Mucus
is secreted by randomly distributed secretory cells located in the
nasal epithelium and in other mucosal epithelia. The structural
unit of mucus is mucin. This glycoprotein is mainly responsible for
the viscoelastic nature of mucus, although other macromolecules may
also contribute to this property. In airway mucus, such
macromolecules include locally produced secretory IgA, IgM, IgE,
lysozyme, and bronchotransferrin, which also play an important role
in host defense mechanisms.
[0073] The coordinate administration methods of the instant
disclosure optionally incorporate effective mucolytic or
mucus-clearing agents, which serve to degrade, thin or clear mucus
from intranasal mucosal surfaces to facilitate absorption of
intranasally administered biotherapeutic agents. Within these
methods, a mucolytic or mucus-clearing agent is coordinately
administered as an adjunct compound to enhance intranasal delivery
of the biologically active agent. Alternatively, an effective
amount of a mucolytic or mucus-clearing agent is incorporated as a
processing agent within a multi-processing method of this
disclosure, or as an additive within a combinatorial formulation of
this disclosure, to provide an improved formulation that enhances
intranasal delivery of biotherapeutic compounds by reducing the
barrier effects of intranasal mucus.
[0074] A variety of mucolytic or mucus-clearing agents are
available for incorporation within the methods and compositions of
this disclosure. Based on their mechanisms of action, mucolytic and
mucus clearing agents can often be classified into the following
groups: proteases (e.g., pronase, papain) that cleave the protein
core of mucin glycoproteins; sulfhydryl compounds that split
mucoprotein disulfide linkages; and detergents (e.g., Triton X-100,
Tween 20) that break non-covalent bonds within the mucus.
Additional compounds in this context include, but are not limited
to, bile salts and surfactants, for example, sodium deoxycholate,
sodium taurodeoxycholate, sodium glycocholate, and
lysophosphatidylcholine.
[0075] The effectiveness of bile salts in causing structural
breakdown of mucus is in the order
deoxycholate>taurocholate>glycocholate. Other effective
agents that reduce mucus viscosity or adhesion to enhance
intranasal delivery according to the methods of this disclosure
include, e.g., short-chain fatty acids, and mucolytic agents that
work by chelation, such as N-acylcollagen peptides, bile acids, and
saponins (the latter function in part by chelating Ca.sup.2+ and/or
Mg.sup.2- which play an important role in maintaining mucus layer
structure).
[0076] Additional mucolytic agents for use within the methods and
compositions of this disclosure include N-acetyl-L-cysteine (ACS),
a potent mucolytic agent that reduces both the viscosity and
adherence of bronchopulmonary mucus and is reported to modestly
increase nasal bioavailability of human growth hormone in
anesthetized rats (from 7.5 to 12.2%). These and other mucolytic or
mucus-clearing agents are contacted with the nasal mucosa,
typically in a concentration range from about 0.2 to about 20 mM,
coordinately with administration of the biologically active agent,
to reduce the polar viscosity and/or elasticity of intranasal
mucus.
[0077] Still other mucolytic or mucus-clearing agents may be
selected from a range of glycosidase enzymes, which are able to
cleave glycosidic bonds within the mucus glycoprotein.
.alpha.-amylase and .beta.-amylase are representative of this class
of enzymes, although their mucolytic effect may be limited. In
contrast, bacterial glycosidases allow these microorganisms to
permeate mucus layers of their hosts.
[0078] For combinatorial use with most biologically active agents
within this disclosure, including peptide and protein therapeutics,
non-ionogenic detergents are generally also useful as mucolytic or
mucus-clearing agents. These agents typically will not modify or
substantially impair the activity of therapeutic polypeptides.
Ciliostatic Agents and Methods
[0079] Because the self-cleaning capacity of certain mucosal
tissues (e.g., nasal mucosal tissues) by mucociliary clearance is
necessary as a protective function (e.g., to remove dust,
allergens, and bacteria), it is appreciated that this function
should not be substantially impaired by mucosally administered
medications. Mucociliary transport in the respiratory tract is a
particularly important defense mechanism against infections. To
achieve this function, ciliary beating in the nasal and airway
passages moves a layer of mucus along the mucosa to removing
inhaled particles and microorganisms.
[0080] Ciliostatic agents find use within the methods and
compositions of this disclosure to increase the residence time of a
mucosally (e.g., intranasally) administered formulation comprising
an insulin, analog and mimetic, and other biologically active agent
disclosed herein. In particular, the delivery of such agents within
the methods and compositions of this disclosure is significantly
enhanced in certain aspects by the coordinate administration or
combinatorial formulation of one or more ciliostatic agents that
function to reversibly inhibit the ciliary activity of mucosal
cells, and thereby to provide for a temporary, reversible increase
in the residence time of the mucosally administered
pharmaceutically active agent(s). For use within these aspects of
this disclosure, the ciliostatic factors set forth herein, either
specific or indirect in their activity, are all candidates for
successful employment as a ciliostatic agent in appropriate amounts
(depending on concentration, duration and mode of delivery) such
that they yield a transient (i.e., reversible) reduction or
cessation of mucociliary clearance at a mucosal site of
administration to enhance delivery of an insulin, analogs and/or
mimetics, and other biologically active agents disclosed herein,
without unacceptable adverse side effects.
[0081] Within more detailed aspects, a specific ciliostatic factor
may be employed in a combined formulation or coordinate
administration protocol with one or more insulin protein, analog
and mimetic, and/or one or more other biologically active agent
disclosed herein. Various bacterial ciliostatic factors isolated
and characterized in the literature may be employed within certain
embodiments of this disclosure. For example, ciliostatic factors
from the bacterium Pseudomonas aeruginosa include a phenazine
derivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and a
rhamnolipid (also known as a hemolysin). The pyo compound produced
ciliostasis at concentrations of 50 .mu.g/ml and without obvious
ultrastructural lesions. The phenazine derivative also inhibited
ciliary motility but caused some membrane disruption, although at
substantially greater concentrations of 400 .mu.g/ml. Limited
exposure of tracheal explants to the rhamnolipid resulted in
ciliostasis, which is associated with altered ciliary membranes.
More extensive exposure to rhamnolipid is associated with removal
of dynein arms from axonemes.
Surface Active Agents and Methods
[0082] Within more detailed aspects of this disclosure, one or more
membrane penetration-enhancing agents may be employed within a
mucosal delivery method or formulation of this disclosure to
enhance mucosal delivery of insulin proteins, analogs and mimetics,
and other biologically active agents disclosed herein. Membrane
penetration enhancing agents in this context can be selected from:
(i) a surfactant; (ii) a bile salt; (iii) a phospholipid additive,
mixed micelle, liposome, or carrier; (iv) an alcohol; (v) an
enamine; (vi) an NO donor compound; (vii) a long-chain amphipathic
molecule; (viii) a small hydrophobic penetration enhancer; (ix)
sodium or a salicylic acid derivative; (x) a glycerol ester of
acetoacetic acid; (xi) a cyclodextrin or beta-cyclodextrin
derivative; (xii) a medium-chain fatty acid; (xiii) a chelating
agent; (xiv) an amino acid or salt thereof, (xv) an N-acetylamino
acid or salt thereof, (xvi) an enzyme degradative to a selected
membrane component; (xvii) an inhibitor of fatty acid synthesis;
(xviii) an inhibitor of cholesterol synthesis; or (xix) any
combination of the membrane penetration enhancing agents recited in
(i)-(xviii).
[0083] Certain surface-active agents, also called surfactants, are
readily incorporated within the mucosal delivery formulations and
methods of this disclosure as mucosal absorption enhancing agents.
These agents, which may be coordinately administered or
combinatorially formulated with insulin proteins, analogs and
mimetics, and other biologically active agents disclosed herein,
may be selected from a broad assemblage of known surfactants.
Surfactants, which generally fall into three classes: (1) nonionic
polyoxyethylene ethers; (2) bile salts such as sodium glycocholate
(SGC) and deoxycholate (DOC); and (3) fusidic acid and derivatives
of fusidic acid such as sodium taurodihydrofusidate (STDHF). The
mechanisms of action of these various classes of surface-active
agents typically include solubilization of the biologically active
agent. For proteins and peptides which often form aggregates, the
surface active properties of these absorption promoters can allow
interactions with proteins such that smaller units such as
surfactant coated monomers may be more readily maintained in
solution. These monomers are presumably more transportable units
than aggregates. Examples of other surface-active agents are
L-.alpha.-Phosphatidylcholine Didecanoyl (DDPC), polysorbate 80 and
polysorbate 20. Additional surface-acting agents include
polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, polyvinyl
alcohol, lanolin alcohol, sorbitan monooleate. All surface-acting
agents of the instant disclosure may be present in a pharmaceutical
formulation alone or in any mixture or combination. A second
potential mechanism is the protection of the peptide or protein
from proteolytic degradation by proteases in the mucosal
environment. Both bile salts and some fusidic acid derivatives
reportedly inhibit proteolytic degradation of proteins by nasal
homogenates at concentrations less than or equivalent to those
required to enhance protein absorption. This protease inhibition
may be especially important for peptides with short biological
half-lives.
Thickening Agents
[0084] Thickening or suspending agents may affect the rate of
release of a drug from the dosage formulation and/or absorption.
Some examples of the materials which can serve as pharmaceutically
acceptable thickening agents are gelatin; methylcellulose (MC);
hydroxypropylmethylcellulose (HPMC) and derivatives thereof,
carboxymethylcellulose (CMC); cellulose; starch; heta starch;
poloxamers; pluronics; sodium CMC; sorbitol; acacia; povidone;
carbopol (as used herein, carbopol is a carbomer; carbopol is also
known as Carbomer Homopolymer Type B, or Carbopol.RTM. 974P NF
Polymer); polycarbophil; chitosan; chitosan microspheres; alginate
microspheres; chitosan glutamate; amberlite resin; hyaluronan;
ethyl cellulose; maltodextrin DE; drum-dried way maize starch
(DDWM); degradable starch microspheres (DSM); deoxyglycocholate
(GDC); hydroxyethyl cellulose (HEC); hydroxypropyl cellulose (HPC);
microcrystalline cellulose (MCC); polymethacrylic acid and
polyethylene glycol; sulfobutylether B cyclodextrin; cross-linked
eldexomer starch biospheres; sodiumtaurodihydrofusidate (STDHF);
N-trimethyl chitosan chloride (TMC); degraded starch microspheres;
amberlite resin; chistosan nanoparticles; spray-dried crospovidone;
spray-dried dextran microspheres; spray-dried microcrystalline
cellulose; and cross-linked eldexomer starch microspheres.
[0085] As used herein, a carbomer thickening agent also includes,
but is not limited to, the following: Acrylic acid homopolymer,
Acrylic acid resin, Acrylic acid, polymer, Acrylic polymer, Acrylic
resin, Acrysol A 1, Acrysol A 3, Acrysol A 5, Acrysol AC 5, Acrysol
WS-24, Acrysol ase-75, Antiprex 461, Antiprex A, Arasorb 750,
Arasorb S 100F, Arolon, Aron, Aron A 10H, Atactic poly(acrylic
acid), CCRIS 3234, Carbomer 1342, Carbomer 910, Carbopol 1342,
Carbopol 910, Carbopol 934, Carbopol 934P, Carbopol 940, Carbopol
941, Carbopol 960, Carbopol 961, Carbopol 971P, Carbopol 974P,
Carbopol 980, Carbopol 981, Carboset 515, Carboset Resin No. 515,
Carboxy vinyl polymer, Carboxypolymethylene, Carpolene, Colloids
119/50, Cyguard 266, Dispex C40, Dow Latex 354, G-Cure, Good-rite K
37, Good-rite K 702, Good-rite K 732, Good-rite K-700, Good-rite
K727, Good-rite WS 801, Haloflex 202, Haloflex 208, Joncryl 678,
Junlon 110, Jurimer AC 10H, Jurimer AC 10P, NSC 106034, NSC 106035,
NSC 106036,NSC 106037,NSC 112122,NSC 112123,NSC 114472,NSC
165257,Nalfloc 636, Neocryl A-1038, OLD 01, P 11H, P 11H, P-11H, PA
11M, PAA-25, Pemulen TR-1, Pemulen TR-2, Poly(acrylic acid),
Polyacrylate, Polyacrylate elastomers, Polymer of acrylic acid,
cross-linked with allyl ethers of pentaerythritol, Polymer of
acrylic acid, cross-linked with allyl ethers of pentaerythritol.
Those where the molecular weight is approximately 1,250,000 such as
Polymer of acrylic acid, cross-linked with allyl ethers of
pentaerythritol. Those where the molecular weight is approximately
750,000 such as Polymer of acrylic acid, cross-linked with allyl
ethers of sucrose or pentaerythritol, Polymer of acrylic acid,
cross-linked with allyl ethers of sucrose or pentaerythritol. Those
where the molecular weight is approximately 3,000,000 such as
Polymer of acrylic acid, cross-linked with allyl ethers of sucrose.
Those where the molecular weight is approximately 3,000,000
Polymer, carboxy vinyl Polymerized acrylic acid, Polytex 973,
Primal ASE 60, Propenoic acid polymer, R968, Racryl, Revacryl A
191, Rohagit SD 15, Sokalan PAS, Solidokoll N, Synthemul 90-588, TB
1131, Tecpol, Texcryl, Versicol E 7, Versicol E15, Versicol E9,
Versicol K 11, Versicol S 25, Viscalex HV 30, Viscon 103, WS 24, WS
801, XPA and the like.
[0086] Other thickening agents in Ugwoke et al., Adv. Drug Deliv.
Rev. 29:1656-57, 1998, are incorporated by reference. Any one
thickening agent or any combination or mixture of thickening
increasing agents may be contained in a pharmaceutical formulation
disclosed herein.
Degradation Enzymes and Inhibitors of Fatty Acid and Cholesterol
Synthesis
[0087] In related aspects of this disclosure, insulin proteins,
analogs and mimetics, and other biologically active agents for
mucosal administration are formulated or coordinately administered
with a penetration enhancing agent selected from a degradation
enzyme, or a metabolic stimulatory agent or inhibitor of synthesis
of fatty acids, sterols or other selected epithelial barrier
components, U.S. Pat. No. 6,190,894. For example, degradative
enzymes such as phospholipase, hyaluronidase, neuraminidase, and
chondroitinase may be employed to enhance mucosal penetration of
insulin proteins, analogs and mimetics, and other biologically
active agent without causing irreversible damage to the mucosal
barrier. In one embodiment, chondroitinase is employed within a
method or composition as provided herein to alter glycoprotein or
glycolipid constituents of the permeability barrier of the mucosa,
thereby enhancing mucosal absorption of insulin proteins, analogs
and mimetics, and other biologically active agents disclosed
herein.
[0088] With regard to inhibitors of synthesis of mucosal barrier
constituents, it is noted that free fatty acids account for 20-25%
of epithelial lipids by weight. Two rate-limiting enzymes in the
biosynthesis of free fatty acids are acetyl CoA carboxylase and
fatty acid synthetase. Through a series of steps, free fatty acids
are metabolized into phospholipids. Thus, inhibitors of free fatty
acid synthesis and metabolism for use within the methods and
compositions of this disclosure include, but are not limited to,
inhibitors of acetyl CoA carboxylase such as
5-tetradecyloxy-2-furancarboxylic acid (TOFA); inhibitors of fatty
acid synthetase; inhibitors of phospholipase A such as gomisin A,
2-(p-amylcinnamyl)amino-4-chlorobenzoic acid, bromophenacyl
bromide, monoalide, 7,7-dimethyl-5,8-eicosadienoic acid,
nicergoline, cepharanthine, nicardipine, quercetin,
dibutyryl-cyclic AMP, R-24571, N-oleoylethanolamine,
N-(7-nitro-2,1,3-benzoxadiazol-4-yl) phosphostidyl serine,
cyclosporine A, topical anesthetics, including dibucaine,
prenylamine, retinoids, such as all-trans and 13-cis-retinoic acid,
W-7, trifluoperazine, R-24571 (calmidazolium),
1-hexadocyl-3-trifluoroethyl glycero-sn-2-phosphomenthol (MJ33);
calcium channel blockers including nicardipine, verapamil,
diltiazem, nifedipine, and nimodipine; antimalarials including
quinacrine, mepacrine, chloroquine and hydroxychloroquine; beta
blockers including propanalol and labetalol; calmodulin
antagonists; EGTA; thimersol; glucocorticosteroids including
dexamethasone and prednisolone; and nonsteroidal antiinflammatory
agents including indomethacin and naproxen.
[0089] Free sterols, primarily cholesterol, account for 20-25% of
the epithelial lipids by weight. The rate limiting enzyme in the
biosynthesis of cholesterol is 3-hydroxy-3-methylglutaryl (HMG) CoA
reductase. Inhibitors of cholesterol synthesis for use within the
methods and compositions of this disclosure include, but are not
limited to, competitive inhibitors of (HMG) CoA reductase, such as
simvastatin, lovastatin, fluindostatin (fluvastatin), pravastatin,
mevastatin, as well as other HMG CoA reductase inhibitors, such as
cholesterol oleate, cholesterol sulfate and phosphate, and
oxygenated sterols, such as 25--OH-- and 26--OH-- cholesterol;
inhibitors of squalene synthetase; inhibitors of squalene
epoxidase; inhibitors of DELTA7 or DELTA24 reductases such as
22,25-diazacholesterol, 20,25-diazacholestenol, AY9944, and
triparanol.
[0090] Each of the inhibitors of fatty acid synthesis or the sterol
synthesis inhibitors may be coordinately administered or
combinatorially formulated with one or more insulin proteins,
analogs and mimetics, and other biologically active agents
disclosed herein to achieve enhanced epithelial penetration of the
active agent(s). An effective concentration range for the sterol
inhibitor in a therapeutic or adjunct formulation for mucosal
delivery is generally from about 0.0001% to about 20% by weight of
the total, more typically from about 0.01% to about 5%.
Nitric Oxide Donor Agents and Methods
[0091] Within other related aspects of this disclosure, a nitric
oxide (NO) donor is selected as a membrane penetration-enhancing
agent to enhance mucosal delivery of one or more insulin proteins,
analogs and mimetics, and other biologically active agents
disclosed herein. Various NO donors are known in the art and are
useful in effective concentrations within the methods and
formulations of this disclosure. Exemplary NO donors include, but
are not limited to, nitroglycerine, nitropruside, NOC5
[3-(2-hydroxy-1-(methyl-ethyl)-2-nitrosohydrazino)-1-propanamine],
NOC12 [N-ethyl-2-(1-ethyl-hydroxy-2-nitrosohydrazino)-ethanamine],
SNAP [S-nitroso-N-acetyl-DL-penicillamine], NOR1 and NOR4. Within
the methods and compositions of this disclosure, an effective
amount of a selected NO donor is coordinately administered or
combinatorially formulated with one or more insulin proteins,
analogs and mimetics, and/or other biologically active agents
disclosed herein, into or through the mucosal epithelium.
Agents for Modulating Epithelial Junction Structure and/or
Physiology
[0092] The present disclosure provides pharmaceutical compositions
that contain one or more insulin protein, analog or mimetic, and/or
other biologically active agents in combination with one or more
mucosal delivery enhancing agent disclosed herein formulated in
such pharmaceutical preparation for mucosal delivery.
[0093] The permeabilizing agent reversibly enhances mucosal
epithelial paracellular transport, typically by modulating
epithelial junctional structure and/or physiology at a mucosal
epithelial surface in the subject. This effect typically involves
inhibition by the permeabilizing agent of homotypic or heterotypic
binding between epithelial membrane adhesive proteins of
neighboring epithelial cells. Target proteins for this blockade of
homotypic or heterotypic binding can be selected from various
related junctional adhesion molecules (JAMs), occludins, or
claudins. Examples of this are antibodies, antibody fragments or
single-chain antibodies that bind to the extracellular domains of
these proteins.
[0094] In yet additional detailed embodiments, this disclosure
provides permeabilizing peptides and peptide analogs and mimetics
for enhancing mucosal epithelial paracellular transport. The
subject peptides and peptide analogs and mimetics typically work
within the compositions and methods of this disclosure by
modulating epithelial junctional structure and/or physiology in a
mammalian subject. In certain embodiments, the peptides and peptide
analogs and mimetics effectively inhibit homotypic and/or
heterotypic binding of an epithelial membrane adhesive protein
selected from ajunctional adhesion molecule (JAM), occludin, or
claudin.
[0095] One such agent that has been extensively studied is the
bacterial toxin from Vibrio cholerae known as the "zonula occludens
toxin" (ZOT). This toxin mediates increased intestinal mucosal
permeability and causes disease symptoms including diarrhea in
infected subjects. Fasano et al., Proc. Nat. Acad. Sci., U.S.A.
8:5242-5246, 1991. When tested on rabbit ileal mucosa, ZOT
increased the intestinal permeability by modulating the structure
of intercellular tight junctions. More recently, it has been found
that ZOT is capable of reversibly opening tight junctions in the
intestinal mucosa. It has also been reported that ZOT is capable of
reversibly opening tight junctions in the nasal mucosa. U.S. Pat.
No. 5,908,825.
[0096] Within the methods and compositions of this disclosure, ZOT,
as well as various analogs and mimetics of ZOT that function as
agonists or antagonists of ZOT activity, are useful for enhancing
intranasal delivery of biologically active agents-by increasing
paracellular absorption into and across the nasal mucosa. In this
context, ZOT typically acts by causing a structural reorganization
of tight junctions marked by altered localization of the junctional
protein ZO1. Within these aspects of this disclosure, ZOT is
coordinately administered or combinatorially formulated with the
biologically active agent in an effective amount to yield
significantly enhanced absorption of the active agent, by
reversibly increasing nasal mucosal permeability without
substantial adverse side effects.
Vasodilator Agents and Methods
[0097] Yet another class of absorption-promoting agents that shows
beneficial utility within the coordinate administration and
combinatorial formulation methods and compositions of this
disclosure are vasoactive compounds, more specifically
vasodilators. These compounds function within the present
disclosure to modulate the structure and physiology of the
submucosal vasculature, increasing the transport rate of insulin,
analogs and mimetics thereof, and other biologically active agents
into or through the mucosal epithelium and/or to specific target
tissues or compartments (e.g., the systemic circulation or central
nervous system).
[0098] Vasodilator agents for use within this disclosure typically
cause submucosal blood vessel relaxation by either a decrease in
cytoplasmic calcium, an increase in nitric oxide (NO) or by
inhibiting myosin light chain kinase. They are generally divided
into 9 classes: calcium antagonists, potassium channel openers, ACE
inhibitors, angiotensin-II receptor antagonists, .alpha.-adrenergic
and imidazole receptor antagonists, .beta.1-adrenergic agonists,
phosphodiesterase inhibitors, eicosanoids and NO donors.
[0099] Despite chemical differences, the pharmacokinetic properties
of calcium antagonists are similar. Absorption into the systemic
circulation is high, and these agents therefore undergo
considerable first-pass metabolism by the liver, resulting in
individual variation in pharmacokinetics. Except for the newer
drugs of the dihydropyridine type (amlodipine, felodipine,
isradipine, nilvadipine, nisoldipine and nitrendipine), the
half-life of calcium antagonists is short. Therefore, to maintain
an effective drug concentration for many of these may require
delivery by multiple dosing, or controlled release formulations, as
described elsewhere herein. Treatment with the potassium channel
opener minoxidil may also be limited in manner and level of
administration due to potential adverse side effects.
[0100] ACE inhibitors prevent conversion of angiotensin-I to
angiotensin-II, and are most effective when renin production is
increased. Since ACE is identical to kininase-II, which inactivates
the potent endogenous vasodilator bradykinin, ACE inhibition causes
a reduction in bradykinin degradation. ACE inhibitors provide the
added advantage of cardioprotective and cardioreparative effects,
by preventing and reversing cardiac fibrosis and ventricular
hypertrophy in animal models. The predominant elimination pathway
of most ACE inhibitors is via renal excretion. Therefore, renal
impairment is associated with reduced elimination and a dosage
reduction of 25 to 50% is recommended in patients with moderate to
severe renal impairment.
[0101] With regard to NO donors, these compounds are particularly
useful within this disclosure for their additional effects on
mucosal permeability. In addition to the above-noted NO donors,
complexes of NO with nucleophiles called NO/nucleophiles, or
NONOates, spontaneously and nonenzymatically release NO when
dissolved in aqueous solution at physiologic pH. In contrast, nitro
vasodilators such as nitroglycerin require specific enzyme activity
for NO release. NONOates release NO with a defined stoichiometry
and at predictable rates ranging from <3 minutes for
diethylamine/NO to approximately 20 hours for diethylenetriamine/NO
(DETANO).
[0102] Within certain methods and compositions of this disclosure,
a selected vasodilator agent is coordinately administered (e.g.,
systemically or intranasally, simultaneously or in combinatorially
effective temporal association) or combinatorially formulated with
one or more insulin, analogs and mimetics, and other biologically
active agent(s) in an amount effective to enhance the mucosal
absorption of the active agent(s) to reach a target tissue or
compartment in the subject (e.g., the liver, hepatic portal vein,
CNS tissue or fluid, or blood plasma).
Selective Transport-Enhancing Agents and Methods
[0103] The compositions and delivery methods of this disclosure
optionally incorporate a selective transport-enhancing agent that
facilitates transport of one or more biologically active agents.
These transport-enhancing agents may be employed in a combinatorial
formulation or coordinate administration protocol with one or more
of the insulin proteins, analogs and mimetics disclosed herein, to
coordinately enhance delivery of one or more additional
biologically active agent(s) across mucosal transport barriers, to
enhance mucosal delivery of the active agent(s) to reach a target
tissue or compartment in the subject (e.g., the mucosal epithelium,
liver, CNS tissue or fluid, or blood plasma). Alternatively, the
transport-enhancing agents may be employed in a combinatorial
formulation or coordinate administration protocol to directly
enhance mucosal delivery of one or more of the insulin proteins,
analogs and mimetics, with or without enhanced delivery of an
additional biologically active agent.
[0104] Exemplary selective transport-enhancing agents for use
within this aspect of this disclosure include, but are not limited
to, glycosides, sugar-containing molecules, and binding agents such
as lectin binding agents, which are known to interact specifically
with epithelial transport barrier components. For example, specific
"bioadhesive" ligands, including various plant and bacterial
lectins, which bind to cell surface sugar moieties by
receptor-mediated interactions can be employed as carriers or
conjugated transport mediators for enhancing mucosall e.g., nasal
delivery of biologically active agents within this disclosure.
Certain bioadhesive ligands within this disclosure will mediate
transmission of biological signals to epithelial target cells that
trigger selective uptake of the adhesive ligand by specialized
cellular transport processes (endocytosis or transcytosis). These
transport mediators can therefore be employed as a "carrier system"
to stimulate or direct selective uptake of one or more insulin
proteins, analogs and mimetics, and other biologically active
agent(s) into and/or through mucosal epithelia. These and other
selective transport-enhancing agents significantly enhance mucosal
delivery of macromolecular biopharmaceuticals (particularly
peptides, proteins, oligonucleotides and polynucleotide vectors)
within this disclosure. Lectins are plant proteins that bind to
specific sugars found on the surface of glycoproteins and
glycolipids of eukaryotic cells. Concentrated solutions of lectins
have a `mucotractive` effect, and various studies have demonstrated
rapid receptor mediated endocytocis (RME) of lectins and lectin
conjugates (e.g., concanavalin A conjugated with colloidal gold
particles) across mucosal surfaces. Additional studies have
reported that the uptake mechanisms for lectins can be utilized for
intestinal drug targeting in vivo. In certain of these studies,
polystyrene nanoparticles (500 nm) were covalently coupled to
tomato lectin and reported yielded improved systemic uptake after
oral administration to rats.
[0105] In addition to plant lectins, microbial adhesion and
invasion factors provide a rich source of candidates for use as
adhesive/selective transport carriers within the mucosal delivery
methods and compositions of this disclosure. Two components are
necessary for bacterial adherence processes, a bacterial `adhesin`
(adherence or colonization factor) and a receptor on the host cell
surface. Bacteria causing mucosal infections need to penetrate the
mucus layer before attaching themselves to the epithelial surface.
This attachment is usually mediated by bacterial fimbriae or pilus
structures, although other cell surface components may also take
part in the process. Adherent bacteria colonize mucosal epithelia
by multiplication and initiation of a series of biochemical
reactions inside the target cell through signal transduction
mechanisms (with or without the help of toxins). Associated with
these invasive mechanisms, a wide diversity of bioadhesive proteins
(e.g., invasin, internalin) originally produced by various bacteria
and viruses are known. These allow for extracellular attachment of
such microorganisms with an impressive selectivity for host species
and even particular target tissues. Signals transmitted by such
receptor-ligand interactions trigger the transport of intact,
living microorganisms into, and eventually through, epithelial
cells by endo- and transcytotic processes. Such naturally occurring
phenomena may be harnessed (e.g., by complexing biologically active
agents such as insulin with an adhesin) according to the teachings
herein for enhanced delivery of biologically active compounds into
or across mucosal epithelia and/or to other designated target sites
of drug action.
[0106] Various bacterial and plant toxins that bind epithelial
surfaces in a specific, lectin-like manner are also useful within
the methods and compositions of this disclosure. For example,
diptheria toxin (DT) enters host cells rapidly by RME. Likewise,
the B subunit of the E. coli heat labile toxin binds to the brush
border of intestinal epithelial cells in a highly specific,
lectin-like manner. Uptake of this toxin and transcytosis to the
basolateral side of the enterocytes has been reported in vivo and
in vitro. Other researches have expressed the transmembrane domain
of diphtheria toxin in E. coli as a maltose-binding fusion protein
and coupled it chemically to high-Mw poly-L-lysine. The resulting
complex is successfully used to mediate internalization of a
reporter gene in vitro. In addition to these examples,
Staphylococcus aureus produces a set of proteins (e.g.,
staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin
1 (TSST-1) which act both as superantigens and toxins. Studies
relating to these proteins have reported dose-dependent,
facilitated transcytosis of SEB and TSST-1 in Caco-2 cells.
[0107] Viral haemagglutinins comprise another type of transport
agent to facilitate mucosal delivery of biologically active agents
within the methods and compositions of this disclosure. The initial
step in many viral infections is the binding of surface proteins
(haemagglutinins) to mucosal cells. These binding proteins have
been identified for most viruses, including rotaviruses, varicella
zoster virus, semliki forest virus, adenoviruses, potato leafroll
virus, and reovirus. These and other exemplary viral hemagglutinins
can be employed in a combinatorial formulation (e.g., a mixture or
conjugate formulation) or coordinate administration protocol with
one or more of the insulin, analogs and mimetics disclosed herein,
to coordinately enhance mucosal delivery of one or more additional
biologically active agent(s). Alternatively, viral hemagglutinins
can be employed in a combinatorial formulation or coordinate
administration protocol to directly enhance mucosal delivery of one
or more of the insulin proteins, analogs and mimetics, with or
without enhanced delivery of an additional biologically active
agent.
[0108] A variety of endogenous, selective transport-mediating
factors are also available for use within this disclosure.
Mammalian cells have developed an assortment of mechanisms to
facilitate the internalization of specific substrates and target
these to defined compartments. Collectively, these processes of
membrane deformations are termed `endocytosis` and comprise
phagocytosis, pinocytosis, receptor-mediated endocytosis
(clathrin-mediated RME), and potocytosis (non-clathrin-mediated
RME). RME is a highly specific cellular biologic process by which,
as its name implies, various ligands bind to cell surface receptors
and are subsequently internalized and trafficked within the cell.
In many cells the process of endocytosis is so active that the
entire membrane surface is internalized and replaced in less than a
half hour. Two classes of receptors are proposed based on their
orientation in the cell membrane; the amino terminus of Type I
receptors is located on the extracellular side of the membrane,
whereas Type II receptors have this same protein tail in the
intracellular milieu.
[0109] Still other embodiments of this disclosure utilize
transferrin as a carrier or stimulant of RME of mucosally delivered
biologically active agents. Transferrin, an 80 kDa
iron-transporting glycoprotein, is efficiently taken up into cells
by RME. Transferrin receptors are found on the surface of most
proliferating cells, in elevated numbers on erythroblasts and on
many kinds of tumors. The transcytosis of transferrin (Tf) and
transferrin conjugates is reportedly enhanced in the presence of
Brefeldin A (BFA), a fungal metabolite. In other studies, BFA
treatment has been reported to rapidly increase apical endocytosis
of both ricin and HRP in MDCK cells. Thus, BFA and other agents
that stimulate receptor-mediated transport can be employed within
the methods of this disclosure as combinatorially formulated (e.g.,
conjugated) and/or coordinately administered agents to enhance
receptor-mediated transport of biologically active agents,
including insulin proteins, analogs and mimetics.
Polymeric Delivery Vehicles and Methods
[0110] Within certain aspects of the disclosure, insulin proteins,
analogs and mimetics, other biologically active agents disclosed
herein, and delivery-enhancing agents as described herein, are,
individually or combinatorially, incorporated within a mucosally
(e.g., nasally) administered formulation that includes a
biocompatible polymer functioning as a carrier or base. Such
polymer carriers include polymeric powders, matrices or
microparticulate delivery vehicles, among other polymer forms. The
polymer can be of plant, animal, or synthetic origin. Often the
polymer is crosslinked. Additionally, in these delivery systems the
insulin, analog or mimetic, can be functionalized in a manner where
it can be covalently bound to the polymer and rendered inseparable
from the polymer by simple ishing. In other embodiments, the
polymer is chemically modified with an inhibitor of enzymes or
other agents which may degrade or inactivate the biologically
active agent(s) and/or delivery enhancing agent(s). In certain
formulations, the polymer is a partially or completely water
insoluble but water swellable polymer, e.g., a hydrogel. Polymers
in this aspect of this disclosure are desirably water interactive
and/or hydrophilic in nature to absorb significant quantities of
water, and they often form hydrogels when placed in contact with
water or aqueous media for a period of time sufficient to reach
equilibrium with water. In more detailed embodiments, the polymer
is a hydrogel which, when placed in contact with excess water,
absorbs at least two times its weight of water at equilibrium when
exposed to water at room temperature, U.S. Pat. No. 6,004,583.
[0111] Drug delivery systems based on biodegradable polymers are
preferred in many biomedical applications because such systems are
broken down either by hydrolysis or by enzymatic reaction into
non-toxic molecules. The rate of degradation is controlled by
manipulating the composition of the biodegradable polymer matrix.
These types of systems can therefore be employed in certain
settings for long-term release of biologically active agents.
Biodegradable polymers such as poly(glycolic acid) (PGA),
poly-(lactic acid) (PLA), and poly(D,L-lactic-co-glycolic acid)
(PLGA), have received considerable attention as possible drug
delivery carriers, since the degradation products of these polymers
have been found to have low toxicity. During the normal metabolic
function of the body these polymers degrade into carbon dioxide and
water. These polymers have also exhibited excellent
biocompatibility.
[0112] For prolonging the biological activity of insulin, analogs
and mimetics, and other biologically active agents disclosed
herein, as well as optional delivery-enhancing agents, these agents
may be incorporated into polymeric matrices, e.g., polyorthoesters,
polyanhydrides, or polyesters. This yields sustained activity and
release of the active agent(s), e.g., as determined by the
degradation of the polymer matrix. Although the encapsulation of
biotherapeutic molecules inside synthetic polymers may stabilize
them during storage and delivery, the largest obstacle of
polymer-based release technology is the activity loss of the
therapeutic molecules during the formulation processes that often
involve heat, sonication or organic solvents.
[0113] Absorption-promoting polymers contemplated for use within
this disclosure may include derivatives and chemically or
physically modified versions of the foregoing types of polymers, in
addition to other naturally occurring or synthetic polymers, gums,
resins, and other agents, as well as blends of these materials with
each other or other polymers, so long as the alterations,
modifications or blending do not adversely affect the desired
properties, such as water absorption, hydrogel formation, and/or
chemical stability for useful application. In more detailed aspects
of this disclosure, polymers such as nylon, acrylan and other
normally hydrophobic synthetic polymers may be sufficiently
modified by reaction to become water swellable and/or form stable
gels in aqueous media.
[0114] Absorption-promoting polymers of this disclosure may include
polymers from the group of homo- and copolymers based on various
combinations of the following vinyl monomers: acrylic and
methacrylic acids, acrylamide, methacrylamide, hydroxyethylacrylate
or methacrylate, vinylpyrrolidones, as well as polyvinylalcohol and
its co- and terpolymers, polyvinylacetate, its co- and terpolymers
with the above listed monomers and
2-acrylamido-2-methyl-propanesulfonic acid (AMPS.RTM.). Very useful
are copolymers of the above listed monomers with copolymerizable
functional monomers such as acryl or methacryl amide acrylate or
methacrylate esters where the ester groups are derived from
straight or branched chain alkyl, aryl having up to four aromatic
rings which may contain alkyl substituents of 1 to 6 carbons;
steroidal, sulfates, phosphates or cationic monomers such as
N,N-dimethylaminoalkyl(meth)acrylamide,
dimethylaminoalkyl(meth)acrylate,
(meth)acryloxyalkyltrimethylammonium chloride,
(meth)acryloxyalkyldimethylbenzyl ammonium chloride.
[0115] Additional absorption-promoting polymers within this
disclosure are those classified as dextrans, dextrins, and from the
class of materials classified as natural gums and resins, or from
the class of natural polymers such as processed collagen, chitin,
chitosan, pullalan, zooglan, alginates and modified alginates such
as "Kelcoloid" (a polypropylene glycol modified alginate) gellan
gums such as "Kelocogel," Xanathan gums such as "Keltrol,"
estastin, alpha hydroxy butyrate and its copolymers, hyaluronic
acid and its derivatives, polylactic and glycolic acids.
[0116] A very useful class of polymers applicable within the
instant disclosure are olefinically-unsaturated carboxylic acids
containing at least one activated carbon-to-carbon olefinic double
bond, and at least one carboxyl group; that is, an acid or
functional group readily converted to an acid containing an
olefinic double bond which readily functions in polymerization
because of its presence in the monomer molecule, either in the
alpha-beta position with respect to a carboxyl group, or as part of
a terminal methylene grouping. Olefinically-unsaturated acids of
this class include such materials as the acrylic acids typified by
the acrylic acid itself, alpha-cyano acrylic acid, beta
methylacrylic acid (crotonic acid), alpha-phenyl acrylic acid,
beta-acryloxy propionic acid, cinnamic acid, p-chloro cinnamic
acid, 1-carboxy-4-phenyl butadiene-1,3, itaconic acid, citraconic
acid, mesaconic acid, glutaconic acid, aconitic acid, maleic acid,
fumaric acid, and tricarboxy ethylene. As used herein, the term
"carboxylic acid" includes the polycarboxylic acids and those acid
anhydrides, such as maleic anhydride, wherein the anhydride group
is formed by the elimination of one molecule of water from two
carboxyl groups located on the same carboxylic acid molecule.
[0117] Representative acrylates useful as absorption-promoting
agents within this disclosure include methyl acrylate, ethyl
acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate,
isobutyl acrylate, methyl methacrylate, methyl ethacrylate, ethyl
methacrylate, octyl acrylate, heptyl acrylate, octyl methacrylate,
isopropyl methacrylate, 2-ethylhexyl methacrylate, nonyl acrylate,
hexyl acrylate, n-hexyl methacrylate, and the like. Higher alkyl
acrylic esters are decyl acrylate, isodecyl methacrylate, lauryl
acrylate, stearyl acrylate, behenyl acrylate and melissyl acrylate
and methacrylate versions thereof Mixtures of two or three or more
long chain acrylic esters may be successfully polymerized with one
of the carboxylic monomers. Other comonomers include olefins,
including alpha olefins, vinyl ethers, vinyl esters, and mixtures
thereof.
[0118] Other vinylidene monomers, including the acrylic nitriles,
may also be used as absorption-promoting agents within the methods
and compositions of this disclosure to enhance delivery and
absorption of one or more insulin proteins, analogs and mimetics,
and other biologically active agent(s), including to enhance
delivery of the active agent(s) to a target tissue or compartment
in the subject (e.g., the liver, hepatic portal vein, CNS tissue or
fluid, or blood plasma). Useful alpha, beta-olefinically
unsaturated nitriles are preferably monoolefinically unsaturated
nitriles having from 3 to 10 carbon atoms such as acrylonitrile,
methacrylonitrile, and the like. Most preferred are acrylonitrile
and methacrylonitrile. Acrylic amides containing from 3 to 35
carbon atoms including monoolefinically unsaturated amides also may
be used. Representative amides include acrylamide, methacrylamide,
N-t-butyl acrylamide, N-cyclohexyl acrylamide, higher alkyl amides,
where the alkyl group on the nitrogen contains from 8 to 32 carbon
atoms, acrylic amides including N-alkylol amides of alpha,
beta-olefinically unsaturated carboxylic acids including those
having from 4 to 10 carbon atoms such as N-methylol acrylamide,
N-propanol acrylamide, N-methylol methacrylamide, N-methylol
maleimide, N-methylol maleamic acid esters, N-methylol-p-vinyl
benzamide, and the like.
[0119] Yet additional useful absorption promoting materials are
alpha-olefins containing from 2 to 18 carbon atoms, more preferably
from 2 to 8 carbon atoms; dienes containing from 4 to 10 carbon
atoms; vinyl esters and allyl esters such as vinyl acetate; vinyl
aromatics such as styrene, methyl styrene and chloro-styrene; vinyl
and allyl ethers and ketones such as vinyl methyl ether and methyl
vinyl ketone; chloroacrylates; cyanoalkyl acrylates such as
alpha-cyanomethyl acrylate, and the alpha-, beta-, and
gamma-cyanopropyl acrylates; alkoxyacrylates such as methoxy ethyl
acrylate; haloacrylates as chloroethyl acrylate; vinyl halides and
vinyl chloride, vinylidene chloride and the like; divinyls,
diacrylates and other polyfunctional monomers such as divinyl
ether, diethylene glycol diacrylate, ethylene glycol
dimethacrylate, methylene-bis-acrylamide, allylpentaerythritol, and
the like; and bis (beta-haloalkyl) alkenyl phosphonates such as
bis(beta-chloroethyl) vinyl phosphonate and the like as are known
to those skilled in the art. Copolymers wherein the carboxy
containing monomer is a minor constituent, and the other vinylidene
monomers present as major components are readily prepared in
accordance with the methods disclosed herein.
[0120] When hydrogels are employed as absorption promoting agents
within this disclosure, these may be composed of synthetic
copolymers from the group of acrylic and methacrylic acids,
acrylamide, methacrylamide, hydroxyethylacrylate (HEA) or
methacrylate (HEMA), and vinylpyrrolidones which are water
interactive and swellable. Specific illustrative examples of useful
polymers, especially for the delivery of peptides or proteins, are
the following types of polymers: (meth)acrylamide and 0.1 to 99 wt.
% (meth)acrylic acid; (meth)acrylamides and 0.1-75 wt %
(meth)acryloxyethyl trimethyammonium chloride; (meth)acrylamide and
0.1-75 wt % (meth)acrylamide; acrylic acid and 0.1-75 wt %
alkyl(meth)acrylates; (meth)acrylamide and 0.1-75 wt % AMPS.RTM.
(trademark of Lubrizol Corp.); (meth)acrylamide and 0 to 30 wt %
alkyl(meth)acrylamides and 0.1-75 wt % AMPS.RTM.; (meth)acrylamide
and 0.1-99 wt % HEMA; (metb)acrylamide and 0.1 to 75 wt % HEMA and
0.1 to 99%(meth)acrylic acid; (meth)acrylic acid and 0.1-99 wt %
HEMA; 50 mole % vinyl ether and 50 mole % maleic anhydride;
(meth)acrylamide and 0.1 to 75 wt % (meth)acryloxyalky dimethyl
benzylammonium chloride; (meth)acrylamide and 0.1 to 99 wt % vinyl
pyrrolidone; (meth)acrylamide and 50 wt % vinyl pyrrolidone and
0.1-99.9 wt % (meth)acrylic acid; (meth)acrylic acid and 0.1 to 75
wt % AMPS.RTM. and 0.1-75 wt % alkyl(meth)acrylamide. In the above
examples, alkyl means C.sub.1 to C.sub.30, preferably C.sub.1 to
C.sub.22, linear and branched and C.sub.4 to C.sub.16 cyclic; where
(meth) is used, it means that the monomers with and without the
methyl group are included. Other very useful hydrogel polymers are
swellable, but insoluble versions of poly(vinyl pyrrolidone)
starch, carboxymethyl cellulose and polyvinyl alcohol.
[0121] Additional polymeric hydrogel materials within this
disclosure include (poly) hydroxyalkyl (meth)acrylate: anionic and
cationic hydrogels: poly(electrolyte) complexes; poly(vinyl
alcohols) having a low acetate residual: a swellable mixture of
crosslinked agar and crosslinked carboxymethyl cellulose: a
swellable composition comprising methyl cellulose mixed with a
sparingly crosslinked agar; a water swellable copolymer produced by
a dispersion of finely divided copolymer of maleic anhydride with
styrene, ethylene, propylene, or isobutylene; a water swellable
polymer of N-vinyl lactams; swellable sodium salts of carboxymethyl
cellulose; and the like.
[0122] Other gelable, fluid imbibing and retaining polymers useful
for forming the hydrophilic hydrogel for mucosal delivery of
biologically active agents include pectin; polysaccharides such as
agar, acacia, karaya, tragacenth, algins and guar and their
crosslinked versions; acrylic acid polymers, copolymers and salt
derivatives, polyacrylamides; water swellable indene maleic
anhydride polymers; starch graft copolymers; acrylate type polymers
and copolymers with water absorbability of about 2 to 400 times its
original weight; diesters of polyglucan; a mixture of crosslinked
poly(vinyl alcohol) and poly(N-vinyl-2-pyrrolidone);
polyoxybutylene-polyethylene block copolymer gels; carob gum;
polyester gels; poly urea gels; polyether gels; polyamide gels;
polyimide gels; polypeptide gels; polyamino acid gels; poly
cellulosic gels; crosslinked indene-maleic anhydride acrylate
polymers; and polysaccharides.
[0123] Synthetic hydrogel polymers for use within this disclosure
may be made by an infinite combination of several monomers in
several ratios. The hydrogel can be crosslinked and generally
possesses the ability to imbibe and absorb fluid and swell or
expand to an enlarged equilibrium state. The hydrogel typically
swells or expands upon delivery to the nasal mucosal surface,
absorbing about 2-5, 5-10, 10-50, up to 50-100 or more times fold
its weight of water. The optimum degree of swellability for a given
hydrogel will be determined for different biologically active
agents depending upon such factors as molecular weight, size,
solubility and diffusion characteristics of the active agent
carried by or entrapped or encapsulated within the polymer, and the
specific spacing and cooperative chain motion associated with each
individual polymer.
[0124] Hydrophilic polymers within this disclosure are water
insoluble but water swellable. Such water-swollen polymers as
typically referred to as hydrogels or gels. Such gels may be
conveniently produced from water-soluble polymer by the process of
cross-linking the polymers by a suitable cross-linking agent.
However, stable hydrogels may also be formed from specific polymers
under defined conditions of pH, temperature and/or ionic
concentration, according to know methods in the art. Typically the
polymers are cross-linked, that is, cross-linked to the extent that
the polymers possess good hydrophilic properties, have improved
physical integrity (as compared to non cross-linked polymers of the
same or similar type) and exhibit improved ability to retain within
the gel network both the biologically active agent of interest and
additional compounds for coadministration therewith such as a
cytokine or enzyme inhibitor, while retaining the ability to
release the active agent(s) at the appropriate location and
time.
[0125] Generally hydrogel polymers within this disclosure are
cross-linked with a difunctional cross-linking in the amount of
from 0.01 to 25 weight percent, based on the weight of the monomers
forming the copolymer, and more preferably from 0.1 to 20 weight
percent and more often from 0.1 to 15 weight percent of the
cross-linking agent. Another useful amount of a cross-linking agent
is 0.1 to 10 weight percent. Tri, tetra or higher multifunctional
cross-linking agents may also be employed. When such reagents are
utilized, lower amounts may be required to attain equivalent
crosslinking density, i.e., the degree of cross-linking, or network
properties that are sufficient to contain effectively the
biologically active agent(s).
[0126] The cross-links can be covalent, ionic or hydrogen bonds
with the polymer possessing the ability to swell in the presence of
water containing fluids. Such crosslinkers and cross-linking
reactions are known to those skilled in the art and in many cases
are dependent upon the polymer system. Thus a crosslinked network
may be formed by free radical copolymerization of unsaturated
monomers. Polymeric hydrogels may also be formed by cross-linking
preformed polymers by reacting functional groups found on the
polymers such as alcohols, acids, amines with such groups as
glyoxal, formaldehyde or glutaraldehyde, bis anhydrides and the
like.
[0127] The polymers also may be cross-linked with any polyene,
e.g., decadiene or trivinyl cyclohexane; acrylamides, such as
N,N-methylene-bis (acrylamide); polyfunctional acrylates, such as
trimethylol propane triacrylate; or polyfunctional vinylidene
monomer containing at least 2 terminal CH.sub.2<groups,
including, for example, divinyl benzene, divinyl naphthlene, allyl
acrylates and the like. In certain embodiments, cross-linking
monomers for use in preparing the copolymers are polyalkenyl
polyethers having more than one alkenyl ether grouping per
molecule, which may optionally possess alkenyl groups in which an
olefinic double bond is present attached to a terminal methylene
grouping (e.g., made by the etherification of a polyhydric alcohol
containing at least 2 carbon atoms and at least 2 hydroxyl groups).
Compounds of this class may be produced by reacting an alkenyl
halide, such as allyl chloride or allyl bromide, with a strongly
alkaline aqueous solution of one or more polyhydric alcohols. The
product may be a complex mixture of polyethers with varying numbers
of ether groups. Efficiency of the polyether cross-linking agent
increases with the number of potentially polymerizable groups on
the molecule. Typically, polyethers containing an average of two or
more alkenyl ether groupings per molecule are used. Other
cross-linking monomers include for example, diallyl esters,
dimethallyl ethers, allyl or methallyl acrylates and acrylamides,
tetravinyl silane, polyalkenyl methanes, diacrylates, and
dimethacrylates, divinyl compounds such as divinyl benzene,
polyallyl phosphate, diallyloxy compounds and phosphite esters and
the like. Typical agents are allyl pentaerythritol, allyl sucrose,
trimethylolpropane triacrylate, 1,6-hexanediol diacrylate,
trimethylolpropane diallyl ether, pentaerythritol triacrylate,
tetramethylene dimethacrylate, ethylene diacrylate, ethylene
dimethacrylate, triethylene glycol dimethacrylate, and the like.
Allyl pentaerythritol, trimethylolpropane diallylether and allyl
sucrose provide suitable polymers. When the cross-linking agent is
present, the polymeric mixtures usually contain from about 0.01 to
about 20 weight percent, e.g., 1%, 5%, or 10% or more by weight of
cross-linking monomer based on the total of carboxylic acid
monomer, plus other monomers.
[0128] In more detailed aspects of this disclosure, mucosal
delivery of insulin, analogs and mimetics, and other biologically
active agents disclosed herein, is enhanced by retaining the active
agent(s) in a slow-release or enzymatically or physiologically
protective carrier or vehicle, for example a hydrogel that shields
the active agent from the action of the degradative enzymes. In
certain embodiments, the active agent is bound by chemical means to
the carrier or vehicle, to which may also be admixed or bound
additional agents such as enzyme inhibitors, cytokines, etc. The
active agent may alternately be immobilized through sufficient
physical entrapment within the carrier or vehicle, e.g., a polymer
matrix.
[0129] Polymers such as hydrogels within this disclosure may
incorporate functional linked agents such as glycosides chemically
incorporated into the polymer for enhancing intranasal
bioavailability of active agents formulated therewith. Examples of
such glycosides are glucosides, fructosides, galactosides,
arabinosides, mannosides and their alkyl substituted derivatives
and natural glycosides such as arbutin, phlorizin, amygdalin,
digitonin, saponin, and indican. There are several ways in which a
typical glycoside may be bound to a polymer. For example, the
hydrogen of the hydroxyl groups of a glycoside or other similar
carbohydrate may be replaced by the alkyl group from a hydrogel
polymer to form an ether. Also, the hydroxyl groups of the
glycosides may be reacted to esterify the carboxyl groups of a
polymeric hydrogel to form polymeric esters in situ. Another
approach is to employ condensation of acetobromoglucose with
cholest-5-en-3beta-ol on a copolymer of maleic acid. N-substituted
polyacrylamides can be synthesized by the reaction of activated
polymers with omega-aminoalkylglycosides: (1)
(carbohydrate-spacer)(n)-polyacrylamide, `pseudopolysaccharides`;
(2) (carbohydrate
spacer)(n)-phosphatidylethanolamine(m)-polyacrylamide,
neoglycolipids, derivatives of phosphatidylethanolamine; and (3)
(carbohydrate-spacer)(n)-biotin(m)-polyacrylamide. These
biotinylated derivatives may attach to lectins on the mucosal
surface to facilitate absorption of the biologically active
agent(s), e.g., a polymer-encapsulated insulin.
[0130] Within more detailed aspects of this disclosure, one or more
insulin, analogs and mimetics, and/or other biologically active
agents, disclosed herein, optionally including secondary active
agents such as protease inhibitor(s), cytokine(s), additional
modulator(s) of intercellular junctional physiology, etc., are
modified and bound to a polymeric carrier or matrix. For example,
this may be accomplished by chemically binding a peptide or protein
active agent and other optional agent(s) within a crosslinked
polymer network. It is also possible to chemically modify the
polymer separately with an interactive agent such as a glycosidal
containing molecule. In certain aspects, the biologically active
agent(s), and optional secondary active agent(s), may be
functionalized, i.e., wherein an appropriate reactive group is
identified or is chemically added to the active agent(s). Most
often an ethylenic polymerizable group is added, and the
functionalized active agent is then copolymerized with monomers and
a crosslinking agent using a standard polymerization method such as
solution polymerization (usually in water), emulsion, suspension or
dispersion polymerization. Often, the functionalizing agent is
provided with a high enough concentration of functional or
polymerizable groups to insure that several sites on the active
agent(s) are functionalized. For example, in a polypeptide
comprising 16 amine sites, it is generally desired to functionalize
at least 2, 4, 5, 7, and up to 8 or more of the sites.
[0131] After functionalization, the functionalized active agent(s)
is/are mixed with monomers and a crosslinking agent that comprise
the reagents from which the polymer of interest is formed.
Polymerization is then induced in this medium to create a polymer
containing the bound active agent(s). The polymer is then washed
with water or other appropriate solvents and otherwise purified to
remove trace unreacted impurities and, if necessary, ground or
broken up by physical means such as by stirring, forcing it through
a mesh, ultrasonication or other suitable means to a desired
particle size. The solvent, usually water, is then removed in such
a manner as to not denature or otherwise degrade the active
agent(s). One desired method is lyophilization (freeze drying) but
other methods are available and may be used (e.g., vacuum drying,
air drying, spray drying, etc.).
[0132] To introduce polymerizable groups in peptides, proteins and
other active agents within this disclosure, it is possible to react
available amino, hydroxyl, thiol and other reactive groups with
electrophiles containing unsaturated groups. For example,
unsaturated monomers containing N-hydroxy succinimidyl groups,
active carbonates such as p-nitrophenyl carbonate, trichlorophenyl
carbonates, tresylate, oxycarbonylimidazoles, epoxide, isocyanates
and aldehyde, and unsaturated carboxymethyl azides and unsaturated
orthopyridyl-disulfide belong to this category of reagents.
Illustrative examples of unsaturated reagents are allyl glycidyl
ether, allyl chloride, allylbromide, allyl iodide, acryloyl
chloride, allyl isocyanate, allylsulfonyl chloride, maleic
anhydride, copolymers of maleic anhydride and allyl ether, and the
like.
[0133] All of the lysine active derivatives, except aldehyde, can
generally react with other amino acids such as imidazole groups of
histidine and hydroxyl groups of tyrosine and the thiol groups of
cystine if the local environment enhances nucleophilicity of these
groups. Aldehyde containing functionalizing reagents are specific
to lysine. These types of reactions with available groups from
lysines, cysteines, tyrosine have been extensively documented in
the literature and are known to those skilled in the art.
[0134] In the case of biologically active agents that contain amine
groups, it is convenient to react such groups with an acyloyl
chloride, such as acryloyl chloride, and introduce the
polymerizable acrylic group onto the reacted agent. Then during
preparation of the polymer, such as during the crosslinking of the
copolymer of acrylamide and acrylic acid, the functionalized active
agent, through the acrylic groups, is attached to the polymer and
becomes bound thereto.
[0135] In additional aspects of this disclosure, biologically
active agents, including peptides, proteins, nucleosides, and other
molecules which are bioactive in vivo, are conjugation-stabilized
by covalently bonding one or more active agent(s) to a polymer
incorporating as an integral part thereof both a hydrophilic
moiety, e.g., a linear polyalkylene glycol, a lipophilic moiety
(see, e.g., U.S. Pat. No. 5,681,811). In one aspect, a biologically
active agent is covalently coupled with a polymer comprising (i) a
linear polyalkylene glycol moiety, and (ii) a lipophilic moiety,
wherein the active agent, linear polyalkylene glycol moiety, and
the lipophilic moiety are conformationally arranged in relation to
one another such that the active therapeutic agent has an enhanced
in vivo resistance to enzymatic degradation (i.e., relative to its
stability under similar conditions in an unconjugated form devoid
of the polymer coupled thereto). In another aspect, the
conjugation-stabilized formulation has a three-dimensional
conformation comprising the biologically active agent covalently
coupled with a polysorbate complex comprising (i) a linear
polyalkylene glycol moiety, and (ii) a lipophilic moiety, wherein
the active agent, the linear polyalkylene glycol moiety and the
lipophilic moiety are conformationally arranged in relation to one
another such that (a) the lipophilic moiety is exteriorly available
in the three-dimensional conformation, and (b) the active agent in
the composition has an enhanced in vivo resistance to enzymatic
degradation.
[0136] In a further related aspect, a multiligand conjugated
complex is provided which comprises a biologically active agent
covalently coupled with a triglyceride backbone moiety through a
polyalkylene glycol spacer group bonded at a carbon atom of the
triglyceride backbone moiety, and at least one fatty acid moiety
covalently attached either directly to a carbon atom of the
triglyceride backbone moiety or covalently joined through a
polyalkylene glycol spacer moiety (see, e.g., U.S. Pat. No.
5,681,811). In such a multiligand conjugated therapeutic agent
complex, the alpha' and beta carbon atoms of the triglyceride
bioactive moiety may have fatty acid moieties attached by
covalently bonding either directly thereto, or indirectly
covalently bonded thereto through polyalkylene glycol spacer
moieties. Alternatively, a fatty acid moiety may be covalently
attached either directly or through a polyalkylene glycol spacer
moiety to the alpha and alpha' carbons of the triglyceride backbone
moiety, with the bioactive therapeutic agent being covalently
coupled with the gamma-carbon of the triglyceride backbone moiety,
either being directly covalently bonded thereto or indirectly
bonded thereto through a polyalkylene spacer moiety. It will be
recognized that a wide variety of structural, compositional, and
conformational forms are possible for the multiligand conjugated
therapeutic agent complex comprising the triglyceride backbone
moiety, within the scope of this disclosure. It is further noted
that in such a multiligand conjugated therapeutic agent complex,
the biologically active agent(s) may advantageously be covalently
coupled with the triglyceride modified backbone moiety through
alkyl spacer groups, or alternatively other acceptable spacer
groups, within the scope of this disclosure. As used in such
context, acceptability of the spacer group refers to steric,
compositional, and end use application specific acceptability
characteristics.
[0137] In yet additional aspects of this disclosure, a
conjugation-stabilized complex is provided which comprises a
polysorbate complex comprising a polysorbate moiety including a
triglyceride backbone having covalently coupled to alpha, alpha'
and beta carbon atoms thereof functionalizing groups including (i)
a fatty acid group; and (ii) a polyethylene glycol group having a
biologically active agent or moiety covalently bonded thereto,
e.g., bonded to an appropriate functionality of the polyethylene
glycol group. Such covalent bonding may be either direct, e.g., to
a hydroxy terminal functionality of the polyethylene glycol group,
or alternatively, the covalent bonding may be indirect, e.g., by
reactively capping the hydroxy terminus of the polyethylene glycol
group with a terminal carboxy functionality spacer group, so that
the resulting capped polyethylene glycol group has a terminal
carboxy functionality to which the biologically active agent or
moiety may be covalently bonded.
[0138] In yet additional aspects of this disclosure, a stable,
aqueously soluble, conjugation-stabilized complex is provided which
comprises one or more insulin proteins, analogs and mimetics,
and/or other biologically active agent(s) disclosed herein
covalently coupled to a physiologically compatible polyethylene
glycol (PEG) modified glycolipid moiety. In such complex, the
biologically active agent(s) may be covalently coupled to the
physiologically compatible PEG modified glycolipid moiety by a
labile covalent bond at a free amino acid group of the active
agent, wherein the labile covalent bond is scissionable in vivo by
biochemical hydrolysis and/or proteolysis. The physiologically
compatible PEG modified glycolipid moiety may advantageously
comprise a polysorbate polymer, e.g., a polysorbate polymer
comprising fatty acid ester groups selected from the group
consisting of monopalmitate, dipalmitate, monolaurate, dilaurate,
trilaurate, monoleate, dioleate, trioleate, monostearate,
distearate, and tristearate. In such complex, the physiologically
compatible PEG modified glycolipid moiety may suitably comprise a
polymer selected from the group consisting of polyethylene glycol
ethers of fatty acids, and polyethylene glycol esters of fatty
acids, wherein the fatty acids for example comprise a fatty acid
selected from the group consisting of lauric, palmitic, oleic, and
stearic acids.
Storage of Material
[0139] In certain aspects of this disclosure, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of peptides and proteins which may
adhere to charged glass thereby reducing the effective
concentration in the container. Silanized containers, for example,
silanized glass containers, are used to store the finished product
to reduce adsorption of the polypeptide or protein to a glass
container. In other aspects, non-silanized Type 1 glass containers
may be used herein.
[0140] In yet additional aspects of this disclosure, a kit for
treatment of a mammalian subject comprises a stable pharmaceutical
composition of one or more insulin compound(s) formulated for
mucosal delivery to the mammalian subject wherein the composition
is effective to alleviate one or more symptom(s) of obesity,
cancer, or malnutrition or wasting related to cancer in said
subject without unacceptable adverse side effects. The kit further
comprises a pharmaceutical reagent vial to contain the one or more
insulin compounds. The pharmaceutical reagent vial is composed of
pharmaceutical grade polymer, glass or other suitable material. The
pharmaceutical reagent vial is, for example, a silanized glass
vial. The kit further comprises an aperture for delivery of the
composition to a nasal mucosal surface of the subject. The delivery
aperture is composed of a pharmaceutical grade polymer, glass or
other suitable material. The delivery aperture is, for example, a
silanized glass.
[0141] A silanization technique combines a special cleaning
technique for the surfaces to be silanized with a silanization
process at low pressure. The silane is in the gas phase and at an
enhanced temperature of the surfaces to be silanized. The method
provides reproducible surfaces with stable, homogeneous and
functional silane layers having characteristics of a monolayer. The
silanized surfaces prevent binding to the glass of polypeptides or
mucosal delivery enhancing agents of the present disclosure.
[0142] The procedure is useful to prepare silanized pharmaceutical
reagent vials to hold insulin compositions of the present
disclosure. Glass trays are cleaned by rinsing with double
distilled water (ddH.sub.2O) before using. The silane tray is then
be rinsed with 95% EtOH, and the acetone tray is rinsed with
acetone. Pharmaceutical reagent vials are sonicated in acetone for
10 minutes. After the acetone sonication, reagent vials are washed
in ddH.sub.2O tray at least twice. Reagent vials are sonicated in
0.1M NaOH for 10 minutes. While the reagent vials are sonicating in
NaOH, the silane solution is made under a hood. (Silane solution:
800 mL of 95% ethanol; 96 L of glacial acetic acid; 25 mL of
glycidoxypropyltrimethoxy silane). After the NaOH sonication,
reagent vials are washed in ddH.sub.2O tray at least twice. The
reagent vials are sonicated in silane solution for 3 to 5 minutes.
The reagent vials are ished in 100% EtOH tray. The reagent vials
are dried with prepurified N.sub.2 gas and stored in a 100.degree.
C. oven for at least 2 hours before using.
Bioadhesive Delivery Vehicles and Methods
[0143] In certain aspects of the disclosure, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of a nontoxic bioadhesive as an
adjunct compound or carrier to enhance mucosal delivery of one or
more biologically active agent(s). Bioadhesive agents in this
context exhibit general or specific adhesion to one or more
components or surfaces of the targeted mucosa. The bioadhesive
maintains a desired concentration gradient of the biologically
active agent into or across the mucosa to ensure penetration of
even large molecules (e.g., peptides and proteins) into or through
the mucosal epithelium. Use of a bioadhesive within the methods and
compositions of this disclosure yields from about a two- to about
five-fold, often from about a five- to about a ten-fold increase in
permeability for peptides and proteins into or through the mucosal
epithelium. This enhancement of epithelial permeation often permits
effective transmucosal delivery of large macromolecules, for
example to the basal portion of the nasal epithelium or into the
adjacent extracellular compartments or a blood plasma or CNS tissue
or fluid.
[0144] This enhanced delivery provides for greatly improved
effectiveness of delivery of bioactive peptides, proteins and other
macromolecular therapeutic species. These results will depend in
part on the hydrophilicity of the compound, whereby greater
penetration will be achieved with hydrophilic species compared to
water insoluble compounds. In addition to these effects, employment
of bioadhesives to enhance drug persistence at the mucosal surface
can elicit a reservoir mechanism for protracted drug delivery,
whereby compounds not only penetrate across the mucosal tissue but
also back-diffuse toward the mucosal surface once the material at
the surface is depleted.
[0145] A variety of suitable bioadhesives are disclosed in the art
for oral administration, U.S. Pat. Nos. 3,972,995; 4,259,314;
4,680,323; 4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092;
4,855,142; 4,250,163; 4,226,848; 4,948,580; and U.S. Pat. Reissue
No. 33,093, which find use within the novel methods and
compositions of this disclosure. The potential of various
bioadhesive polymers as a mucosal, e.g., nasal, delivery platform
within the methods and compositions of this disclosure can be
readily assessed by determining their ability to retain and release
insulin, as well as by their capacity to interact with the mucosal
surfaces following incorporation of the active agent therein. In
addition, well known methods are applied to determine the
biocompatibility of selected polymers with the tissue at the site
of mucosal administration. When the target mucosa is covered by
mucus (i.e., in the absence of mucolytic or mucus-clearing
treatment), it can serve as a connecting link to the underlying
mucosal epithelium. Therefore, the term "bioadhesive" as used
herein also covers mucoadhesive compounds useful for enhancing
mucosal delivery of biologically active agents within this
disclosure. However, adhesive contact to mucosal tissue mediated
through adhesion to a mucus gel layer may be limited by incomplete
or transient attachment between the mucus layer and the underlying
tissue, particularly at nasal surfaces where rapid mucus clearance
occurs. In this regard, mucin glycoproteins are continuously
secreted and, immediately after their release from cells or glands,
form a viscoelastic gel. The luminal surface of the adherent gel
layer, however, is continuously eroded by mechanical, enzymatic
and/or ciliary action. Where such activities are more prominent or
where longer adhesion times are desired, the coordinate
administration methods and combinatorial formulation methods of
this disclosure may further incorporate mucolytic and/or
ciliostatic methods or agents as disclosed herein above.
[0146] Typically, mucoadhesive polymers for use within the present
disclosure are natural or synthetic macromolecules which adhere to
wet mucosal tissue surfaces by complex, but non-specific,
mechanisms. In addition to these mucoadhesive polymers, this
disclosure also describes methods and compositions incorporating
bioadhesives that adhere directly to a cell surface, rather than to
mucus, by means of specific, including receptor-mediated,
interactions. One example of bioadhesives that function in this
specific manner is the group of compounds known as lectins. These
are glycoproteins with an ability to specifically recognize and
bind to sugar molecules, e.g., glycoproteins or glycolipids, which
form part of intranasal epithelial cell membranes and can be
considered as "lectin receptors."
[0147] In certain aspects of this disclosure, bioadhesive materials
for enhancing intranasal delivery of biologically active agents
comprise a matrix of a hydrophilic, e.g., water soluble or
swellable, polymer or a mixture of polymers that can adhere to a
wet mucous surface. These adhesives may be formulated as ointments,
hydrogels (see above) thin films, and other application forms.
Often, these adhesives have the biologically active agent mixed
therewith to effectuate slow release or local delivery of the
active agent. Some are formulated with additional ingredients to
facilitate penetration of the active agent through the nasal
mucosa, e.g., into the circulatory system of the individual.
[0148] Various polymers, both natural and synthetic ones, show
significant binding to mucus and/or mucosal epithelial surfaces
under physiological conditions. The strength of this interaction
can readily be measured by mechanical peel or shear tests. When
applied to a humid mucosal surface, many dry materials will
spontaneously adhere, at least slightly. After such an initial
contact, some hydrophilic materials start to attract water by
adsorption, swelling or capillary forces, and if this water is
absorbed from the underlying substrate or from the polymer-tissue
interface, the adhesion may be sufficient to achieve the goal of
enhancing mucosal absorption of biologically active agents. Such
`adhesion by hydration` can be quite strong, but formulations
adapted to employ this mechanism must account for swelling which
continues as the dosage transforms into a hydrated mucilage. This
is projected for many hydrocolloids useful within this disclosure,
especially some cellulose-derivatives, which are generally
non-adhesive when applied in pre-hydrated state. Nevertheless,
bioadhesive drug delivery systems for mucosal administration are
effective within this disclosure when such materials are applied in
the form of a dry polymeric powder, microsphere, or film-type
delivery form.
[0149] Other polymers adhere to mucosal surfaces not only when
applied in dry, but also in fully hydrated state, and in the
presence of excess amounts of water. The selection of a
mucoadhesive thus requires due consideration of the conditions,
physiological as well as physico-chemical, under which the contact
to the tissue will be formed and maintained. In particular, the
amount of water or humidity usually present at the intended site of
adhesion, and the prevailing pH, are known to largely affect the
mucoadhesive binding strength of different polymers.
[0150] Several polymeric bioadhesive drug delivery systems have
been fabricated and studied in the past 20 years, not always with
success. A variety of such carriers are, however, currently used in
clinical applications involving dental, orthopedic,
ophthalmological, and surgical uses. For example, acrylic-based
hydrogels have been used extensively for bioadhesive devices.
Acrylic-based hydrogels are well suited for bioadhesion due to
their flexibility and nonabrasive characteristics in the partially
swollen state, which reduce damage-causing attrition to the tissues
in contact. Furthermore, their high permeability in the swollen
state allows unreacted monomer, un-crosslinked polymer chains, and
the initiator to be ished out of the matrix after polymerization,
which is an important feature for selection of bioadhesive
materials within this disclosure. Acrylic-based polymer devices
exhibit very high adhesive bond strength. For controlled mucosal
delivery of peptide and protein drugs, the methods and compositions
of this disclosure optionally include the use of carriers, e.g.,
polymeric delivery vehicles that function in part to shield the
biologically active agent from proteolytic breakdown, while at the
same time providing for enhanced penetration of the peptide or
protein into or through the nasal mucosa. In this context,
bioadhesive polymers have demonstrated considerable potential for
enhancing oral drug delivery. As an example, the bioavailability of
9-desglycinamide, 8-arginine vasopressin (DGAVP) intraduodenally
administered to rats together with a 1% (w/v) saline dispersion of
the mucoadhesive poly(acrylic acid) derivative polycarbophil, is
3-5-fold increased compared to an aqueous solution of the peptide
drug without this polymer.
[0151] Mucoadhesive polymers of the poly(acrylic acid)-type are
potent inhibitors of some intestinal proteases. The mechanism of
enzyme inhibition is explained by the strong affinity of this class
of polymers for divalent cations, such as calcium or zinc, which
are essential cofactors of metallo-proteinases, such as trypsin and
chymotrypsin. Depriving the proteases of their cofactors by
poly(acrylic acid) is reported to induce irreversible structural
changes of the enzyme proteins which were accompanied by a loss of
enzyme activity. At the same time, other mucoadhesive polymers
(e.g., some cellulose derivatives and chitosan) may not inhibit
proteolytic enzymes under certain conditions. In contrast to other
enzyme inhibitors contemplated within this disclosure (e.g.,
aprotinin, bestatin), which are relatively small molecules, the
trans-nasal absorption of inhibitory polymers is likely to be
minimal in light of the size of these molecules, and thereby
eliminate possible adverse side effects. Thus, mucoadhesive
polymers, particularly of the poly(acrylic acid)-type, may serve
both as an absorption-promoting adhesive and enzyme-protective
agent to enhance controlled delivery of peptide and protein drugs,
especially when safety concerns are considered.
[0152] In addition to protecting against enzymatic degradation,
bioadhesives and other polymeric or non-polymeric
absorption-promoting agents within this disclosure may directly
increase mucosal permeability to biologically active agents. To
facilitate the transport of large and hydrophilic molecules, such
as peptides and proteins, across the nasal epithelial barrier,
mucoadhesive polymers and other agents have been postulated to
yield enhanced permeation effects beyond what is accounted for by
prolonged premucosal residence time of the delivery system. The
time course of drug plasma concentrations reportedly suggested that
the bioadhesive microspheres caused an acute, but transient
increase of insulin permeability across the nasal mucosa. Other
mucoadhesive polymers within this disclosure, for example chitosan,
reportedly enhance the permeability of certain mucosal epithelia
even when they are applied as an aqueous solution or gel. Another
mucoadhesive polymer reported to directly affect epithelial
permeability is hyaluronic acid and ester derivatives thereof. A
particularly useful bioadhesive agent within the coordinate
administration, and/or combinatorial formulation methods and
compositions of this disclosure is chitosan, as well as its analogs
and derivatives. Chitosan is a non-toxic, biocompatible and
biodegradable polymer that is widely used for pharmaceutical and
medical applications because of its favorable properties of low
toxicity and good biocompatibility. It is a natural
polyaminosaccharide prepared from chitin by N-deacetylation with
alkali. As used within the methods and compositions of this
disclosure, chitosan increases the retention (i.e., residence time)
of insulin proteins, analogs and mimetics, and other biologically
active agents disclosed herein at a mucosal site of application.
This mode of administration can also improve patient compliance and
acceptance. As further provided herein, the methods and
compositions of this disclosure will optionally include a novel
chitosan derivative or chemically modified form of chitosan. One
such novel derivative within this disclosure is denoted as a
.beta.-[1.fwdarw.4]-2-guanidino-2-deoxy-D-glucose polymer
(poly-GuD). Chitosan is the N-deacetylated product of chitin, a
naturally occurring polymer that has been used extensively to
prepare microspheres for oral and intra-nasal formulations. The
chitosan polymer has also been proposed as a soluble carrier for
parenteral drug delivery. Within one aspect of this disclosure,
o-methylisourea is used to convert a chitosan amine to its
guanidinium moiety. The guanidinium compound is prepared, for
example, by the reaction between equi-normal solutions of chitosan
and o-methylisourea at pH above 8.0.
[0153] Additional compounds classified as bioadhesive agents within
the present disclosure act by mediating specific interactions,
typically classified as "receptor-ligand interactions" between
complementary structures of the bioadhesive compound and a
component of the mucosal epithelial surface. Many natural examples
illustrate this form of specific binding bioadhesion, as
exemplified by lectin-sugar interactions. Lectins are (glyco)
proteins of non-immune origin which bind to polysaccharides or
glycoconjugates.
[0154] Several plant lectins have been investigated as possible
pharmaceutical absorption-promoting agents. One plant lectin,
Phaseolus vulgaris hemagglutinin (PHA), exhibits high oral
bioavailability of more than 10% after feeding to rats. Tomato
(Lycopersicon esculeutum) lectin (TL) appears safe for various
modes of administration.
[0155] In summary, the bioadhesive agents herein disclosed are
useful in the combinatorial formulations and coordinate
administration methods of the instant disclosure, which optionally
incorporate an effective amount and form of a bioadhesive agent to
prolong persistence or otherwise increase mucosal absorption of one
or more insulin proteins, analogs and mimetics, and other
biologically active agents. The bioadhesive agents may be
coordinately administered as adjunct compounds or as additives
within the combinatorial formulations of this disclosure. In
certain embodiments, the bioadhesive agent acts as a
`pharmaceutical glue,` whereas in other embodiments adjunct
delivery or combinatorial formulation of the bioadhesive agent
serves to intensify contact of the biologically active agent with
the nasal mucosa, in some cases by promoting specific
receptor-ligand interactions with epithelial cell "receptors," and
in others by increasing epithelial permeability to significantly
increase the drug concentration gradient measured at a target site
(e.g., liver, blood plasma, or CNS tissue or fluid). Yet additional
bioadhesive agents within this disclosure act as enzyme (e.g.,
protease) inhibitors to enhance the stability of mucosally
administered biotherapeutic agents delivered coordinately or in a
combinatorial formulation with the bioadhesive agent.
Liposomes and Micellar Delivery Vehicles
[0156] The coordinate administration methods and combinatorial
formulations of the instant disclosure optionally incorporate
effective lipid or fatty acid based carriers, processing agents, or
delivery vehicles, to provide improved formulations for mucosal
delivery of insulin proteins, analogs and mimetics, and other
biologically active agents. For example, a variety of formulations
and methods are provided for mucosal delivery which comprise one or
more of these active agents, such as a peptide or protein, admixed
or encapsulated by, or coordinately administered with, a liposome,
mixed micellar carrier, or emulsion, to enhance chemical and
physical stability and increase the half life of the biologically
active agents (e.g., by reducing susceptibility to proteolysis,
chemical modification and/or denaturation) upon mucosal
delivery.
[0157] Within certain aspects of this disclosure, specialized
delivery systems for biologically active agents comprise small
lipid vesicles known as liposomes. These may be made from natural,
biodegradable, non-toxic, and non-immunogenic lipid molecules, and
can efficiently entrap or bind drug molecules, including peptides
and proteins, into, or onto, their membranes. The attractiveness of
liposomes as a peptide and protein delivery system within this
disclosure is increased by the fact that the encapsulated proteins
can remain in their preferred aqueous environment within the
vesicles, while the liposomal membrane protects them against
proteolysis and other destabilizing factors. Even though not all
liposome preparation methods known are feasible in the
encapsulation of peptides and proteins due to their unique physical
and chemical properties, several methods allow the encapsulation of
these macromolecules without substantial deactivation.
[0158] A variety of methods are available for preparing liposomes
within this disclosure, U.S. Pat. Nos. 4,235,871; 4,501,728; and
4,837,028. For use with liposome delivery, the biologically active
agent is typically entrapped within the liposome, or lipid vesicle,
or is bound to (i.e., associated with) the outside of the
vesicle.
[0159] Like liposomes, unsaturated long chain fatty acids, which
also have enhancing activity for mucosal absorption, can form
closed vesicles with bilayer-like structures (so-called
"ufasomes"). These can be formed, for example, using oleic acid to
entrap biologically active peptides and proteins for mucosal, e.g.,
intranasal, delivery within this disclosure.
[0160] Other delivery systems within this disclosure combine the
use of polymers and liposomes to ally the advantageous properties
of both vehicles such as encapsulation inside the natural polymer
fibrin. In addition, release of biotherapeutic compounds from this
delivery system is controllable through the use of covalent
crosslinking and the addition of antifibrinolytic agents to the
fibrin polymer.
[0161] More simplified delivery systems within this disclosure
include the use of cationic lipids as delivery vehicles or
carriers, which can be effectively employed to provide an
electrostatic interaction between the lipid carrier and such
charged biologically active agents as proteins and polyanionic
nucleic acids. This allows efficient packaging of the drugs into a
form suitable for mucosal administration and/or subsequent delivery
to systemic compartments.
[0162] Additional delivery vehicles within this disclosure include
long and medium chain fatty acids, as well as surfactant mixed
micelles with fatty acids. Most naturally occurring lipids in the
form of esters have important implications with regard to their own
transport across mucosal surfaces. Free fatty acids and their
monoglycerides which have polar groups attached have been
demonstrated in the form of mixed micelles to act on the intestinal
barrier as penetration enhancers. This discovery of barrier
modifying function of free fatty acids (carboxylic acids with a
chain length varying from 12 to 20 carbon atoms) and their polar
derivatives has stimulated extensive research on the application of
these agents as mucosal absorption enhancers.
[0163] For use within the methods of this disclosure, long chain
fatty acids, especially fusogenic lipids (unsaturated fatty acids
and monoglycerides such as oleic acid, linoleic acid, linoleic
acid, monoolein, etc.) provide useful carriers to enhance mucosal
delivery of insulin, analogs and mimetics, and other biologically
active agents disclosed herein. Medium chain fatty acids (C6 to
C12) and monoglycerides have also been shown to have enhancing
activity in intestinal drug absorption and can be adapted for use
within the mocosal delivery formulations and methods of this
disclosure. In addition, sodium salts of medium and long chain
fatty acids are effective delivery vehicles and
absorption-enhancing agents for mucosal delivery of biologically
active agents within this disclosure. Thus, fatty acids can be
employed in soluble forms of sodium salts or by the addition of
non-toxic surfactants, e.g., polyoxyethylated hydrogenated castor
oil, sodium taurocholate, etc. Other fatty acid and mixed micellar
preparations that are within this disclosure include, but are not
limited to, Na caprylate (C8), Na caprate (C 10), Na laurate (C 12)
or Na oleate (C18), optionally combined with bile salts, such as
glycocholate and taurocholate.
Pegylation
[0164] Additional methods and compositions provided within this
disclosure involve chemical modification of biologically active
peptides and proteins by covalent attachment of polymeric
materials, for example dextrans, polyvinyl pyrrolidones,
glycopeptides, polyethylene glycol and polyamino acids. The
resulting conjugated peptides and proteins retain their biological
activities and solubility for mucosal administration. In alternate
embodiments, insulins, proteins, analogs and mimetics, and other
biologically active peptides and proteins, are conjugated to
polyalkylene oxide polymers, particularly polyethylene glycols
(PEG). U.S. Pat. No. 4,179,337.
[0165] Amine-reactive PEG polymers within this disclosure include
SC-PEG with molecular masses of 2000, 5000, 10000, 12000, and
20000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000; T-PEG-12000;
and TPC-PEG-5000. PEGylation of biologically active peptides and
proteins may be achieved by modification of carboxyl sites (e.g.,
aspartic acid or glutamic acid groups in addition to the carboxyl
terminus). The utility of PEG-hydrazide in selective modification
of carbodiimide-activated protein carboxyl groups under acidic
conditions has been described. Alternatively, bifunctional PEG
modification of biologically active peptides and proteins can be
employed. In some procedures, charged amino acid residues,
including lysine, aspartic acid, and glutamic acid, have a marked
tendency to be solvent accessible on protein surfaces.
Other Stabilizing Modifications of Active Agents
[0166] In addition to PEGylation, biologically active agents such
as peptides and proteins within this disclosure can be modified to
enhance circulating half-life by shielding the active agent via
conjugation to other known protecting or stabilizing compounds, for
example by the creation of fusion proteins with an active peptide,
protein, analog or mimetic linked to one or more carrier proteins,
such as one or more immunoglobulin chains.
Formulation and Administration
[0167] Mucosal delivery formulations of the present disclosure
comprise insulin, analogs and mimetics, which may be combined
together with one or more pharmaceutically acceptable carriers and,
optionally, other therapeutic ingredients. The carrier(s) must be
"pharmaceutically acceptable" in the sense of being compatible with
the other ingredients of the formulation and not eliciting an
unacceptable deleterious effect in the subject. Such carriers are
described herein or are otherwise well known to those skilled in
the art of pharmacology. Desirably, the formulation should not
include substances such as enzymes or oxidizing agents with which
the biologically active agent to be administered is known to be
incompatible. The formulations may be prepared by any of the
methods well known in the art of pharmacy.
[0168] Within the compositions and methods of this disclosure, the
insulin proteins, analogs and mimetics, and other biologically
active agents disclosed herein may be administered to subjects by a
variety of mucosal administration modes, including by oral, rectal,
vaginal, intranasal, intrapulmonary, or transdermal delivery, or by
topical delivery to the eyes, ears, skin or other mucosal surfaces.
Optionally, insulin proteins, analogs and mimetics, and other
biologically active agents disclosed herein can be coordinately or
adjunctively administered by non-mucosal routes, including by
intramuscular, subcutaneous, intravenous, intra-atrial,
intra-articular, intraperitoneal, or parenteral routes. In other
alternative embodiments, the biologically active agent(s) can be
administered ex vivo by direct exposure to cells, tissues or organs
originating from a mammalian subject, for example as a component of
an ex vivo tissue or organ treatment formulation that contains the
biologically active agent in a suitable, liquid or solid
carrier.
[0169] Compositions according to the present disclosure are often
administered in an aqueous solution as a nasal or pulmonary spray
and may be dispensed in spray form by a variety of methods known to
those skilled in the art. Preferred systems for dispensing liquids
as a nasal spray are disclosed in U.S. Pat. No. 4,511,069. The
formulations may be presented in multi-dose containers, for example
in the sealed dispensing system disclosed in U.S. Pat. No.
4,511,069. Additional aerosol delivery forms may include, e.g.,
compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers,
which deliver the biologically active agent dissolved or suspended
in a pharmaceutical solvent, e.g., water, ethanol, or a mixture
thereof.
[0170] Nasal and pulmonary spray solutions of the present
disclosure typically comprise the drug or drug to be delivered,
optionally formulated with a surface-active agent, such as a
nonionic surfactant (e.g., polysorbate-80), and one or more
buffers. In some embodiments of the present invention, the nasal
spray solution further comprises a propellant. The pH of the nasal
spray solution is optionally from about pH 2.0 to about 8,
preferably 4.5.+-.0.5. Suitable buffers for use within these
compositions are as described above or as otherwise known in the
art. Other components may be added to enhance or maintain chemical
stability, including preservatives, surfactants, dispersants, or
gases. Suitable preservatives include, but are not limited to,
phenol, methyl paraben, propyl paraben, butyl paraben, paraben,
m-cresol, ortho-cresol, meta-cresol, par-cresol, thiomersal,
chlorobutanol, benzylalkonimum chloride, benzethonium chloride,
sodium benzoate, sorbic acid, and the like. Pharmaceutical
formulations within the context of this disclosure, may include any
one preservative or any combination or mixture of more than one
preservative. Suitable surfactants include, but are not limited to,
oleic acid, sorbitan trioleate, polysorbates, lecithin,
phosphotidyl cholines, and various long chain diglycerides and
phospholipids. Suitable dispersants include, but are not limited
to, ethylenediaminetetraacetic acid, and the like. Suitable gases
include, but are not limited to, nitrogen, helium,
chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon
dioxide, air, and the like.
[0171] Within alternate embodiments, mucosal formulations are
administered as dry powder formulations comprising the biologically
active agent in a dry, usually lyophilized, form of an appropriate
particle size, or within an appropriate particle size range, for
intranasal delivery. Minimum particle size appropriate for
deposition within the nasal or pulmonary passages is often about
0.5.mu. mass median equivalent aerodynamic diameter (MMEAD),
commonly about 1.mu. MMEAD, and more typically about 2.mu. MMEAD.
Maximum particle size appropriate for deposition within the nasal
passages is often about 10.mu. MMEAD, commonly about 8.mu. MMEAD,
and more typically about 4.mu. MMEAD. Intranasally respirable
powders within these size ranges can be produced by a variety of
conventional techniques, such as jet milling, spray drying, solvent
precipitation, supercritical fluid condensation, and the like.
These dry powders of appropriate MMEAD can be administered to a
patient via a conventional dry powder inhaler (DPI), which rely on
the patient's breath, upon pulmonary or nasal inhalation, to
disperse the power into an aerosolized amount. Alternatively, the
dry powder may be administered via air-assisted devices that use an
external power source to disperse the powder into an aerosolized
amount, e.g., a piston pump.
[0172] Dry powder devices typically require a powder mass in the
range from about 1 mg to 20 mg to produce a single aerosolized dose
("puff"). If the required or desired dose of the biologically
active agent is lower than this amount, the powdered active agent
will typically be combined with a pharmaceutical dry bulking powder
to provide the required total powder mass. Preferred dry bulking
powders include sucrose, lactose, dextrose, mannitol, glycine,
trehalose, human serum albumin (HSA), and starch. Other suitable
dry bulking powders include cellobiose, dextrans, maltotriose,
pectin, sodium citrate, sodium ascorbate, and the like.
[0173] To formulate compositions for mucosal delivery within the
present disclosure, the biologically active agent can be combined
with various pharmaceutically acceptable additives, as well as a
base or carrier for dispersion of the active agent(s). Desired
additives include, but are not limited to, pH control agents, such
as arginine, sodium hydroxide, glycine, hydrochloric acid, citric
acid, acetic acid, etc. In addition, local anesthetics (e.g.,
benzyl alcohol), isotonizing agents (e.g., sodium chloride,
mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),
solubility enhancing agents (e.g., cyclodextrins and derivatives
thereof), stabilizers (e.g., serum albumin), and reducing agents
(e.g., glutathione) can be included. When the composition for
mucosal delivery is a liquid, the tonicity of the formulation, as
measured with reference to the tonicity of 0.9% (w/v) physiological
saline solution taken as unity, is typically adjusted to a value at
which no substantial, irreversible tissue damage will be induced in
the nasal mucosa at the site of administration. Generally, the
tonicity of the solution is adjusted to a value of about 1/3 to
about 3, more typically about 1/2 to about 2, and most often about
3/4 to about 1.7.
[0174] The biologically active agent may be dispersed in a base or
vehicle, which may comprise a hydrophilic compound having a
capacity to disperse the active agent and any desired additives.
The base may be selected from a wide range of suitable carriers,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with
other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.),
hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives such as
hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural
polymers such as chitosan, collagen, sodium alginate, gelatin,
hyaluronic acid, and nontoxic metal salts thereof Often, a
biodegradable polymer is selected as a base or carrier, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters, etc., can be employed
as carriers. Hydrophilic polymers and other carriers can be used
alone or in combination, and enhanced structural integrity can be
imparted to the carrier by partial crystallization, ionic bonding,
crosslinking and the like. The carrier can be provided in a variety
of forms, including, fluid or viscous solutions, gels, pastes,
powders, microspheres and films for direct application to the nasal
mucosa. The use of a selected carrier in this context may result in
promotion of absorption of the biologically active agent.
[0175] The biologically active agent can be combined with the base
or carrier according to a variety of methods, and release of the
active agent may be by diffusion, disintegration of the carrier, or
associated formulation of water channels. In some circumstances,
the active agent is dispersed in microcapsules (microspheres) or
nanocapsules (nanospheres) prepared from a suitable polymer, e.g.,
isobutyl 2-cyanoacrylate and dispersed in a biocompatible
dispersing medium applied to the nasal mucosa, which yields
sustained delivery and biological activity over a protracted
time.
[0176] To further enhance mucosal delivery of pharmaceutical agents
within this disclosure, formulations comprising the active agent
may also contain a hydrophilic low molecular weight compound as a
base or excipient. Such hydrophilic low molecular weight compounds
provide a passage medium through which a water-soluble active
agent, such as a physiologically active peptide or protein, may
diffuse through the base to the body surface where the active agent
is absorbed. The hydrophilic low molecular weight compound
optionally absorbs moisture from the mucosa or the administration
atmosphere and dissolves the water-soluble active peptide. The
molecular weight of the hydrophilic low molecular weight compound
is generally not more than 10000 and preferably not more than 3000.
Exemplary hydrophilic low molecular weight compound include polyol
compounds, such as oligo-, di- and monosaccarides such as sucrose,
mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose,
D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose,
gentibiose, glycerin and polyethylene glycol. Other examples of
hydrophilic low molecular weight compounds useful as carriers
within this disclosure include N-methylpyrrolidone, and alcohols
(e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene
glycol, etc.). These hydrophilic low molecular weight compounds can
be used alone or in combination with one another or with other
active or inactive components of the intranasal formulation.
[0177] The compositions of this disclosure may alternatively
contain as pharmaceutically acceptable carriers substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, triethanolamine oleate, etc. A tabulation of
ingredients listed by the above categories can be found in the U.S.
Pharmacopeia National Formulary, 1990, 1857-1859, which is
incorporated by reference. For solid compositions, conventional
nontoxic pharmaceutically acceptable carriers can be used which
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like.
[0178] Therapeutic compositions for administering the biologically
active agent can also be formulated as a solution, microemulsion,
or other ordered structure suitable for high concentration of
active ingredients. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. Proper
fluidity for solutions can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of a desired
particle size in the case of dispersible formulations, and by the
use of surfactants. In many cases, it will be desirable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the biologically active agent can be
brought about by including in the composition an agent which delays
absorption, for example, monostearate salts and gelatin.
[0179] In certain embodiments of the instant disclosure, the
biologically active agent is administered in a time-release
formulation, for example in a composition which includes a slow
release polymer. The active agent can be prepared with carriers
that will protect against rapid release, for example a controlled
release vehicle such as a polymer, microencapsulated delivery
system or bioadhesive gel. Prolonged delivery of the active agent,
in various compositions of this disclosure can be brought about by
including in the composition agents that delay absorption, for
example, aluminum monosterate hydrogels and gelatin. When
controlled release formulations of the biologically active agent is
desired, controlled release binders suitable for use in accordance
with this disclosure include any biocompatible controlled-release
material which is inert to the active agent and which is capable of
incorporating the biologically active agent. Numerous such
materials are known in the art. Useful controlled-release binders
are materials that are metabolized slowly under physiological
conditions following their intranasal delivery (e.g., at the nasal
mucosal surface, or in the presence of bodily fluids following
transmucosal delivery). Appropriate binders include but are not
limited to biocompatible polymers and copolymers previously used in
the art in sustained release formulations. Such biocompatible
compounds are non-toxic and inert to surrounding tissues, and do
not trigger significant adverse side effects such as nasal
irritation, immune response, inflammation, or the like. They are
metabolized into metabolic products that are also biocompatible and
easily eliminated from the body.
[0180] Exemplary polymeric materials for use in this context
include, but are not limited to, polymeric matrices derived from
copolymeric and homopolymeric polyesters having hydrolysable ester
linkages. A number of these are known in the art to be
biodegradable and to lead to degradation products having no or low
toxicity. Exemplary polymers include polyglycolic acids (PGA) and
polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL
PLGA), poly(D-lactic acid-coglycolic acid)(D PLGA) and
poly(L-lactic acid-co-glycolic acid)(L PLGA). Other useful
biodegradable or bioerodable polymers include but are not limited
to such polymers as poly(epsilon-caprolactone),
poly(epsilon-aprolactone-CO-lactic acid),
poly(.epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy
butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels such as
poly(hydroxyethyl methacrylate), polyamides, poly(amino acids)
(i.e., L-leucine, glutamic acid, L-aspartic acid and the like),
poly (ester urea), poly (2-hydroxyethyl DL-aspartamide), polyacetal
polymers, polyorthoesters, polycarbonate, polymaleamides,
polysaccharides and copolymers thereof. Many methods for preparing
such formulations are generally known to those skilled in the art.
Other useful formulations include controlled-release compositions
e.g., microcapsules, U.S. Pat. Nos. 4,652,441 and 4,917,893, lactic
acid-glycolic acid copolymers useful in making microcapsules and
other formulations, U.S. Pat. Nos. 4,677,191 and 4,728,721, and
sustained-release compositions for water-soluble peptides, U.S.
Pat. No. 4,675,189.
[0181] Sterile solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated herein, as
required, followed by filtered sterilization. Dispersions may be
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile powders, methods of preparation include vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof. The prevention of the action of
microorganisms can be accomplished by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like.
[0182] Mucosal administration according to the present disclosure
allows effective self-administration of treatment by patients,
provided that sufficient safeguards are in place to control and
monitor dosing and side effects. Mucosal administration also
overcomes certain drawbacks of other administration forms, such as
injections, that are painful and expose the patient to possible
infections and may present drug bioavailability problems. For nasal
and pulmonary delivery, systems for controlled aerosol dispensing
of therapeutic liquids as a spray are well known. In one
embodiment, metered doses of active agent are delivered by means of
a specially constructed mechanical pump valve, U.S. Pat. No.
4,511,069.
Dosage
[0183] For prophylactic and treatment purposes, the biologically
active agent(s) disclosed herein may be administered to the subject
in a single bolus delivery, via continuous delivery (e.g.,
continuous transdermal, mucosal, or intravenous delivery) over an
extended time period, or in a repeated administration protocol
(e.g., by an hourly, daily or weekly, repeated administration
protocol). In this context, a therapeutically effective amount
(i.e., dosage) of a insulin may include repeated doses within a
prolonged prophylaxis or treatment regimen that will yield
clinically significant results to alleviate one or more symptoms or
detectable conditions associated with a targeted disease or
condition as set forth herein. Determination of effective dosages
in this context is typically based on animal model studies followed
up by human clinical trials and is guided by determining effective
dosages and administration protocols that significantly reduce the
occurrence or severity of targeted disease symptoms or conditions
in the subject. Suitable models in this regard include, for
example, murine, rat, porcine, feline, non-human primate, and other
accepted animal model subjects known in the art. Alternatively,
effective dosages can be determined using in vitro models (e.g.,
immunologic and histopathologic assays). Using such models, only
ordinary calculations and adjustments are typically required to
determine an appropriate concentration and dose to administer a
therapeutically effective amount of the biologically active
agent(s) (e.g., amounts that are intranasally effective,
transdermally effective, intravenously effective, or
intramuscularly effective to elicit a desired response).
[0184] In an alternative embodiment, this disclosure provides
compositions and methods for intranasal delivery of insulin,
wherein the insulin compound(s) is/are repeatedly administered
through an intranasal effective dosage regimen that involves
multiple administrations of the insulin to the subject during a
daily or weekly schedule to maintain a therapeutically effective
elevated and lowered pulsatile level of insulin during an extended
dosing period. The compositions and method provide insulin
compound(s) that are self-administered by the subject in a nasal
formulation between one and six times daily to maintain a
therapeutically effective elevated and lowered pulsatile level of
insulin during an 8 hour to 24 hour extended dosing period.
Kits
[0185] The instant disclosure also includes kits, packages and
multicontainer units containing the herein described pharmaceutical
compositions, active ingredients, and/or means for administering
the same for use in the prevention and treatment of diseases and
other conditions in mammalian subjects. Briefly, these kits include
a container or formulation that contains one or more insulin
proteins, analogs or mimetics, and/or other biologically active
agents in combination with mucosal delivery enhancing agents
disclosed herein formulated in a pharmaceutical preparation for
mucosal delivery.
[0186] The intranasal formulations of the present disclosure can be
administered using any spray bottle or syringe, or by instillation.
An example of a nasal spray bottle is the, "Nasal Spray Pump w/
Safety Clip," Pfeiffer SAP #60548, which delivers a dose of 0.1 mL
per squirt and has a diptube length of 36.05 mm. It can be
purchased from Pfeiffer of America of Princeton, N.J.
Aerosol Nasal Administration of an Insulin
[0187] We have discovered that one or more GRP can be administered
intranasally using a nasal spray or aerosol. This is surprising
because many proteins and peptides have been shown to be sheared or
denatured due to the mechanical forces generated by the actuator in
producing the spray or aerosol. In this area the following
definitions are useful.
[0188] 1. Aerosol--A product that is packaged under pressure and
contains therapeutically active ingredients that are released upon
activation of an appropriate valve system.
[0189] 2. Metered aerosol--A pressurized dosage form comprised of
metered dose valves, which allow for the delivery of a uniform
quantity of spray upon each activation.
[0190] 3. Powder aerosol--A product that is packaged under pressure
and contains therapeutically active ingredients in the form of a
powder, which are released upon activation of an appropriate valve
system.
[0191] 4. Spray aerosol--An aerosol product that utilizes a
compressed gas as the propellant to provide the force necessary to
expel the product as a wet spray; it generally applicable to
solutions of medicinal agents in aqueous solvents.
[0192] 5. Spray--A liquid minutely divided as by a jet of air or
steam. Nasal spray drug products contain therapeutically active
ingredients dissolved or suspended in solutions or mixtures of
excipients in nonpressurized dispensers.
[0193] 6. Metered spray--A non-pressurized dosage form consisting
of valves that allow the dispensing of a specified quantity of
spray upon each activation.
[0194] 7. Suspension spray--A liquid preparation containing solid
particles dispersed in a liquid vehicle and in the form of course
droplets or as finely divided solids.
[0195] The fluid dynamic characterization of the aerosol spray
emitted by metered nasal spray pumps as a drug delivery device
("DDD"). Spray characterization is an integral part of the
regulatory submissions necessary for Food and Drug Administration
("FDA") approval of research and development, quality assurance and
stability testing procedures for new and existing nasal spray
pumps.
[0196] Thorough characterization of the spray's geometry has been
found to be the best indicator of the overall performance of nasal
spray pumps. In particular, measurements of the spray's divergence
angle (plume geometry) as it exits the device; the spray's
cross-sectional ellipticity, uniformity and particle/droplet
distribution (spray pattern); and the time evolution of the
developing spray have been found to be the most representative
performance quantities in the characterization of a nasal spray
pump. During quality assurance and stability testing, plume
geometry and spray pattern measurements are key identifiers for
verifying consistency and conformity with the approved data
criteria for the nasal spray pumps.
Definitions
[0197] Plume Height--the measurement from the actuator tip to the
point at which the plume angle becomes non-linear because of the
breakdown of linear flow. Based on a visual examination of digital
images, and to establish a measurement point for width that is
consistent with the farthest measurement point of spray pattern, a
height of 30 mm is defined for this study:
[0198] Major Axis--the largest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm).
[0199] Minor Axis--the smallest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm).
[0200] Ellipticity Ratio--the ratio of the major axis to the minor
axis, preferably between 1.0 and 1.5, and most preferably between
1.0 and 1.3.
[0201] D.sub.10--the diameter of droplet for which 10% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m).
[0202] D.sub.50--the diameter of droplet for which 50% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m), also known as the mass median diameter.
[0203] D.sub.90--the diameter of droplet for which 90% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m).
[0204] Span--measurement of the width of the distribution, the
smaller the value, the narrower the distribution. Span is
calculated as:
( D 90 - D 10 ) D 50 . ##EQU00001##
[0205] % RSD--percent relative standard deviation, the standard
deviation divided by the mean of the series and multiplied by 100,
also known as % CV.
[0206] Volume--the volume of liquid or powder discharged from the
delivery device with each actuation, preferably between 0.01 mL and
about 2.5 mL and most preferably between 0.02 mL and 0.25 mL.
Example
[0207] The above disclosure generally describes the present
invention, which is further exemplified by the following examples.
These examples are described solely for purposes of illustration,
and are not intended to limit the scope of the invention. Although
specific terms and values have been employed herein, such terms and
values will likewise be understood as exemplary and non-limiting to
the scope of the invention.
Example 1
[0208] Intranasally Administered Insulin Pharmacokinetic Results in
Rabbits Pharmacokinetic (PK; e.g., insulin measurement) values were
measured for insulin treated New Zealand White Rabbits at specified
time-points up to 240 minutes following administration. All data
calculations are dose normalized and the pharmacokinetic data was
baseline corrected.
[0209] Intranasal peptide delivery formulations, "PDF" (shown in
Table 1), were compared with a SC NovoLog rapid-acting formulation
(NovoLog diluent consists of: 16 mg/mL glycerin, 1.5 mg/mL phenol,
1.72 mg/mL m-cresol, 19.6 .mu.g/mL zinc, 1.25 mg/mL disodium
hydrogen phosphate dihydrate, and 0.58 mg/mL NaCl, pH 7.2-7.6).
"PDF" as used herein is a formulation consisting of 45 mg/mL
Me-.beta.-CD, 1 mg/mL DDPC, 1 mg/mL EDTA, 10 mM arginine pH 7.0
with NaCl added to achieve about 220 mOsm/kg, and with or without
preservative. 2.times. PDF is a formulation consisting of 90 mg/mL
Me-.beta.-CD, 2 mg/mL DDPC, 2 mg/mL EDTA (other components remain
same as in PDF). As used herein, -DDPC is a PDF without DDPC;
Polysorabte 80 (Tween) was added to various PDF at 1%, 2%, 5% (10,
20, or 50 mg/mL) as indicated.
TABLE-US-00001 TABLE 1 PDF Components and Dosage in Rabbits Insulin
Me-.beta.- Arg Formulation Dose CD DDPC EDTA Tween 80 Buffer NaCl
(Group) (IU/kg) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mM) (mg/mL) pH
IN/1X PDF 6 45 1 1 10 10 4 7 1% Tween IN/1X PDF- 6 45 0 1 10 10 4 7
DDPC IN/1X PDF 6 45 1 1 20 10 4 7 2% Tween IN/1X PDF 6 45 1 1 50 10
4 7 5% Tween IN/2X PDF 6 90 2 2 10 10 4 7 1% Tween IN 2X PDF 6 90 2
2 20 10 4 7 2% Tween SC-PDF 0.6 45 1 1 10 10 4 7 SC-NovoLog 0.6
IU/kg (3 U/mL) NovoLog in NovoLog Dilutent 7.4
[0210] Shown in Table 2 are the T.sub.max, % C.sub.max,
AUC.sub.last, AUC.sub.inf, and % bioavailability relative to
SC-NovoLog results (with pharmacokinetic baseline subtracted); and
results for the IN/1.times. PDF 1% Tween and SC-Regular
formulations. The pharmacokinetic curves of these formulations are
similar, showing that IN/1.times. PDF results in a unique
pharmacokinetic profile for IN insulin.
TABLE-US-00002 TABLE 2 Pharmacokinetic Results after Intranasal
Administration of Insulin PDF in Rabbits T.sub.max % C.sub.max
AUCl.sub.ast AUC.sub.inf % BA Formulation (min) (.mu.lU/mL) (min *
.mu.lU/mL) (min * .mu.lU/mL) (insulin) IN/1X PDF 1% Tween 30 73.84
1766.20 3445.22 2.2 IN/1X PDF 1% Tween* 18 81.00 2397.00 4192.93
2.9 IN/1X PDF-DDPC 19 56.32 1549.00 2868.44 1.9 IN/1X PDF 2% Tween
27 97.65 4106.48 2436.22 5.0 IN/1X PDF 5% Tween 24 65.30 1412.40
2253.16 1.7 IN/2X PDF 1% Tween 15 79.24 2744.00 4173.69 3.4 IN 2X
PDF 2% Tween 22.5 73.28 2283.34 7819.15 2.8 SC-Regular* 30 128.38
7750.15 8982.12 95.0 SC-PDF 29 141.60 5830.50 8821.04 71.4 SC
NovoLog 23 168.84 8160.70 12338.64 *Results from separate data
set
[0211] The results in Table 2 show that the IN/1.times. PDF 2%
Tween had the highest % bioavailability, C.sub.max and AUC.sub.last
of the intranasal formulations tested. The % bioavailability,
C.sub.max and AUC.sub.last were decreased when DDPC was removed.
Regular, SC-NovoLog, and SC-PDF insulin resulted in similar
bioavailability. For the % bioavailability, intranasal formulations
resulted in approximately 2-5% bioavailability. IN/1.times. PDF 2%
Tween showed the highest bioavailability at 5%.
[0212] Table 3 shows another group of PDF dosed in rabbits. Some of
the formulations in Table 3 contained a combination of
preservatives: 10 mg/mL propylene glycol, 0.33 mg/mL methylparaben,
and 0.17 mg/mL propylparaben. The formulations labeled "-Pre" are
the PDF formulations without a preservative. Two SC groups were
dosed, one with regular insulin in absence of enhancers, and one
with regular insulin in presence of PDF.
TABLE-US-00003 TABLE 3 PDF Dosage in Rabbits Formulation Regular
Insulin Dose (IU/kg) 1XPDF 1% Tween 6 1XPDF 1% Tween (-DDPC) 6
1XPDF 2% Tween 6 1XPDF 2% Tween (-DDPC) 6 1XPDF 1% Tween (-Pre) 6
1XPDF 1% Tween (-PreDDPC) 6 SC-Regular PDF 0.6 SC-Regular Saline
0.6
[0213] The pharmacokinetic data for the groups shown in Table 3 are
shown in Table 4, Table 5 and Table 6. Within the % CV for the
various pharmacokinetic parameters the pharmacokinetic data are
similar for the various groups, with a bioavailability relative to
SC regular insulin control about 2-6% and T.sub.max in the range of
12-36 minutes.
TABLE-US-00004 TABLE 4 PK Results after Intranasal Administration
of Insulin PDF (Table 3) in Rabbits AUClast Group Tmax Cmax (min *
Formulation # (min) (uIU/mL) uIU/mL) 1XPDF 1% Tween 1 29.0 108.4
2504.2 1XPDF 1% Tween (-DDPC) 2 16.3 95.7 2284.8 1XPDF 2% Tween 3
36.3 88.1 2122.7 1XPDF 2% Tween (-DDPC) 4 12.0 138.5 3387.4 1XPDF
1% Tween (-Pre) 5 29.0 79.0 1174.5 1XPDF 1% Tween (-PreDDPC) 6 13.0
94.7 2453.3 SC Regular PDF 7 19.0 129.7 5014.3 SC Regular Saline 8
17.0 144.2 5885.5
TABLE-US-00005 TABLE 5 Bioavailability Results after Intranasal
Administration of Insulin PDF (Table 3) in Rabbits Formulation
Group # Bioavailability 1XPDF 1% Tween 1 4.3 1XPDF 1% Tween (-DDPC)
2 3.9 1XPDF 2% Tween 3 3.6 1XPDF 2% Tween (-DDPC) 4 5.8 1XPDF 1%
Tween (-Pre) 5 2.0 1XPDF 1% Tween (-PreDDPC) 6 4.2 SC Regular PDF 7
85.2 SC Regular Saline 8 NA
TABLE-US-00006 TABLE 6 % CV Results after Intranasal Administration
of Insulin PDF (Table 3) in Rabbits AUClast Group Tmax Cmax (min *
uIU/ Formulation # (min) (uIU/mL) mL) 1XPDF 1% Tween 1 56.4 84.2
84.7 1XPDF 1% Tween (-DDPC) 2 58.2 90.8 124.5 1XPDF 2% Tween 3 75.9
81.4 105.9 1XPDF 2% Tween (-DDPC) 4 22.8 87.9 105.2 1XPDF 1% Tween
(-Pre) 5 97.8 54.4 95.8 1XPDF 1% Tween 6 34.4 68.2 72.7 (-PreDDPC)
SC Regular PDF 7 57.1 58.3 64.2 SC Regular Saline 8 73.8 28.7
62.5
[0214] Pharmarmacodynamic (PD; e.g., glucose measurements) were
measured for insulin treated New Zealand White Rabbits at specified
time-points up to 240 minutes following administration of the
formulations shown in Table 3. Glucose was measured at every
time-point in duplicate with a Glucometer (One-Touch Ultra). The
pharmacodynamic data, change in glucose, are shown in Table 7.
TABLE-US-00007 TABLE 7 PD Results after Intranasal Administration
of Insulin PDF (Table 3) in Rabbits Dose Formulation (IU/kg) Tmin %
Cmin 1XPDF 1% Tween 6 30 49.8 1XPDF 1% Tween (-DDPC) 6 30 54.6
1XPDF 2% Tween 6 30 49.5 1XPDF 2% Tween (-DDPC) 6 30 48.4 1XPDF 1%
Tween (-Pre) 6 30 55.6 1XPDF 1% Tween (-PreDDPC) 6 30 57.3
SC-Regular PDF 0.6 45 36.4 SC-Regular Saline 0.6 60 38.4
[0215] The data in Table 7 shows that the time to onset of glucose
fall (as indicated by T.sub.min) is faster for regular insulin in
the intranasal PDF (45 min for SC; 30 min for intranasal) compared
to the control formulation (60 min for SC). All intranasal groups
demonstrated about the same pharmacodynamic effect (T.sub.min and %
C.sub.min). Presence or absence of DDPC in the formulation did not
affect the pharmacodynamic results. As used herein, C.sub.min means
a pharmacodynamic measurement representing the minimum
concentration of glucose (i.e., a glucose trough) occurring at time
T.sub.min, following the administration of insulin.
[0216] Table 8 describes intranasal, oral, and SC regular insulin
formulations. TDM is a PDF further consisting of 2.5 mg/mL
tetradecylmaltoside. Polysorbate 80 (Tween) was added to various
formulations at 1% (10 mg/mL) as indicated. Propylene glycol (PG)
was added to various formulations at 1% or 2.5% (10 or 25 mg/ml).
The effect of gelatin at 0.2% was tested. Three oral groups were
dosed, one with regular insulin in absence of enhancers (#8), one
with regular insulin in presence of PDF (#9), and one with regular
insulin in presence of PDF without DDPC (#7). An SC regular insulin
group was dosed for comparison.
TABLE-US-00008 TABLE 8 Description of IN, Oral and SC Groups Dosed
Dose Level Group # Formulation Route (IU/kg) 1 IXPDF 1% Tween (-PG)
IN 6 2 1XPDF 1% Tween (2.5% PG) IN 6 3 TDM hypotonic IN 6 4 TDM
Isotonic IN 6 5 1XPDF 1% Tween (1% PG) IN 6 6 1XPDF 1% Tween (0.2%
Gelatin) IN 6 7 1XPDF Oral (-DDPC + PG) Oral 6 8 1XPDF Oral (-DDPC
- PG-Tween) Oral 6 9 1XPDF Oral (+DDPC + PG) Oral 6 10 SC Regular
Insulin SC 0.6
[0217] The pharmacokinetic data in rabbits for the groups shown in
Table 8 are presented in Tables 9, Table 10 and Table 11.
TABLE-US-00009 TABLE 9 PK Parameters for IN, Oral and SC Groups
(Table 8) AUClast AUCinf (min * (min * Tmax Cmax .mu.IU/ .mu.IU/
Formulation (min) (.mu.IU/mL) mL) mL) 1XPDF 1% Tween (-PG) 59
125.06 5001.45 2565.5917 1XPDF 1% Tween 18 95.2 3178 5192.0496
(2.5% PG) TDM hypotonic 33 206.58 3971 9828.6486 TDM Isotonic 23
179.52 5663 9788.9524 1XPDF 1% Tween 34 108 6218 62759.0604 (1% PG)
1XPDF 1% Tween 13 373.6 8755.5 9067.4665 (0.2% Gelatin) 1XPDFOral
(-DDPC + 5 24.56 111.9 N/A PG) 1XPDFOral (-DDPC - 5 6.6 16.5 N/A
PG-Tween) 1XPDFOral (+DDPC + 5 3.08 64 408.0042 PG) SC Regular
Insulin 17 144.2 5885.5 3358.285
TABLE-US-00010 TABLE 10 PK Data (bioavailability) for IN, Oral and
SC Groups (Table 8) AUClast bioavailability Formulation (min *
uIU/mL) (insulin) 1XPDF 1% Tween (-PG) 5001.45 8.5 1XPDF 1% Tween
(2.5% PG) 3178 5.4 TDM hypotonic 3971 6.7 TDM Isotonic 5663 9.6
1XPDF 1% Tween (1% PG) 6218 10.6 1XPDF 1% Tween (0.2% Gelatin)
8755.5 14.9 1XPDFOral (-DDPC + PG) 111.9 0.2 1XPDFOral (-DDPC -
PG-Tween) 16.5 0.0 1XPDFOral (+DDPC + PG) 64 0.1 SC Regular Insulin
5885.5 N/A
TABLE-US-00011 TABLE 11 % CV for IN, Oral and SC Groups (Table 8)
Tmax Cmax AUClast Formulation (min) (uIU/mL) (min * uIU/mL) 1XPDF
1% Tween (-PG) 67.4 59.9 111.1 1XPDF 1% Tween (2.5% PG) 87.0 75.4
77.1 TDM hypotonic 59.3 41.3 56.6 TDM Isotonic 42.4 73.4 91.4 1XPDF
1% Tween (1% PG) 142.0 51.7 95.9 1XPDF 1% Tween (0.2% Gelatin) 34.4
21.3 35.3 1XPDFOral (-DDPC + PG) 0.0 164.5 190.0 1XPDFOral (-DDPC -
PG- 0.0 199.2 199.2 Tween) 1XPDFOral (+DDPC + PG) 0.0 116.6 178.3
SC Regular Insulin 73.8 28.7 62.5
[0218] For the intranasal groups containing PDF with or without PG
(and no gelatin), as well as for the groups containing TDM, the
pharmacokinetic data were similar, with a bioavailability compared
to SC regular insulin at about 5.4-10.6% and T.sub.max in the range
of from about 18 to about 59 minutes. In the case of 1.times. PDF
with 1% Tween in the presence of 0.2% gelatin, bioavailability
increased to about 14.9%. % CV for C.sub.max and AUC were between
50-111% for the intranasal groups in Table 8 containing PDF with or
without PG (and no gelatin), as well as the groups containing TDM.
In contrast, for 1.times. PDF with 1% Tween in the presence of 0.2%
gelatin, there was a decrease in % CV for C.sub.max and AUC to
21.3% and 35.3%, respectively. It was noted that the % CV for Cmax
and AUC of the 1.times. PDF with 1% Tween in the presence of 0.2%
gelatin formulation were lower than those observed for the SC
injection.
[0219] Pharmacodynamic data was similar between all intranasal
formulations, but SC dosing had an extended pharmacodynamic effect
compared to intranasal. No pharmacodynamic effect was observed for
the oral dose groups.
[0220] The pharmacokinetic and pharmacodynamic data show that
regular insulin administered in intranasal PDF is consistent with
an ultra-rapid acting insulin profile. It is surprising that an
intranasal administration of the pharmaceutical formulations
disclosed herein provides a more rapid acting insulin profile than
previously attained, for example, following SC administration of a
selectively designed insulin analogue or derivative. These data
show that the onset (maximum drop in glucose concentration as
indicated by T.sub.min) is faster for intranasally administered
regular insulin in the PDF compared to SC formulations. The
addition of gelatin, a thickening agent, enhanced the
pharmacodynamic and pharmacokinetic (14.9% bioavailability relative
to SC control) effect for intranasally administered insulin in
PDF.
Example 2
PK and PD Results for Intranasal Administration of Insulin
Formulations Containing Thickening Agents in Rabbits
[0221] Pharmacokinetic and pharmacodynamic data were evaluated for
rabbits dosed with intranasal insulin formulations containing
different thickening agents. Abbreviations include the following:
Me-.beta.-CD=methyl-beta-cyclodextrin, EDTA=disodium edetate, Tween
or TW=polysorbate 80, HPMC=hydroxypropyl methylcellulose (100 cps),
MC=methylcellulose (15 cps), CMC=carboxymethylcellulose sodium (low
viscosity), MP=methylparaben sodium, PP=propylparaben sodium,
PG=propylene glycol, NaCl=sodium chloride. Small amounts of 2N HCl
or NaOH were added to the formulation when necessary to achieve the
desired pH. The regular insulin used in the study was at a
concentration of approximately 28 IU/mg. Table 12 shows the
intranasal formulations used in this Example.
TABLE-US-00012 TABLE 12 Intranasal Insulin Formulations Containing
a Thickening Agent Regular Tween Arginine Thickening Insulin
Me-.beta.- EDTA 80 Buffer Agent MP PP PG NaCl # (IU/mL) CD (mg/mL)
(mg/mL) (mg/mL) (mM) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) pH 1
400 45 1 10 10 0 0.33 0.17 10 0 7.3 2 400 45 1 10 10 Gelatin 0.33
0.17 10 0 7.3 (2 mg/mL) 3 400 45 1 10 10 Gelatin 0.33 0.17 10 0 7.3
(4 mg/mL) 4 400 45 1 10 10 HPMC 0.33 0.17 10 0 7.3 (2.5 mg/mL) 5
400 45 1 10 10 MC 0.33 0.17 10 0 7.3 (2.5 mg/mL) 6 400 45 1 10 10
Carbopol 0.33 0.17 10 0 7.3 974 P (2.5 mg/mL) 7 400 45 1 10 10 CMC
0.33 0.17 10 0 7.3 (1 mg/mL) 8 400 45 1 10 10 Gelatin 0.33 0.17 10
3 7.3 (2 mg/mL)
[0222] In this example, 15 mL of each formulation was manufactured
and stored in clear non-silanized glass vials at 2-8.degree. C. All
formulations were dosed at 6.0 IU/kg. Table 13 describes the
dosages for used for the Table 12 formulations.
TABLE-US-00013 TABLE 13 Thickening Agent Dosage Groups Group #
Formulation Dose IU/kg 1 1XPDF 1% Tween 6.0 2 1XPDF 1% Tween (0.2%
Gelatin) 6.0 3 1XPDF 1% Tween (0.4% Gelatin) 6.0 4 1XPDF 1% Tween
(0.25% HPMC) 6.0 5 1XPDF 1% Tween (0.25% MC) 6.0 6 1XPDF 1% Tween
(0.25% Carbopol) 6.0 7 1XPDF 1% Tween (0.1% CMC) 6.0 8 1XPDF 1% TW
(0.2% Gelatin) 6.0
[0223] The pharmacokinetic results for the mean concentration of
insulin (.mu.IU/mL) over time is shown in FIG. 2. FIG. 2 shows that
C.sub.max was greatest for Group 6, 1.times. PDF 1% Tween (25%
Carbopol), compared to the other formulations. Peak serum insulin
levels for the 8 Groups occurred within 13-37 minutes. The
pharmacokinetic parameters are summarized in Table 14.
TABLE-US-00014 TABLE 14 PK Parameter Results after Administration
of Insulin in Formulations Containing a Thickening Agent (Table 12)
in Rabbits Group # Formulation Tmax (min) Cmax (.mu.IU/mL) AUClast
(min * .mu.IU/mL) AUCinf (min * .mu.IU/mL) 1 1XPDF 1% Tween 13.00
243.68 7409.6 7546.2311 2 1XPDF 1% Tween (0.2% Gelatin) 18.00
119.28 3487.6 3756.8904 3 1XPDF 1% Tween (0.4% Gelatin) 22.00
280.64 6617.8 10094.2851 4 1XPDF 1% Tween (0.25% HPMC) 37.00 212.74
6570.05 8149.3682 5 1XPDF 1% Tween (0.25% MC) 14.00 114.16 3383.2
4536.5694 6 1XPDF 1% Tween (0.25% Carbopol) 15.00 460.48 11583.6
12107.2492 7 1XPDF 1% Tween (0.1% CMC) 24.00 320.2 10482.5
11361.0313 8 1XPDF 1% TW (0.2% Gelatin) 29.00 231.48 6497.95
12461.998
[0224] The % CV results are shown in Table 15.
TABLE-US-00015 TABLE 15 % CV Results after Administration of
Insulin in Formulations Containing a Thickening Agent (Table 12) in
Rabbits Group # Formulation Tmax Cmax AUClast 1 1XPDF 1% Tween 21.1
68.4 73.2 2 1XPDF 1% Tween (0.2% Gelatin) 37.3 27.5 48.1 3 1XPDF 1%
Tween (0.4% Gelatin) 98.5 79.3 69.1 4 1XPDF 1% Tween (0.25% HPMC)
127.3 74.7 84.0 5 1XPDF 1% Tween (0.25% MC) 16.0 48.2 60.7 6 1XPDF
1% Tween (0.25% Carbopol) 0.0 62.0 47.6 7 1XPDF 1% Tween (0.1% CMC)
55.9 76.4 60.0 8 1XPDF 1% TW (0.2% Gelatin) 76.5 95.0 76.1
[0225] The bioavailability results are shown in Table 16.
TABLE-US-00016 TABLE 16 Bioavailability (insulin) Results after
Administration of Insulin in Formulations Containing a Thickening
Agent (Table 12) in Rabbits AUClast Dose (min * Group # Formulation
IU/kg uIU/mL) % F 1 1XPDF 1% Tween 6.0 7409.6 12.6 2 1XPDF 1% Tween
(0.2% Gelatin) 6.0 3487.6 5.9 3 1XPDF 1% Tween (0.4% Gelatin) 6.0
6617.8 11.2 4 1XPDF 1% Tween (0.25% HPMC) 6.0 6570.05 11.2 5 1XPDF
1% Tween (0.25% MC) 6.0 3383.2 5.7 6 1XPDF 1% Tween (0.25%
Carbopol) 6.0 11583.6 19.7 7 1XPDF 1% Tween (0.1% CMC) 6.0 10482.5
17.8 8 1XPDF 1% TW (0.2% Gelatin) 6.0 6497.95 11.0 SC Regular
Insulin 0.6 5885.5
[0226] The pharmacodynamic results are shown in FIG. 3. Glucose was
measured at regular time-points with a Glucometer (One-Touch
Ultra). FIG. 3 shows the mean change in % glucose over time for the
eight groups tested. Group 6, a formulation consisting of 1.times.
PDF 1% Tween (0.25% Carbopol), showed the greatest reduction in %
glucose from initial compared to all other groups. Glucose troughs
for the 8 Groups occurred within 60 minutes. Group 8 (which
contained a tonicity adjusting agent, NaCl) had the greatest
reduction in % glucose from initial compared to the other gelatin
formulations. The formulations containing Carbopol and CMC had the
greatest reduction in % glucose from initial compared to the other
non-gelatin formulations.
[0227] The pharmacokinetic and pharmacodynamic results in rabbits
show that the intranasal insulin formulations tested had
ultra-rapid acting insulin profiles, with peak serum insulin levels
in less than 60 minutes and glucose troughs in less than 90
minutes. Bioavailability was increased when thickening agents were
added to PDF intranasal insulin formulations. Isotonic formulations
containing gelatin showed an increase in bioavailability. The
formulation containing gelatin showed improved performance with
isotonic conditions (Group #8; 0.2% Gelatin including NaCl)
compared to hypotonic conditions (Group #2; 0.2% Gelatin without
NaCl). The formulations containing Carbopol and CMC showed the
greatest increase in pharmacokinetic and pharmacodynamic results
for intranasal insulin formulations (compare to bioavailability
shown in Tables 2, 5 and 10). The bioavailability for formulations
from Table 12 was 19.7% and 17.8% for Carbopol and CMC,
respectively. The pharmacodynamic effect as shown by % glucose from
initial was improved with the addition of thickening agents, such
as Carbopol and CMC, to the intranasal insulin formulations.
[0228] Additional intranasal formulations comprising a thickening
agent such as Carbopol or CMC, were tested and are described in
Table 17.
TABLE-US-00017 TABLE 17 Insulin Formulations Containing Carbopol or
CMC as Thickening Agent Regular Tween Arginine Thickening Insulin
Me-.beta.- EDTA 80 Buffer Agent MP PP PG # (IU/mL) CD (mg/mL)
(mg/mL) (mg/mL) (mM) (mg/mL) (mg/mL) (mg/mL) (mg/mL) pH 1 800 45 1
10 10 CMC LV, 1 mg/mL 0.33 0.17 10 7.3 2 800 45 1 10 10 CMC LV,
0.33 0.17 10 7.3 10 mg/mL 3 800 45 1 10 10 Carbopol 0.33 0.17 10
7.3 974P, 2.5 mg/mL 4 800 45 1 10 10 CMC MV, 0.33 0.17 10 7.3 10
mg/mL 5 800 22.5 1 10 10 0.25% 0.33 0.17 10 7.3 6 800 10 1 10 10
Carbopol 0.33 0.17 10 7.3 7 800 45 1 5 10 974P 0.33 0.17 10 7.3 8
800 45 1 1 10 0.33 0.17 10 7.3 Abbreviations: LV means low
viscosity; MV means medium viscosity
[0229] The pharmacokinetic and pharmacodynamic results in rabbits
testing these alternative carbopol and CMC thickening agent
modified formulations, shown in Table 17, are shown in Tables 18,
19, and 20.
TABLE-US-00018 TABLE 18 PK Results after Intranasal Administration
of Carbopol and CMC Thickening Agent Modified Insulin Formulations
(Table 17) in Rabbits Dose (IU/ Formulation kg) Tmax Cmax AUClast
1XPDF 1% Tween 0.1% CMCLV 12 22.50 573.66 25468.22 1XPDF 1% Tween
0.1% CMCLV 12 31.88 411.46 18547.22 1XPDF 1% Tween 0.25% Carbopol
12 43.13 370.40 13774.06 1XPDF 1% Tween 1% CMC MV 12 27.50 409.32
15797.13 0.5XPDF 1% Tween 0.25% Carbopol 12 31.88 408.66 19360.03
0.22XPDF 1% Tween 0.25% 12 29.29 340.46 16721.11 Carbopol 1XPDF
0.5% Tween 0.25% Carbopol 12 22.50 324.25 9595.94 1XPDF 0.1% Tween
0.25% Carbopol 12 35.00 703.69 12845.09 SC Regular Saline 0.6 17.00
144.20 5885.50
TABLE-US-00019 TABLE 19 % CV Results after Intranasal
Administration of Carbopol and CMC Thickening Agent Modified
Insulin Formulations (Table 17) in Rabbits Dose Formulation (IU/kg)
Tmax Cmax AUClast 1XPDF 1% Tween 0.1% CMCLV 12 35.6 63.2 79.4 1XPDF
1% Tween 1% CMCLV 12 113.7 90.2 83.4 1XPDF 1% Tween 0.25% Carbopol
12 84.1 63.6 60.5 1XPDF 1% Tween 1% CMC MV 12 92.5 54.3 87.6
0.5XPDF 1% Tween 0.25% Carbopol 12 58.7 86.5 103.3 0.22XPDF 1%
Tween 0.25% 12 54.4 83.7 88.0 Carbopol 1XPDF 0.5% Tween 0.25%
Carbopol 12 50.4 68.1 69.1 1XPDF 0.1% Tween 0.25% Carbopol 12 106.1
154.0 74.3 SC Regular Saline 0.6 73.8 28.7 62.5
TABLE-US-00020 TABLE 20 Bioavailability after Intranasal
Administration of Carbopol and CMC Thickening Agent Modified
Insulin Formulations (Table 17) in Rabbits Formulation Dose (IU/kg)
AUClast % F 1XPDF 1% Tween 0.1% CMCLV 12 25468.22 21.6 1XPDF 1%
Tween 1% CM CLV 12 18547.22 15.8 1XPDF 1% Tween 0.25% Carbopol 12
13774.06 11.7 1XPDF 1% Tween 1% CMC MV 12 15797.13 13.4 0.5XPDF 1%
Tween 0.25% Carbopol 12 19360.03 16.4 0.22XPDF 1% Tween 0.25%
Carbopol 12 16721.11 14.2 1XPDF 0.5% Tween 0.25% Carbopol 12
9595.94 8.2 1XPDF 0.1% Tween 0.25% Carbopol 12 12845.09 10.9 SC
Regular Saline 0.6 5885.50
[0230] The 0.5.times. PDF/1% Tween/0.25% Carbopol and 1.times. PDF
1%/Tween 1%/CMC (LV) formulations resulted in good bioavailability
at 16.4% and 15.8%, respectively. The 1.times. PDF/1% Tween/0.1%
CMC (LV) resulted in the highest insulin bioavailability (21.6%).
These data indicate that the addition of thickening agents (e.g.,
carbopol and CMC) significantly and surprisingly enhance the
percent bioavailability of an insulin contained within exemplary
pharmaceutical formulations disclosed herein. Such an increase in
percent bioavailability is also associated with a T.sub.max from
about 22 to about 30 minutes in rabbits.
[0231] A further rabbit study was performed in which blood insulin
and glucose levels were determined at specified time points up to
240 minutes. In addition to thickening agents, these formulations
also contained the preservatives methylparaben and/or propylparaben
and/or phenylethanol. Glucose concentration was measured in
duplicate with a glucometer (e.g., One-Touch Ultra). The
formulations tested are listed in Table 21.
TABLE-US-00021 TABLE 21: PK and PD Rabbit Study Thickening Agents
Plus Preservatives Group Number Formulation Code 1 0.1% CMC; 0.033%
MP; IDFCMC-LDMPPP 0.017% PP 2 0.1% CMC; 0.333% MP; IDFCMC-MPPP
0.17% PP 3 0.1% CMC; 033% MP; IDFCMC-MPPPPE 0.017% PP; 0.2% PE 4
Insulin isotonic saline SC 5 0.25% Carbopol; IDF- 0.033% MP; 0.017%
PP 0.25% CHLDMPPP 6 0.1% Carbopol; IDF-0.1% 0.033% MP; 0.017% PP
CHLDMPPP 7 0.25% Carbopol; IDF-0.25% 0.33% MP; 0.17% PP; CHMPPPPE
0.2% PE 8 0.1% Carbopol; IDF-0.1% CHMPPPPE 0.33% MP; 0.17% PP; 0.2%
PE * 4.5% MBCD; 0.1% No Thickening Agent EDTA; 1.0% Tween; 1%
Modified Insulin PG; Arginine Formulation Abbreviations: PP is
Propylparaben; MP is Methylparaben; PE is Phenylethanol; IDF is
Insulin delivery Formulation which is 4.5% Me-.beta.-CD, 0.1% EDTA,
1.0% Tween, 1% Propylene Glycol, Arginine
[0232] The pharmacokinetic results for this rabbit experiment are
shown in Tables 22 and 23.
TABLE-US-00022 TABLE 22 PK Results after Intranasal Administration
of Thickening Agent Plus Preservative Insulin Formulations (Table
21) in Rabbits Group Tmax AUC.sub.Last C.sub.max % Number
Formulation (min) (min * .mu.IU/ml) (.mu.IU/ml) Bioavailability 1
0.1% CMC; 0.033% MP; 23.6 17738.8 337.7 6.9 0.017% PP 2 0.1% CMC;
0.333% MP; 20.0 26481.0 448.5 10.3 0.17% PP 3 0.1% CMC; 033% MP;
77.5 34782.9 817.8 13.5 0.017% PP; 0.2% PE 4 Insulin isotonic
saline 29.4 12891.3 173.0 5 0.25% Carbopol; 29.2 42958.7 1405.6
16.7 0.033% MP; 0.017% PP 6 0.1% Carbopol; 22.5 64953.6 1604.0 25.2
0.033% MP; 0.017% PP 7 0.25% Carbopol; 41.3 60030.2 843.4 23.3
0.33% MP; 0.17% PP; 0.2% PE 8 0.1% Carbopol; 31.4 50373.7 980.9
19.5 0.33% MP; 0.17% PP; 0.2% PE
TABLE-US-00023 TABLE 23 % CV Results after Intranasal
Administration of Thickening Agent Plus Preservative Insulin
Formulations (Table 21) in Rabbits Group Number Formulation Tmax
AUC.sub.Last C.sub.max 1 0.1% CMC; 0.033% MP; 72.2 81.6 32.9 0.017%
PP 2 0.1% CMC; 0.333% MP; 47.9 98.3 58.5 0.17% PP 3 0.1% CMC; 033%
MP; 116.3 42.2 47.0 0.017% PP; 0.2% PE 4 Insulin isotonic saline
57.1 34.1 22.2 5 0.25% Carbopol; 87.7 65.1 55.9 0.033% MP; 0.017%
PP 6 0.1% Carbopol; 71.3 120.3 113.1 0.033% MP; 0.017% PP 7 0.25%
Carbopol; 84.2 131.5 63.5 0.33% MP; 0.17% PP; 0.2% PE 8 0.1%
Carbopol; 46.6 80.9 60.6 0.33% MP; 0.17% PP; 0.2% PE
[0233] The pharmacokinetic results for formulations in Table 21
showed T.sub.max ranging from 20.0 minutes to 77.5 minutes, with
AUC.sub.Last ranging from 12891.3 .mu.lU/ml to 64953.6 .mu.lU/ml.
Bioavailability was increased when thickening agents were added to
PDF intranasal insulin formulations. The formulation containing
0.1% Carbopol plus the preservatives MP and PP (Group #6), and the
formulation containing 0.25% Carbopol plus preservatives MP, PP and
PE (Group #7) provided the highest bioavailability, 25.2% and
23.3%, respectively; and, representing a further increase in
bioavailability over the data from experiments performed using
formulations manufactured without addition of a thickening agent as
presented in Tables 2, 5, and 10.
Example 3
PK and PD Results for Intranasal Administration of Insulin in
Humans
[0234] Human subjects participated in a seven treatment group study
in which the treatment groups included the following: one treatment
of a nasal placebo, four regular human insulin (25 IU, 50 IU, 100
IU, and 25 IU/1% PG) intranasal formulations without a thickening
agent (shown in Table 24), one treatment of 3 mg rapid-acting
insulin aspart subcutaneous injection (NovoLog), and one treatment
with human insulin inhalation powder (EXUBERA, 3 mg).
[0235] IU is the unit of measurement for the amount of insulin
based on measured biological effect (1 IU=0.04167 mg or 23.9 IU=1
mg). The intransal insulin formulations were 250 to 1000 IU/mL and
were delivered in a volume of 0.1 mL. For comparison, Exubera was a
dose of about 70 IU.
TABLE-US-00024 TABLE 24 Intranasal Insulin Formulations Without a
Thickening Agent for Human PK/PD Study Nasal Nasal Nasal Nasal
Nasal 1% PG Placebo 25 IU 50 IU 100 IU 25 IU Formulation Component
(STD) (STD) (STD) (STD) (STD) Insulin (IU/mL) 0 250 500 1000 250
Me-.beta.-CD (mg/mL) 45 (4.5%) 45 (4.5%) 45 (4.5%) 45 (4.5%) 45
(4.5%) DDPC (mg/mL) 1 1 1 1 0 EDTA (mg/mL) 1 (0.1%) 1 (0.1%) 1
(0.1%) 1 (0.1%) 1 (0.1%) Polysorbate 80 (mg/mL) 10 (1%) 10 (1%) 10
(1%) 10 (1%) 10 (1%) Arginine (mM) 10 10 10 10 10 Sodium Chloride
(mg/mL) 4 4 4 4 0 Propylparaben Sodium 0.17 (0.1%) 0.17 (0.1%) 0.17
(0.1%) 0.17 (0.1%) 0.17 (1%) (mg/mL) Methylparaben Sodium 0.33 0.33
0.33 0.33 0.33 (mg/mL) Propylene Glycol (mg/mL) 1 1 1 1 10 Sodium
Hydroxide TAP TAP TAP TAP TAP Purified Water quantity quantity
quantity quantity quantity sufficient sufficient sufficient
sufficient sufficient pH 7.0-7.6 7.0-7.6 7.0-7.6 7.0-7.6 7.0-7.6
Abbreviation: TAP means to adjust pH
[0236] Plasma insulin and glucose levels were measured at 12 time
points up to six hours. Pharmacokinetic parameters, including
T.sub.max, C.sub.max, and AUC.sub.last were calculated based on
plasma concentrations of insulin for each subject. FIG. 4 shows the
pharmacokinetic profile for the four intranasal doses (25 IU, 50
IU, 100 IU, and 25 IU/1% PG), EXUBERA, NovoLog, and the control
(Nasal Placebo). Pharmacokinetic calculations were performed using
commercial software (WinNonlin). AUCO.sub.0-.infin., K.sub.e, and
t.sub.1/2 were calculated when the data permitted accurate
estimation. Statistical analysis of bioavailability data was
calculated.
[0237] The pharmacokinetic profile (mean serum insulin) and
relative percent bioavailability for the IN formulations and
EXUBERA compared to NovoLog of the described formulations are shown
in FIG. 4 and also embodied below in Table 25.
TABLE-US-00025 TABLE 25 Pharmacokinetic Results after
Administration of Intranasal Insulin Formulations Without a
Thickening Agent to Human Subjects Pharmacokinetic Parameters
Relative % BA Compared T.sub.max C.sub.max AUCl.sub.ast to (min)
(.mu.U/mL) (min * .mu.U/mL) AUC.sub.inf NovoLog Formulation (STD)
(STD) (STD) (min * .mu.U/mL) (STD) Nasal Placebo 63.8 2.8 (3.98)
273.1 (452.11) 5106.7 -- (control) (30.92) NovoLog 38.3 52 (31.35)
2484.7 (998.4) 3502.4 -- (30.92) EXUBERA (3 mg) 23.4 14.5 (4.21)
1194.2 (699.05) 3703.7 7.1 (6.3) (14.68) Nasal (25 IU) 19.2 21
(8.61) 724.8 (469.55) 1834.5 10.1 (8.0) (6.95) Nasal (50 IU) 16.2
23.5 (16.99) 670.8 (598.94) 1366.3 5.2 (5.39) (4.94) Nasal (100 IU)
16.8 43.6 (44.66) 1368 (1898.56) 2634.5 4.8 (4.8) (6.81) Nasal 25
IU 1% PG 24.4 17.8 (10.57) 724.1 (652.72) 3494.9 14.8 (22.6)
(26.52)
[0238] With respect to time to maximum plasma level for insulin or
T.sub.max, the four intranasal doses (25 IU, 50 IU, and 100 IU, and
25 IU/1% PG insulin) had T.sub.max values of about 16 to about 19
minutes, which provided the shortest T.sub.max values compared to
the rapid-acting insulin aspart (NovoLog) and inhaled insulin
(EXUBERA). With respect to plasma insulin levels (C.sub.max),
rapid-acting insulin aspart injection (NovoLog) had the highest
concentration, followed by the four nasal formulations, with
inhaled insulin (EXUBERA) having the lowest. With respect to the
extent of absorption, rapid-acting insulin aspart injection
(NovoLog) had the greatest total exposure or AUC.sub.last, with the
highest dose of four nasal formulations next (100 IU), followed by
the inhaled insulin (EXUBERA) and then the lower doses of the three
nasal spray formulations (25 IU, 25 IU 1% PG and 50 IU). The
intranasal formulations resulted in quicker return to baseline
insulin levels compared to Exubera.
[0239] The bioavailability of the insulin intranasal formulations
ranged between about 4.8-14.8% (relative to SC NovoLog). 25 IU and
25 IU/1% PG formulations had a higher mean % bioavailability than
Exubera (7.1%) in this study. The highest bioavailablity was
achieved by the intranasal 25 IU/1% PG formulation (14.8%).
[0240] The % CV results are shown in Table 26.
TABLE-US-00026 TABLE 26 % CV Results after Administration of
Intranasal Insulin Formulations Without a Thickening Agent to Human
Subjects % CV Results T.sub.max C.sub.max AUC.sub.last AUC.sub.inf
Formulation (min) (.mu.U/mL) (min * .mu.U/mL) (min * .mu.U/mL)
Nasal Placebo 48.5 141.5 162.5 -- (control) NovoLog 26.8 60.3 40.2
23.5 EXUBERA (3 mg) 62.6 29.1 58.5 87.4 Nasal (25 IU) 36.3 41.1
68.8 41.7 Nasal (50 IU) 30.5 72.2 89.5 47.8 Nasal (100 IU) 40.5
102.3 140.3 85.2 Nasal 25 IU 1% PG 108.8 59.5 90.1 63.9
[0241] The AUC intersubject % CV was approximately 70-140% for 25
IU, 50 IU, and 100 IU IN groups, and 90.1% for the 25 IU 1% PG
group. The % CV for Exubera was 60%, and NovoLog was 40%.
[0242] A glucometer was used to measure glucose levels for the
pharmacodynamic data collection. For each sample, the time to
maximum % glucose fall from initial (Tmax) and maximum % glucose
fall from initial (Cmax or % Fall) were calculated. A summary of
the glucose maximum % Fall and Time to maximum % Fall percent
reduction in glucose for each treatment group is shown in Table 27.
The maximum % glucose fall from initial was approximately 55% for
NovoLog and 20-30% for the IN formulations. The incidence of 30%,
20%, and 10% reduction in glucose percent for each treatment group
is shown in Table 28.
TABLE-US-00027 TABLE 27 Glucose maximum % Fall and Time to Maximum
% Fall Results after Administration of Intranasal Insulin
Formulations Without a Thickening Agent to Human Subjects Treatment
Group Glucose Max % Fall Time to Max % Fall (min) Nasal Placebo 6.4
193.6 NovoLog (SC) 55.8 50.5 Exubera 3 mg 22.5 105 Nasal 25 IU 19.8
43.6 Nasal 50 IU 24.7 109.1 Nasal 100 IU 30.5 69.5 Nasal 25 IU 1%
PG 21.9 61.9
TABLE-US-00028 TABLE 28 Incidence of Human Subjects with 30%, 20%,
and 10% Glucose Reduction Results after Administration of
Intranasal Insulin Formulations Without a Thickening Agent to Human
Subjects Subjects with Glucose % Reduction Treatment # of GE 30% GE
20% GE 10% Group Subjects N (%) N (%) N (%) Nasal Placebo 812 0
(0%) 1 (12.58.3%) 4 (5033%) NovoLog (SC) 812 8 (100%) 812 (100%)
812 (100%) 10 (83.3%) Exubera 3 mg 711 1 (14.30 (0%) 4 (57.1%) 8
(72.7 Nasal 25 IU 5 (45.5%) (100%) Nasal 2550 IU 811 1 (12.5%) 4
(506 7 (87.5%) 4 (36.4%) (54.5%) 9 (81.8%) Nasal 50100 IU 811 2
(253 4 (5036.4%) 811 (100%) (27.3%) Nasal 100 IU 86 2 (33.3 5
(62.5%) 86 (100%) Exubera 3 mg (37.5%) 4 (66.7%) Nasal 25 IU 8 2
(25%) 4 (50%) 7 (87.5%) 1% PG
[0243] The results of this pharmacodynamic study demonstrate that
intranasal administration of insulin is effective in reducing a
patient's blood glucose level (reflected as % glucose fall). The
mean % glucose change from baseline results showed a more rapid
glucose fall for intranasally administered insulin compared to
EXUBERA and NovoLog.
[0244] The pharmacokinetic-pharmacodynamic relationship
demonstrated a high correlation between either C.sub.max or
AUC.sub.last and the maximum glucose response. There were no
observed side effects (adverse reactions) resulting from intranasal
administration of insulin, including clinically significant
hypoglycemia. The intranasal insulin doses were well tolerated and
post-dose nasal examinations were normal. There were no clinically
important changes in vital signs (systolic or diastolic blood
pressure and heart rate), ECG, or physical examination during the
course of the study.
Example 4
PK and PD Results for Intranasal Administration of Insulin
Formulations Containing a Thickening Agent in Humans
[0245] This example describes intranasal insulin formulations
containing various thickening agents that were tested in human
subjects. The intranasal insulin formulations containing the
thickening agents carboxymethylcellulose sodium-low viscosity (CMC)
and carbopol, described in Table 29, were tested.
TABLE-US-00029 TABLE 29 Insulin Formulations Containing a
Thickening Agent for Human PK/PD Study Nasal Plus Nasal Plus Nasal
Plus Nasal Plus CMC CMC CH CH Formulation Component (25 IU) (50 IU)
(25 IU) (50 IU) Insulin (IU/mL) 250 500 250 500 Me-.beta.-CD
(mg/mL) 45 (4.5%) 45 (4.5%) 45 (4.5%) 45 (4.5%) EDTA (mg/mL) 1
(0.1%) 1 (0.1%) 1 (0.1%) 1 (0.1%) Polysorbate 80 (mg/mL) 10 (1%) 10
(1%) 10 (1%) 10 (1%) CMC (mg/mL) 1 (0.1%) 1 (0.1%) 0 0 Carbopol
(mg/mL) 0 0 2.5 (0.25%) 2.5 (0.25%) Arginine (mg/mL) 2.1 2.1 2.1
2.1 Propylene Glycol (PG) (mg/mL) 10 (1%) 10 (1%) 10 (1%) 10 (1%)
Propylparaben Sodium (PP) (mg/mL) 0.17 0.17 0.17 0.17 Methylparaben
Sodium (MP) 0.33 0.33 0.33 0.33 (mg/mL) Purified Water quantity
quantity quantity quantity sufficient sufficient sufficient
sufficient Sodium Hydroxide TAP TAP TAP TAP pH 7 7 7 7
Abbreviations: CH means carbomer homopolymer (trade name: Carbopol
974P)
[0246] The methyl-.beta.-cyclodextrin used in these intranasal
formulations was tested in six and nine month toxicity studies in
rats and dogs, respectively with no signs of systemic or nasal
toxicity. In addition, these excipients have been administered to
humans in other formulations with no signs of systemic or nasal
toxicity. The other excipients, i.e., Carbopol 974P (a carbomer
homopolymer), carboxy-methylcellulose, and polysorbate 80 are
either generally recognized as safe (GRAS), listed in the FDA
Inactive Ingredient Guide, or contained in ophthalmic or other
nasal products at the same or higher concentrations.
[0247] Absorption, tolerance and bioavailability data were
collected for insulin (insulin regular) nasal spray formulations
containing a thickening agent and compared to subcutaneous insulin
(NovoLog) in healthy human subjects. The study included 12 healthy
male and female subjects between the ages of 18 and 45 years with
no history of diabetes or hypoglycemia, and a body mass index
between 20-28 kg/m.sup.2. Each subject was administered ascending
doses of insulin starting with nasal placebo, then subcutaneous
administration of NovoLog at a dose of 20% of 0.6 IU/kg (not to
exceed 10 IU) followed by the nasal doses of 25 and 50 IU per
formulation (50 IU dose was given as 25 IU nasal spray per
nostril). Each insulin administration was given at least 24 hours
apart. Subjects were fasted overnight and given a standard meal 5
minutes after dosing. The subjects were monitored for symptoms and
glucose was monitored by glucometer (finger stick).
[0248] Prior to intranasal administration, the assembled nasal
spray pump and bottle (applicator) were primed. Subject was
instructed to gently blow his/her nose. The primed intranasal
applicator was gently inserted into the nostril. The bottle was
tilted to be in a straight line with the nasal passage. The pump
was firmly pressed down once to spray the medication into the
subject's nose while he/she gently inhaled. The subjects were
instructed to remain upright for a minimum of 15 minutes following
dosing. Subject refrained from blowing his/her nose for 1 hour
following intranasal administration.
[0249] Blood samples for analysis of insulin, glucose and C-Peptide
levels were collected at 0 (pre-first dose), 5, 10, 15, 30, 45, 60,
90 minutes and 2, 3, 4, and 5 hours post-dose. The following
pharmacokinetic parameters were calculated based on plasma
concentrations of insulin for each subject: C.sub.max, t.sub.max,
and AUC.sub.0-l. Pharmacokinetic calculations were performed using
commercial software (WinNonlin). AUC.sub.0-.infin., K.sub.e, and
t.sub.1/2 were calculated when the data permitted accurate
estimation. Pharmacokinetic data is shown in Table 30, and mean
serum insulin levels for the groups tested in this example are
shown in FIG. 5. The % CV results are shown in Table 31.
[0250] Statistical analysis of pharmacokinetic/pharmacodynamic data
(bioavailability) was calculated. Differences for all
pharmacokinetic/pharmacodynamic variables, except Tmax between each
formulation of insulin nasal spray versus the reference
(subcutaneous NovoLog) were evaluated using two-sided pair T-test.
A separate analysis was performed for each formulation of insulin
nasal spray versus the reference.
TABLE-US-00030 TABLE 30 Pharmacokinetic Parameters of Intranasal
Insulin Formulations Containing a Thickening Agent Administered to
Human Subjects Relative % BA AUC.sub.last AUC.sub.inf Compared to
T.sub.max min C.sub.max .mu.U/mL) (min * .mu.U/mL) (min * .mu.U/mL)
NovoLog Formulation (STD) (STD) (STD) (STD) (STD) Nasal Placebo 45
(32.4) 2.5 (3.81) 79.6 (163.82) -- -- (control) NovoLog 51.3
(14.94) 24.3 (9.96) 1246.9 (562.46) 2089.1 (658.55) -- Nasal (25
IU) 14.6 (5.42) 23.7 (14.53) 885.6 (637.66) 2779.3 (1693.96) 28.4
(18.8) Plus CMC Nasal (50 IU) 30 (35.99) 31.2 (21.98) 991.6
(964.27) 1584.8 (1044.42) 16.8 (15.11) Plus CMC Nasal (25 IU) 26.7
(22.6) 28.7 (13.53) 915.4 (479) 1468.4 (958.53) 30.6 (16.43) Plus
Carbopol Nasal (50 IU) 14.6 (1.44) 47.2 (34.94) 1319.7 (930.69)
1529 (872.27) 21.5 (14.16) Plus Carbopol
TABLE-US-00031 TABLE 31 % CV Results for Intranasal Insulin
Formulations Containing a Thickening Agent Administered to Human
Subjects % CV Results T.sub.max C.sub.max AUC.sub.last AUC.sub.inf
Formulation (min) (.mu.U/mL) (min * .mu.U/mL) (min * .mu.U/mL)
Nasal Placebo 72 151.9 205.8 -- (control) NovoLog 29.2 41.1 45.1
31.5 Nasal (25 IU) 37.2 61.4 72 60.9 Plus CMC Nasal (50 IU) 120
70.4 97.2 65.9 Plus CMC Nasal (25 IU) 84.7 47.2 52.3 65.3 Plus
Carbopol Nasal (50 IU) 9.9 74 70.5 57 Plus Carbopol
[0251] These results show that addition of a thickening agent to
the intranasal insulin formulation disclosed herein resulted in an
increase in insulin bioavailability compare to formulations without
thickening agent, compare to Table 25. The relative %
bioavailability of the insulin intranasal formulations containing a
thickening agent ranged from about 16.8-30.6%. The highest
bioavailability was achieved with intranasal administration of the
25 IU insulin in the 0.25% carbopol formulation.
[0252] The AUC intersubject % CV was approximately 70-97% and
50-72% for the CMC and Carbopol IN groups, respectively. The AUC
intersubject % CV was approximately 45% for NovoLog. The Cmax
intersubject % CV was approximately 61-70% and 46-74% for the CMC
and Carbopol IN groups, respectively. The Cmax intersubject % CV
was approximately 41% for NovoLog.
[0253] When contrasted to the data presented in Table 25, the
addition of a thickening agent (as presented in Table 30) increased
the extent of insulin absorption. For example, at 50 IU, the extent
of absorption (i.e., total exposure or AUC.sub.last) doubled in the
presence of the thickening agent carbopol (1319.7 .mu.U/ml,
compared to 670.8 .mu.U/ml). Similar increases in extent of
absorption were detected when the thickening agent was CMC. In each
case, there is also a corresponding increase in C.sub.max.
[0254] Analysis for pharmacodynamic parameters for each dose based
on glucose levels was conducted. The mean percent glucose reduction
data is shown in FIG. 6. For each sample, the time to maximum %
glucose fall from initial (Tmax) and maximum % glucose fall from
initial (Cmax or % Fall) were calculated, data is shown in Table 32
and Table 33, respectively.
TABLE-US-00032 TABLE 32 Glucose maximum % Fall and Time to Maximum
% Fall Results for Intranasal Insulin Formulations Containing a
Thickening Agent Administered to Human Subjects Treatment Group
Glucose Max % Fall Time to Max % Fall (min) Nasal Placebo 5.8
(4.93) 101.3 (90.43) (control) NovoLog 47.3 (8.54) 87.5 (50.29)
Nasal (25 IU) 23.7 (16.93) 88.8 (91.68) Plus CMC Nasal (50 IU) 33.2
(20.99) 56.3 (58.31) Plus CMC Nasal (25 IU) 30.6 (17.15) 75 (77.81)
Plus Carbopol Nasal (50 IU) 37 (17.45) 42.5 (12.52) Plus
Carbopol
TABLE-US-00033 TABLE 33 Incidence of Human Subjects with 30%, 20%,
and 10% Glucose Reduction Results for Intranasal Insulin
Formulations Containing a Thickening Agent Administered to Human
Subjects Subjects with Glucose % Reduction Treatment # of GE 30% GE
20% GE 10% Group Subjects N (%) N (%) N (%) Nasal Placebo 12 0 (0%)
0 (0%) 6 (50%) (control) NovoLog 12 12 (100%) 12 (100%) 12 (100%)
Nasal (25 IU) 12 1 (8.3%) 7 (58.3%) 12 (100%) Plus CMC Nasal (50
IU) 12 6 (50%) 8 (66.7%) 10 (83.3%) Plus CMC Nasal (25 IU) 12 5
(41.7%) 8 (66.7%) 10 (83.3%) Plus Carbopol Nasal (50 IU) 12 8
(66.7%) 10 (83.3%) 12 (100%) Plus Carbopol
[0255] The mean glucose change results shown in FIG. 6 illustrates
more rapid glucose fall for intranasally administered insulin
compared to NovoLog. The time to maximum % glucose fall for Nasal
Plus CMC and Nasal Plus Carbopol (both at 50 IU) was faster than
NovoLog. There was a statistical correlation for AUC and Cmax for
maximum % glucose fall levels. The nasal formulations time to
return to baseline glucose levels was quicker (90-120 minutes)
compared to NovoLog (240-300 minutes) and Exubera (>360 minutes,
data not shown).
[0256] A summary of the mean serum insulin levels (pharmacokinetic)
and mean glucose levels adjusted to baseline (PD) for human
subjects dosed with 25 IU and 50 IU doses of intranasal insulin
formulations containing a thickening agent and a control
formulation (NovoLog) is shown in FIG. 7. This figure illustrates
that the intranasal insulin formulations containing a thickening
agent result in an ultra-rapid acting insulin profile compared to
SC NovoLog in humans.
[0257] All IN formulations with a thickening agent were well
tolerated with no signs of nasal irritation.
Example 5
Physical and Chemical Stability of Nasal Spray Formulations of
Regular Human Insulin
[0258] Physical and chemical stability of nasal spray formulation
were tested. Samples were prepared as described in Tables 34
(formulations without a thickening agent) and 35 (formulations
containing a thickening agent). The materials used for manufacture
of the formulations are shown in Table 36. Osmolality, appearance,
density, viscosity, refractive index and UV absorbance were tested
at approximately T=0 for all formulations. Additionally, insulin
and preservatives content and purity were tested by HPLC.
TABLE-US-00034 TABLE 34 Manufacture of Insulin Formulations Without
a Thickening Agent Regular Me-.beta.- Insulin CD EDTA Tween 80
Arginine MP PP PG Sample # (IU/mL) (mg/mL) (mM) (mg/mL) pH Prep 1
100 0 0 0 10 0 0 0 2.0 0.22 .mu.m filtered 2 100 0 0 0 10 0 0 0 3.0
0.22 .mu.m filtered 3 100 0 0 0 10 0 0 0 7.3 0.22 .mu.m filtered 4
250 45 1 0 10 0.33 0.17 10 7.3 0.22 .mu.m filtered 5 500 45 1 0 10
0.33 0.17 10 7.3 0.22 .mu.m filtered 6 250 0 1 10 10 0.33 0.17 10
7.3 0.22 .mu.m filtered 7 500 0 1 10 10 0.33 0.17 10 7.3 0.22 .mu.m
filtered 8 0 45 1 10 10 0.33 0.17 10 7.3 0.22 .mu.m filtered 9 250
45 1 10 10 0.33 0.17 10 7.3 0.22 .mu.m filtered 10 500 45 1 10 10
0.33 0.17 10 7.3 0.22 .mu.m filtered 11 Humulin .RTM. R, 100 U 0.22
.mu.m filtered 12 Humulin .RTM. R, 500 U 0.22 .mu.m filtered 13
NovoLog .RTM., 100 U 0.22 .mu.m filtered
TABLE-US-00035 TABLE 35 Manufacture of Viscosity Enhanced Insulin
Formulations Regular Me-.beta.- Tween Arginine Carbopol CMC Insulin
CD 80 Buffer 974P NaCl LV MP PP PG # (IU/mL) (mg/mL) (mM) (mg/ml) 1
0 0 0 10 0 4 0 0 0 0 2 0 0 0 10 0 4 0 0.33 0.17 0 3 0 0 10 10 0 4 0
0.33 0.17 0 4 0 45 0 10 0 4 0 0.33 0.17 0 5 0 0 0 10 0 0 0 0.33
0.17 10 6 250 0 0 10 0 0 0 0.33 0.17 10 7 500 0 0 10 0 0 0 0.33
0.17 10 8 0 0 0 10 0 4 1 0 0 0 9 0 0 0 10 0 0 1 0.33 0.17 10 10 0 0
10 10 0 0 1 0.33 0.17 10 11 0 45 0 10 0 0 1 0.33 0.17 10 12 250 45
10 10 0 0 1 0.33 0.17 10 13 500 45 10 10 0 0 1 0.33 0.17 10 14 0 0
0 10 2.5 4 0 0 0 0 15 0 0 0 10 2.5 0 0 0.33 0.17 10 16 0 0 10 10
2.5 0 0 0.33 0.17 10 17 0 45 0 10 2.5 0 0 0.33 0.17 10 18 250 45 10
10 2.5 0 0 0.33 0.17 10 19 500 45 10 10 2.5 0 0 0.33 0.17 10 20 0
45 10 10 0 0 0 0.33 0.17 10 21 250 45 10 10 0 0 0 0.33 0.17 10 22
500 45 10 10 0 0 0 0.33 0.17 10
TABLE-US-00036 TABLE 36 Materials Used in Manufacture of Insulin
Nasal Spray Formulations Chemical Grade Vendor Cat # Human Insulin,
Recombinant, GMP USP Diosynth -- Methyl-.beta.-Cyclodextrin Pharma
Wacker 60007006 (Me-.beta.-CD) Edetate Disodium (EDTA) USP JT Baker
8994 Polysorbate 80 (Tween 80) (INS-019) USP JT Baker 4117
Polysorbate 80 (Tween 80) (INS-111) NF Spectrum P0138 L-Arginine
(INS-019) Hydrochloride USP JT Baker 2067 L-Arginine (INS-111)
Hydrochloride USP JT Baker 2067 Carboxymethylcellulose, low
viscosity USP Spectrum CA193 Carbopol 974P (Carbomer Homopolymer)
USP Noveon -- Sodium Chloride (NaCl) USP Spectrum S0155
Methylparaben NF Nastech 6215-18 (JT Baker) Propylparaben NF
Nastech 7624-18 (JT Baker) Propylene Glycol USP JT Baker 9403
Sterile Water For Irrigation (INS-019) USP Spectrum/Braun S1944
Sterile Water For Irrigation (INS-111) USP Spectrum/Braun S1944 2N
Hydrochloric Acid Research JT Baker 5616-02 2N Sodium Hydroxide
Research JT Baker 5633-02 12 mm polystyrene cuvette -- Malvern
Corp. ZEN0112 1 cc sterile disposable syringes -- BD Corp. 309628
0.22 .mu.m PDVF filter -- Millipore SLGV013 SL
[0259] The pH was measured using a Cole Parmer semi-micro NMR tube
glass pH probe (cat #05990-30) or equivalent with Orion 520Aplus pH
meter, Thermo Electron Corp (USA) or equivalent. The pH
specification for insulin nasal spray was 7.3.+-.0.3.
[0260] The osmolality of the formulations were measured with an
Advanced Micro Osmometer, Model 2020, Advanced Instruments Inc.
(Norwood, Mass.). The osmolality specification for insulin nasal
spray was 200-280 mOsm/kg H.sub.2O.
[0261] Density measurements are made using the DMA 5000 Density
Meter, Anton Paar USA (Ashland, Va.). The density is measured based
on the oscillating U-tube principle.
[0262] Formulation viscosities were measured using an AMVn
Automated Micro Viscometer, Anton Paar USA (Ashland, Va.). The AMVn
determined the dynamic and kinematic viscosity of liquids by the
rolling/falling ball principle which is based on Stoke's law.
[0263] Refractive Index (RI) measurements were performed using a
Palm Abbe PA202 Digital Refractometer, Misco Instruments
(Cleveland, Ohio). Light from an LED light was passed through the
sample; some of the light is transmitted through the solution and
lost while the remaining light is reflected onto a linear array of
photodiodes through a sapphire prism. This was then correlated by
the internal software to refractive index and displayed on the LCD
screen.
[0264] UV absorbances of all samples was measured using the
Spectramax M5 and SoftMax Pro v. 5.0 software, Molecular Devices,
Sunnyvale, Calif. The UV absorbance was read at 633 nm (i.e., the
wavelength of the Helium-Neon laser used by the Malvern Zetasizer
Nano ZS for the purposes of particle sizing).
[0265] HPLC analysis was conducted on samples at T=0 to verify
insulin and preservative content. The outputs of the analysis
include Insulin Identification, Insulin Assay, A-21 Desamido
Insulin Content, Total Other Insulin-Related Impurities Content,
Methylparaben Identification, Methylparaben Assay, Propylparaben
Identification, and Propylparaben Assay. The final product
specifications for these measurements for the insulin nasal spray
are listed in Table 37.
TABLE-US-00037 TABLE 37 Specifications for Insulin Nasal Spray
Category Specification Insulin Identification The retention time of
the major peak in the chromatogram corresponds to that of the
standard preparation (pH = 7.3 .+-. 0.3; Osmolality = 200-280
mOsm/kg H.sub.2O) Insulin Assay 80.0-120.0% of Formulation Label
Claim Insulin Related Substances A-21 Desamido Insulin Content:
Assay .ltoreq.10.0% of Insulin Related Peaks Other Insulin Related
Substances: .ltoreq.5.0% of Insulin Related Peaks Methylparaben
Identification The retention time of the major peak in the
chromatogram corresponds to that of the standard preparation
Methylparaben Assay N/A Propylparaben Identification The retention
time of the major peak in the chromatogram corresponds to that of
the standard preparation Propylparaben Assay N/A
[0266] Summaries of physical analyses of formulations from Tables
34 and 35 are shown in below Table 38 and Table 39,
respectively.
TABLE-US-00038 TABLE 38 Physical and Chemical Analysis of Insulin
Formulations Without a Thickening Agent Osmolality Vis- Sam-
(mOsm/kg Density cosity ple # pH H2O) Appearance (g/cc) (mPa-s) 1
2.0 42 clear and colorless solution 0.999 0.888 2 3.0 25 clear and
colorless solution 0.999 0.902 3 7.4 43 clear and colorless
solution 0.999 0.902 4 7.3 242 clear and colorless solution 1.014
0.901 5 7.2 260 clear and colorless solution 1.017 1.094 6 7.4 198
clear and colorless solution 1.003 1.133 7 7.2 211 clear and
colorless solution 1.005 1.006 8 7.3 220 clear and colorless
solution 1.012 1.047 9 7.4 243 clear and colorless solution 1.015
1.060 10 7.4 264 clear and colorless solution 1.017 1.287 11 7.5
213 clear and colorless solution 1.002 1.330 12 7.4 257 clear and
colorless solution 1.006 0.944 13 7.3 267 clear and colorless
solution 1.004 0.991
TABLE-US-00039 TABLE 39 Physical and Chemical Analysis of Insulin
Formulations Containing a Thickening Agent HPLC Analysis Osmolality
Refractive Insulin Preservative (mOsm/kg Density Viscosity Index UV
(% LC) (% LC) # pH H2O) Appearance (g/cc) (mPa-s) (nD) Absorbance
Content Impurities MP PP 1 7.5 160 CC 1.001 0.895 1.334 0.00 0.0
0.0 0.0 0.0 2 7.1 154 CC 1.001 0.927 1.335 0.00 0.0 0.0 158.2 119.2
3 7.4 154 CC 1.001 0.951 1.336 0.00 0.0 0.0 162.1 120.7 4 7.3 202
CC 1.013 0.966 1.340 0.00 0.0 0.0 107.8 117.8 5 7.2 171 CC 0.999
1.045 1.335 0.00 0.0 0.0 109.1 117.4 6 7.2 183 CC 1.002 0.936 1.337
0.00 105.0 0.9 110.4 110.3 7 7.2 189 CC 1.004 0.974 1.338 0.00
102.2 0.9 108.2 122.2 8 7.5 150 CC 1.001 1.013 1.334 0.00 0.0 0.0
0.0 0.0 9 7.4 184 CC 1.000 1.148 1.335 0.00 0.0 0.0 109.9 121.7 10
7.5 195 CC 1.001 1.273 1.336 0.00 0.0 0.0 111.6 122.7 11 7.3 232 CC
1.012 1.349 1.341 0.00 0.0 0.0 111.7 123.4 12 7.3 248 CC 1.016
1.465 1.344 0.00 107.7 1.4 113.8 120.6 13 7.3 256 CC 1.018 1.757
1.346 0.00 107.5 1.4 112.4 134.9 14 7.2 237 ST 1.004 1.810 1.335
0.00 0.0 0.0 0.0 0.0 15 7.2 197 ST 1.001 1.946 1.335 0.09 0.0 0.0
110.6 122.1 16 7.2 192 CC 1.003 9.839 1.337 0.07 0.0 0.0 111.4
152.4 17 7.5 232 CC 1.013 12.103 1.341 0.01 0.0 0.0 106.2 148.5 18
7.2 325 CC 1.017 21.668 1.344 0.02 99.7 2.8 106.2 154.9 19 7.2 257
ST 1.019 4.957 1.347 0.01 106.6 2.3 113.2 160.8 20 7.2 228 CC 1.012
7.693 1.342 0.11 0.0 0.0 110.4 157.4 21 7.5 237 CC 1.015 1.255
1.344 0.00 106.6 1.7 113.4 165.0 22 7.4 245 CC 1.017 1.304 1.346
0.00 106.3 1.4 111.5 174.0 CC = a clear and colorless solution was
observed; ST = a slightly turbid solution was observed; LC =
formulation label claim
[0267] All formulations manufactured were clear and colorless in
appearance except formulations 14, 15 and 19, which contained a
thickening agent and were slightly turbid. These formulations
contained Carbopol 974P, which is a very large molecule (molecular
weight .about.150,000) and is therefore may be difficult to
solubilize without surfactants (e.g., Tween 80).
[0268] All initial and final pH measurements were within the range
of 7.3.+-.0.3, with the exception of formulations 1 and 2 which did
not contain a thickening agent. The target pH for these two
samples, were pH 2.0 and pH 3.0 respectively.
[0269] Osmolality measurements were generally within the range from
about 200 to about 280 mOsm/kg H.sub.2O (i.e., within the range set
forth in the insulin nasal spray final product specification), with
the exception of formulations 1, 2 and 3 which did not contain a
thickening agent and formulations 1, 2, 3, 5, 8 and 18 which did
contain a thickening agent. The osmolality of these formulations
was outside the expected range because they did not contain
propylene glycol (i.e., the component within the insulin nasal
spray formulation that has the largest effect upon tonicity) nor
did they contain adequate sodium chloride to compensate for the
absence of propylene glycol. the thickening agent modified
formulation 18 was measured to have higher than expected osmolality
at 325 mOsm/kg H2O. This observation may be attributed to the
relative high amounts of sodium hydroxide and hydrochloric acid
required to adjust pH and clarify the solution.
[0270] A summary of the chemical analyses performed on formulations
containing a thickening agent are outlined in Table 39 above. HPLC
was conducted on the formulations containing a thickening agent per
the finished product specification insulin nasal spray to determine
insulin identity, insulin assay, A-21 desamido insulin content,
total other insulin related impurities, preservative identification
(if appropriate), and preservative quantitation (if appropriate).
HPLC analysis was not conducted on formulations that did not
contain a thickening agent.
[0271] Insulin identification passed the required specification for
insulin nasal spray for all formulations containing a thickening
agent and insulin.
[0272] Insulin assay was measured to be within the 80.0-120.0% of
label claim set forth in the finished product specification for all
formulations containing a thickening agent and insulin.
[0273] The A-21 desamido content as a percent of insulin related
peaks were .ltoreq.1% for all samples tested, well within the less
than or equal to 10% specification. The total other insulin-related
impurities were measured to be less than 5%, which is within the
specification, for all samples tested.
[0274] All formulations that contained methylparaben and
propylparaben (i.e., the formulation preservatives) passed the
requirements for preservative identification set forth in the
finished product specification for insulin nasal spray.
[0275] The HPLC analysis for preservative recovery of the
formulations containing methylparaben and propylparaben
demonstrated a higher level of preservatives for most samples
evaluated in formulations containing a thickening agent. The
preservative levels trended a bit high within the samples due to
how parabens perform within the formulations. Previous studies have
consistently yielded relative low (i.e., approximately 80% of label
claim) recoveries of methylparaben and propylparaben even though
the correct amounts of each component are added to the formulations
during the manufacturing process. To compensate for this loss, the
formulations containing a thickening agent were manufactured
assuming this 20% loss (i.e., the formulations manufactured
assuming 0.40 mg/mL methylparaben and 0.2 mg/mL propylparaben in
hopes of obtaining final concentrations of 0.33 mg/mL and 0.17
mg/mL of methylparaben and propylparaben respectively).
[0276] In addition, as determined herein, the "in use stability" of
thickening agent modified formulations and those formulations
manufactured without a thickening agent (e.g., with CMC or
Carbopol) was evaluated. All such formulations tested were shown to
be stable upon spraying, including when evaluated for insulin
content, impurities and shot weight. Similarly, formulations
containing a thickening agent were shown to be stable after storage
for eighty days at 25.degree. C. and 60% relative humidity; and,
under accelerated stability conditions of 40.degree. C. and 75%
relative humidity. In addition, at 5.degree. C. and ambient
humidity (referred to as "as sold" stability), all formulations
containing a thickening agent were shown to be stable.
Example 6
Stable Nasal Spray Formulations of Regular Human Insulin in
Monomeric/Dimeric Form
[0277] Particle size characterization studies were conducted to
determine the physical (e.g., oligomeric) state of insulin within
the insulin nasal spray formulations. Such determinations may be
evaluated by determining the particle size distribution of
formulations containing insulin in combination with one or more of
the various pharmaceutically acceptably excipients disclosed herein
(e.g., methyl-.beta.-cyclodextrin, Polysorbate 80, edetate
disodium, propylene glycol, arginine, methylparaben, propylparaben,
carboxymethylcellulose sodium (CMC), and carbomer (e.g., carbopol
974P) using a subtraction style experimental design. For clarity,
as used herein, carbopol is a carbomer; carbopol 974P is also known
as Carbomer Homopolymer Type B, or Carbopol.RTM. 974P NF
Polymer.
[0278] Clinically, two relevant dosage strengths of insulin nasal
spray, 250 and 500 IU/mL, were evaluated (IU=international units).
There are approximately 28 IU per milligram of insulin). The
insulin nasal spray formulations without or with a thickening agent
(e.g., CMC and Carbopol) that were evaluated in this Example are
presented above Table 34 and Table 35. In addition, two different
marketed injectable insulin products were also evaluated as
comparators. Humulin.RTM. R is an injectable product which contains
zinc, which has been shown to stabilize regular human insulin in
the hexameric form. Humulin is known as an "intermediate acting"
injectable insulin. The other marketed injectable product tested,
NovoLog (insulin aspart), contains a chemically modified form of
insulin designed to favor the monomeric state, and thus providing a
rapid acting injectable insulin. The IN formulations including a
thickening agent that were tested are shown in Table 40.
TABLE-US-00040 TABLE 40 Insulin Nasal Spray Formulations Containing
a Thickening Agent Carbopol Formulation CMC Formulation
Concentrations Concentrations Component mg/mL orIU/mL mM mg/mL or
IU/mL mM Human Insulin, Recombinant 250 and 500 IU/mL -- 250 and
500 IU/mL -- Methyl-.beta.-Cyclodextrin (Me-.beta.- 45 mg/mL 33.3
45 mg/mL 33.3 CD) Edetate Disodium Dihydrate 1 mg/mL 2.7 1 mg/mL
2.7 (EDTA) L-Arginine Hydrochloride 2.1 mg/mL 10 2.1 mg/mL 10
Polysorbate 80 10 mg/mL 7.6 10 mg/mL 7.6 Carboxymethylcellulose
Sodium 0 mg/mL 0 1 mg/mL <0.0.sup..dagger. (CMC) Carbopol 974P
(Carbomer) 2.5 mg/mL <0.0.sup..dagger. 0 mg/mL 0 Propylene
Glycol 10 mg/mL 25 10 mg/mL 25 Methylparaben 0.33 mg/mL 2.2 0.33
mg/mL 2.2 Propylparaben 0.17 mg/mL 0.9 0.17 mg/mL 0.9
.sup..dagger.Molecular weights in excess of 100,000 Da
[0279] The Malvern Zetasizer Nano ZS was used to measure particle
size. The instrument uses a Helium-Neon laser and non-invasive
back-scatter technology to determine the hydrodynamic radii of
particles. The size of a particle is indirectly proportional to its
Brownian motion (through its diffusion co-efficient) and this
relation is given by the Stokes-Einstein equation:
d(H)=kT/3 .pi..eta.D where: d(H)=hydrodynamic diameter; D
=translational diffusion coefficient; k=Boltzmann's constant;
T=absolute temperature; and .eta.=viscosity.
[0280] The amount of back-scatter of the particles in Brownian
motion is detected by the instrument and is then auto-correlated to
give the hydrodynamic radius.
[0281] Disposable polystyrene cuvettes (12 mm.sup.2, low-volume
from Malvern Instruments USA, catalog #ZEN0112) are used to read
the samples within the instrument. Each cuvette was first air-blown
to remove any dust or lint particles. 500 .mu.l of the sample was
first loaded into a sterile-packed 1 cc syringe (BD Corp., catalog
#309628) and then filtered into the cleaned cuvette through a low
protein-binding, 13 mm diameter, 0.22 .mu.m pore size PVDF syringe
filter (Millipore Corporation, catalog #SLGV013 SL).
[0282] Control cuvettes were loaded with 1.0 mL of sterile filtered
water (0.02 .mu.m filtered). The standards used to calibrate the
instrument were: lysozyme (.about.3.8 nm diameter), Bovine Serum
Albumin (.about.7.2 nm diameter) 20, 30 and 40 nm diameter
polystyrene Duke Standards. All standards were appropriately
diluted in filtered water before testing as highly concentrated
samples lead to a large amount of light scattering and an
overloaded detector. Each test cuvette was loaded with the
appropriate insulin sample and only those samples that were
observed to be visually clear and colorless in appearance are
assayed.
[0283] The instrument chamber was first equilibrated for at least
30 minutes prior to conducting the first measurement in order to
stabilize the temperature of the sample/cell chamber. The
pre-loaded cuvette was then capped and placed in the cell chamber
to conduct the measurements. Approximately three minutes were
provided for each sample to equilibrate to 25.degree. C. within the
sample chamber before each reading. Three measurements of ten
readings each are taken per cuvette. Each sample involved two
separate cuvettes to ensure reproducibility of the data.
[0284] Standards of known particle size were tested to confirm
validity of the assay. The particle size measurements of the
standards were close to their theoretical values obtained from
literature. Table 41 shows the comparison of the known
(theoretical) values and observed (determined) particle size values
of the standards.
TABLE-US-00041 TABLE 41 Comparison of Theoretical and Determined
Particle Sizes of Standards Theoretical Size Determined Size (nm)
Standard (nm) Study 1 Study 2 Lysozyme 3.8.sup.a 3.83 .+-. 0.7 Not
measured BSA 7.2.sup.a 6.31 .+-. 1.0 6.3 .+-. 1.0 Duke 20 nm 21.0
.+-. 1.5.sup.b 19.2 .+-. 3.0 19.5 .+-. 3.1 Duke 30 nm 33.0 .+-.
1.4.sup.b 33.0 .+-. 4.4 32.2 .+-. 4.4 Duke 40 nm 40.0 .+-.
1.8.sup.b 42.3 .+-. 5.4 41.0 .+-. 5.1 .sup.afrom Malvern technical
notes:
www.malvern.co.uk/LabEng/industry/protein/protein_solutions.htm
.sup.bDuke standards' specifications sheet
[0285] The control samples of insulin in saline at various pH
levels provided results in agreement with previously published
data. As depicted in Table 42 the marketed products tested, insulin
in both the strengths of Humulin.RTM. R appear to be in the
monomer/dimer form, whereas NovoLog appears to be stabilized in the
hexameric state. In addition, the formation of insulin complexes
(i.e., dimer or hexamer) is known to be dependent upon solution pH.
At pH 2.0, the insulin is thought to be stabilized predominantly in
the monomeric form; at pH 3.0, the dimeric form is thought to be
dominant; and at pH 7.0 the molecules are thought to be stabilized
in the hexameric state. These values are based upon the theoretical
monomer, dimer and hexamer sizes obtained from literature. Table 42
compares the observed and theoretical particle sizes of the
controls.
TABLE-US-00042 TABLE 42 Comparison of Theoretical and Determined
Particle Sizes of Insulin Controls Perceived Theoretical Determined
Insulin Size Size Oligomeric Formulation (nm) (nm) State No
Thickening agent 1 (pH 2) 2.7 2.6 .+-. 0.5 Monomer Humulin 100 U/ml
N/A 2.8 .+-. 0.5 Monomer Humulin 500 U/ml N/A 2.9 .+-. 0.5 Monomer
No Thickening Agent 3.4 2.7 .+-. 0.5 Dimer 2 (pH 3) No Thickening
Agent 5.2 4.3 .+-. 0.7 Hexamer 3 (pH 7) NovoLog 100 U/ml N/A 4.4
.+-. 0.7 Hexamer N/A = not available
[0286] Particle sizes consistent with those of insulin monomer,
dimer and hexamer sizes presented in the literature were
experimentally confirmed via the dynamic light scattering
evaluation of samples that contain insulin, saline, and buffers
appropriate for the various pH levels tested (i.e., pH 2, 3, and
7).
[0287] The control formulations, Humulin R and NovoLog, performed
as expected via dynamic light scattering. Both the 100 IU/mL and
the 500 IU/mL concentration formulations of Humulin were found to
contain a single species, likely to be insulin, in the presumed
monomeric state (2.8 nm-2.9 nm). The 100 IU/mL NovoLog, insulin was
found to probably exist in hexamer state.
[0288] In the study of formulations that did not contain a
thickening agent from Table 34, the low insulin concentration nasal
spray formulation (sample 9) was determined to contain particles,
the majority of which (99% by volume) were found to be 3.1.+-.0.7
nm in size, whereas a second population of particles (i.e., the
remaining 1% by volume) were measured to be 33.2.+-.7.8 nm. When
the same formulation was evaluated in the study of formulations
that did contain a thickening agent from Table 35; formulation 21
containing a thickening agent, the particle size distribution was
determined to contain a third population (i.e., 41% by volume) of
particle size 1.4.+-.0.2 nm. The other particle sizes detected were
mostly 3.5.+-.0.6 nm (i.e., 58% by volume) and a small population
(i.e., 1% by volume) of size 29.3.+-.12.6 nm.
[0289] In case of the high insulin concentration formulation
(sample 10 without thickening agent from Table 34 and sample 22
with a thickening agent from Table 35), the particle size
distribution was virtually identical in the two studies.
Approximately one third of the particles detected by volume were
roughly 1.5.+-.0.2 nm in size while the remaining approximate two
thirds were determined to be roughly 3.5.+-.0.6 nm in size. As
observed in the lower concentration formulation (sample 9
manufactured without a thickening agent and sample 21 manufactured
with a thickening agent), approximately 1% by volume was determined
to be 33.5.+-.0.5 nm in size.
[0290] The average particle size distributions of the two strengths
were virtually identical. Three separate species appeared to exist
in each of the strengths, approximately one third of the particles
by volume were of roughly 1.5 nm diameter while the remaining
approximate two-thirds by volume were of roughly 3.5 nm diameter.
Finally, a third population of roughly 33 nm diameter was observed,
but comprised approximately 1% of the population by volume.
[0291] Tables 43 and 44 provide a summary of the particle size
distributions of the samples (formulations manufactured without a
thickening agent from Table 34 and formulations manufactured with a
thickening agent from Table 35, respectively) tested in the two
studies.
TABLE-US-00043 TABLE 43 Summary of Particle Size Distribution of
Insulin Formulations Manufactured Without a Thickening Agent
Determined Size in nm (% Volume) Formulation Peak 1 Peak 2 Peak 3 4
2.2 .+-. 0.6 N/A N/A 5 1.0 .+-. 0.1 (9%) 3.2 .+-. 0.7 (91%) N/A 6
5.2 .+-. 1.0 N/A N/A 7 5.2 .+-. 0.9 N/A N/A 8 8.9 .+-. 1.6 (27%)
32.0 .+-. 8.1 (73%) N/A 9 3.1 .+-. 0.7 (99%) 33.2 .+-. 7.8 (1%) N/A
10 1.5 .+-. 0.2 (31%) 3.5 .+-. 0.6 (68%) 33.5 .+-. 0.5 (1%) N/A =
not applicable (no reading)
TABLE-US-00044 TABLE 44 Summary of Particle Size Distribution of
Insulin Formulations Manufactured With a Thickening Agent For- mu-
Determined Size in nm (% Volume) lation Peak 1 Peak 2 Peak 3 1 0.8
.+-. 0.1 N/A N/A 2 0.8 .+-. 0.1 N/A N/A 3 8.0 .+-. 1.1 N/A N/A 4
1.2 .+-. 0.2 N/A N/A 5 0.7 .+-. 0.1 N/A N/A 6 5.1 .+-. 0.9 N/A N/A
7 5.0 .+-. 0.9 N/A N/A 8 8.8 .+-. 2.5 N/A N/A 9 1.9 .+-. 0.2 (15%)
6.8 .+-. 0.9 (85%) N/A 10 6.1 .+-. 0.9 N/A N/A 11 1.0 .+-. 0.2 N/A
N/A 12 2.2 .+-. 0.5 (99%) 24.5 .+-. 6.3 (1%) N/A 13 0.9 .+-. 0.1
(17%) 2.5 .+-. 0.4 (82%) 19.6 .+-. 5.7 (1%) 14 N/A N/A N/A 15 2.3
.+-. 0.2 (50%) 5590.0 .+-. 323.0 (50%) N/A 16 0.8 .+-. 0.1 N/A N/A
17 3080.0 .+-. 483.5 N/A N/A 18 1.3 .+-. 0.2 (98%) 6.8 .+-. 2.4
(1%) N/A 19 0.6 .+-. 0.0 (99%) 6.5 .+-. 0.5 (1%) N/A 20 30.4 .+-.
6.5 (100%) N/A N/A 21 1.4 .+-. 0.2 (41%) 3.5 .+-. 0.6 (58%) 29.3
.+-. 12.6 (1%) 22 1.5 .+-. 0.2 (33%) 3.6 .+-. 0.7 (66%) 35.7 .+-.
12.2 (1%) N/A = not applicable (no reading)
[0292] When the particle size of the formulations without Tween 80
(i.e., sample 4 without a thickening agent from Table 43,
cross-referencing Table 34) were evaluated, the low insulin
concentration was found to be comprised of a single population of
particles sized 2.2.+-.0.6 nm diameter. In contrast, the particle
size distribution of the high insulin concentration formulation is
found to contain bimodal particle size distribution. The majority
(91% by volume) of the particles were of diameter 3.2.+-.0.7 nm
while the remaining (9% were by volume) are of diameter 1.0.+-.0.1
nm, and may represent a concentration-dependent effect upon
particle formulation in the absence of Tween 80.
[0293] In the absence of methyl-.beta.-cyclodextrin (i.e., samples
6 and 7 manufactured without a thickening agent from Table 34) both
low and high insulin concentrations yielded a single peak of the
same size; 5.2.+-.1.0 nm and 5.2.+-.0.9 nm, respectively.
[0294] Formulations that contain insulin but no Tween 80 or
methyl-.beta.-cyclodextrin were evaluated. A size distribution
similar to that observed in the formulations without
methyl-.beta.-cyclodextrin was observed (i.e., 5.0.+-.0.9 nm). This
result is consistent with the approximate size of insulin hexamers
(i.e., sample 3 manufactured without a thickening agent), which is
the likely complexation state of insulin expected to be prevalent
in pH 7.3 in arginine buffer. These data may indicate that there is
a difference between those formulations that contain insulin,
methyl-.beta.-cyclodextrin and Tween 80, but that the formulations
that contain insulin and Tween 80 or insulin alone are equivalent.
Therefore, it appears that there may be an unexpected molecular
interaction between the three components (i.e., Tween 80,
methyl-.beta.-cyclodextrin, and insulin) that allows for the
formation of particles of a size consistent with insulin
monomer/dimer to be formed.
[0295] A placebo formulation (sample 20 manufactured with a
thickening agent as identified in Table 35) was evaluated and was
found to contain a single species of particle size 30.4.+-.6.5 nm.
A formulation of identical composition was evaluated, sample 8
without a thickening agent (see Table 34), and was observed to
contain two populations of particle sizes 8.9.+-.1.6 nm (27% by
volume) and 32.0.+-.8.1 nm (73% by volume). The difference in the
results obtained for a formulation of identical composition may be
explained by the presence of a potential outlier peak that was
observed in a single measurement in the study presented in Table 43
(without a thickening agent) and was included in the average
particle size calculation. When this outlier is removed from the
average calculation, a monomodal particle size distribution is
observed with average particle size 31.5.+-.7.1 nm, which is very
similar to the average obtained for the equivalent formulation
evaluated in the study presented in Table 35, which includes the
addition of a thickening agent.
[0296] A placebo formulation that contained
methyl-.beta.-cyclodextrin but no Tween 80 showed a single peak at
1.1.+-.0.3 nm, which corresponded with the expected size of
methyl-.beta.-cyclodextrin inclusion complexes. Placebo that
contained Tween 80 but no methyl-.beta.-cyclodextrin was also found
to be comprised of a single species, but of a larger size,
8.1.+-.1.0. This particle size distribution is likely attributed to
the formation of Tween 80 micelles. The formulation concentration
of 10 mg/mL (i.e., 7.6 mM) is several times higher than the
critical micelle concentration of 0.1 mM.
[0297] Formulations that contained propylene glycol were compared
to those that did not. The particle size distributions remain
unchanged in the presence or absence of propylene glycol,
indicating that propylene glycol did not have an effect of the
formation of methyl-.beta.-cyclodextrin complexes or Tween 80
micelles (or the interaction between methyl-.beta.-cyclodextrin,
Tween 80, and insulin interactions) within the formulation.
[0298] Formulations containing carboxymethylcellulose sodium, low
viscosity (CMC LV) were evaluated in the study presented in Table
35 (thickening agent modified formulations). The formulation
containing 1 mg/mL CMC LV in arginine buffer at pH 7.3 was found to
contain a single species of particle size 8.8.+-.2.5 nm. When the
preservatives and propylene glycol were added to this formulation,
the distribution was observed to be bimodal with peaks at particle
size 1.9.+-.0.2 nm (15% by volume) and 6.8.+-.0.9 nm (85% by
volume). Further, when Tween 80 was included in the formulation
(thickening agent containing sample 10, Table 35), a monomodal
distribution of particles was regained with average particle
diameter 6.1.+-.0.9 nm. However, the presence of
methyl-.beta.-cyclodextrin (in the absence of Tween 80) resulted in
a single species but of smaller particle diameter; 1.0.beta.0.2 nm.
Low and high concentration insulin nasal spray formulations
(samples 12 and 13, containing a thickening agent, Table 35)
consisted mostly of particles of average size 2.2.+-.0.5 nm (99% by
volume) and 2.5.+-.0.4 nm (82% by volume) respectively. The high
insulin concentration formulation (sample 13, containing a
thickening agent, Table 35) had an additional peak of particle size
0.9.+-.0.1 nm (17% by volume), as a result of an outlier during one
measurement.
[0299] The sample 17 (see Table 35) containing carbopol 974P as
thickening agent and methyl-.beta.-cyclodextrin was comprised of a
population of particles greater than 3 .mu.m and other populations
that are of size greater than the measuring range of the instrument
(i.e., over 10 .mu.m). A single population of particles of size
0.8.+-.0.1 nm diameter was observed in a formulation containing
Tween 80 and Carbopol 974P in arginine buffer. This observation is
different from the size of Tween 80 micelles observed in the
absence of Carbopol 974P (average particle size 8.1.+-.1.0 nm). Low
and high concentration insulin nasal spray formulations containing
Carbopol 974P had particle sizes 1.3.+-.0.2 nm (98% by volume) and
0.6.+-.0.0 nm (99% by volume) in size, respectively.
[0300] In summary, the identified insulin nasal spray formulations,
including formulations containing a thickening agent such as CMC or
Carbopol 974P, contain particles that are consistent in size with
insulin monomers and/or dimers.
[0301] According to the data presented herein, there was no
difference in particle size distribution when the formulations were
either filtered through a 0.22 um polystyrene syringe filter,
centrifuged at 100 rpm for 5 minutes, or were unfiltered or not
centrifuged.
[0302] Tween 80 is known to form micelles at concentrations of at
least 5 .mu.M, (note that the Insulin Nasal Spray formulation
contains 7 mM Tween 80) and these micelles were observed to be the
approximate size of 8 nm. Methyl-.beta.-cyclodextrin is also known
to forms complexes that are approximately 1 nm in size within the
Insulin Nasal Spray formulation; these approximate 1 nm particles
were observed as expected. The presence or absence of propylene
glycol does not seem to affect either the Tween 80 micelle
formation or the methyl-.beta.-cyclodextrin complex formation.
[0303] Insulin at high and low concentrations in arginine buffer
(i.e., in the absence of Tween 80 and methyl-.beta.-cyclodextrin)
at pH 7.3 was observed to contain particles that are consistent
with insulin molecules in the hexameric state (i.e., .about.5.2
nm). The addition of Tween 80 to the system does not alter the size
of the particles (i.e., the .about.5.2 nm particle population is
observed, with a second population at .about.8 nm, representing the
probable population of Tween 80 micelles). The addition of
methyl-.beta.-cyclodextrin results in a single peak at
approximately 2.2 nm for the low concentration insulin nasal spray
formulation (i.e., a particle consistent with the size of an
insulin monomer or dimer). A bimodal distribution (i.e., 3.2 nm
(91% by volume and 1.0 nm (9% by volume)) was observed for the high
concentration insulin nasal spray formulation. These findings may
indicate a concentration dependence for insulin monomer/dimer
stabilization; i.e., that the formulation excipients may need to be
properly ratioed to account for insulin concentration. The data
disclosed may indicate that methyl-.beta.-cyclodextrin and Tween 80
act synergistically to stabilize insulin in the monomeric or
dimeric (i.e., rather than hexameric) form. The data indicate that
particles consistent in size to the theoretical size of insulin
monomer/dimer are formed within certain of the insulin nasal
formulations disclosed herein independent of insulin
concentration.
[0304] Insulin was present in the monomeric/dimeric form within the
insulin nasal spray formulation containing CMC and there was a
similar size distribution for both low and high concentration
formulations. The data also indicate that insulin is present in the
monomeric/dimeric form within insulin nasal spray formulations
containing Carbopol 974P for both in the low and high concentration
formulations.
[0305] The dynamic light scattering data show the surprising result
that regular human insulin molecules were stabilized in the
monomeric or dimeric form within the nasal spray formulations,
including formulations containing a thickening agent such as CMC or
Carbopol 974P. These results in combination with the biological
data in rabbits and humans show ultra-rapid acting insulin is
achieved with the described nasal spray formulations.
Example 7
Preservative Optimization of Formulations Comprising a Thickening
Agent and Insulin
[0306] This example summarizes studies performed in order to
develop a preservative system for Insulin Nasal Spray formulations
suitable for USP Antimicrobial Effectiveness Testing requirements
and EP Antimicrobial Effectiveness Testing requirements. A variety
of pharmaceutically acceptable preservatives were screened that are
known to be used in currently marketed nasal spray products, and
all levels selected for this study are within the range of
concentrations in those currently marketed products for each
preservative. A list of exemplary preservatives evaluated is shown
in Table 45.
TABLE-US-00045 TABLE 45 Preservatives used in Insulin Nasal Spray
Formulations Preservative Levels Evaluated Benzethonium Chloride
0.1-0.2 mg/mL Methylparaben* 0.33-4.2 mg/mL Propylparaben* 0.17-2
mg/mL Phenylethyl Alcohol 1-2 mg/mL Benzyl Alcohol 1-5 mg/mL
Ethanol 2 mg/mL *Note that both the sodium salts and free base
phenols have been evaluated
[0307] The antimicrobial effectiveness of a formulation is
determined using the USP and EP Antimicrobial Effectiveness Testing
(AET) methodologies, which are described in USP <51> and EP
<5.4.1>. The requirements for each test are represented in
Tables 46 and 47.
TABLE-US-00046 TABLE 46 USP AET Requirements, USP <51> USP
AET Requirements, USP <51> Microorganism P. aeurginosa E.
coli S. aureus C. albicans A. niger Days 14 28 14 28 14 28 14 28 14
28 Log Reduction 2 no 2 no 2 no no no no no (Min) increase increase
increase increase increase increase increase
TABLE-US-00047 TABLE 47 EP AET Requirements, EP <5.4.1> EP
AET Requirements, EP <5.4.1> Microorganism P. aeurginosa S.
aureus C. albicans A. niger Days 2 7 28 2 7 28 14 28 14 28 Log
Reduction 2 3 no 2 3 no 2 no 2 no increase (Min) increase increase
increase
[0308] The quality (physical and chemical analysis) of all
formulations to be evaluated was monitored for pH, osmolality, and
appearance at the time of manufacturing. In addition, HPLC analysis
was performed at T=0 and T=end of study to ensure stability of
insulin and of the preservative, as necessary. The data were used
to identify a combination of preservatives that are successful in
passing USP AET requirements for both the carbomer (e.g., Carbopol
974P) and CMC Insulin Nasal Spray formulations. Table 48
illustrates the tested combinations.
TABLE-US-00048 TABLE 48 Effective Preservative Combination
Formulations (i.e., Passes USP AET Requirements) Insulin
Polysorbate Propylene Conc. Me-.beta.-CD EDTA 80 Arginine Glycol
CMC CH PE ID # (IU/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)
(mg/mL) (mg/mL) MP/PP (mg/L) pH 1 250 45 1 10 2.1 10 0 2.5 3.3/1.7
2 7.3 .+-. 0.3 2 500 45 1 10 2.1 10 0 2.5 3.3/1.7 2 7.3 .+-. 0.3 3
250 45 1 10 2.1 10 1 0 3.3/1.7 2 7.3 .+-. 0.3 4 1000 45 1 10 2.1 10
1 0 3.3/1.7 2 7.3 .+-. 0.3 Abbreviations: Me-.beta.-CD:
methyl-.beta.-cyclodextrin, EDTA = edetate disodium, Polysorbate 80
= Tween 80, CMC = carboxymethylcellulose sodium, CH = carbomer
homopolymer (trade name: Carbopol 974P), MP = methylparaben, PP =
propylparaben, PE = phenylethanol
[0309] The formulations presented in Table 48 underwent release
testing that included appearance, pH, osmolality, and
quantification of insulin content, methylparaben content,
propylparaben content, A-21 desamido insulin content, total other
insulin related substances by Reverse Phase HPLC. The formulations
underwent post-testing at the end of the assay (i.e., one month
post-manufacturing, formulations stored at 2-8.degree. C.) to
ensure stability and product quality. The release data are listed
in Table 49, and the end of study data are listed in Table 50. The
data illustrate that the formulations evaluated are stable over the
1 month duration required for Antimicrobial Effectiveness Testing
per the US and European compendia.
TABLE-US-00049 TABLE 49 Release Testing of Formulations Passing USP
AET Requirements Total other A-21 insulin desamido related Methyl-
Propyl- Formulation Insulin insulin substances paraben paraben
Number Appearance pH Osmolality Assay content content content
content 1 Slightly 7.3 250 101.4 1.0 1.8 100.4 101.5 turbid
solution 2 Slightly 7.3 258 100.9 0.6 1.3 97.6 101.1 turbid
solution 3 Clear and 7.3 242 102.9 0.7 1.2 97.5 100.9 colorless
solution 4 Clear and 7.3 281 103.2 0.8 0.9 97.1 101.8 colorless
solution
TABLE-US-00050 TABLE 50 Post Testing of Formulations Passing USP
AET Requirements Total other A-21 insulin desamido related Methyl-
Propyl- Formulation Insulin insulin substances paraben paraben
Number Appearance pH Osmolality Assay content content content
content 1 Slightly 7.3 245 96.4 0.6 3.2 96.1 99.6 turbid solution 2
Slightly 7.4 258 99.7 0.7 2.2 95.3 102.8 turbid solution 3 Clear
and 7.4 239 101.1 0.9 0.9 97.1 101.9 colorless solution 4 Clear and
7.4 269 103.3 1.0 0.8 101.1 108.1 colorless solution
[0310] The USP AET results for the formulations presented in Tables
48 are listed in Table 51. The corresponding EP AET results are
listed in Table 52.
TABLE-US-00051 TABLE 51 Representative USP AET Data (Log Reduction)
for Specific Formulations Microorganism C. P. aeurginosa E. coli S.
aureus albicans A. niger Days 14 28 14 28 14 28 14 28 14 28
Formulation 1 5.8 5.8 5.9 5.9 5.9 5.9 1.2 2.1 1.2 2.1 Formulation 2
5.8 5.8 5.9 5.9 5.9 5.9 1.2 2.1 1.2 2.1 Formulation 3 5.5 5.5 5.8
5.8 5.7 5.7 1.1 1.9 1.8 3.3 Formulation 4 5.5 5.5 5.8 5.8 5.7 5.7
0.9 2.1 1.7 2.0
TABLE-US-00052 TABLE 52 Representative EP AET Data (Log Reduction)
for Specific Formulations Microorganism P. aeurginosa S. aureus C.
albicans A. niger Days 2 7 28 2 7 28 14 28 14 28 Formulation 5.8
5.8 5.8 0.2* 3.1 5.9 1.2** 2.1 1.2* 2.1 1 Formulation 5.8 5.8 5.8
0.3* 3.4 5.9 1.2** 2.1 1.2** 2.1 2 Formulation 5.5 5.5 5.5 0.3* 3.3
5.7 1.1** 1.9 1.8** 3.3 3 Formulation 5.5 5.5 5.5 0.6* 3.5 5.7 0.9*
2.1 1.7** 2.0 4 *Indicates failing result **Passes EP "Category B"
Requirements (requires a 1-log reduction of C. albicans and A.
niger at T = 14 days rather than the 2-log reduction required by
the "Category A" requirements)
[0311] As presented in Tables 51 and 52, all four formulations
tested pass USP AET requirements. In addition, all formulations are
demonstrated to be stable over the course of this study, providing
additional confidence in the final AET data.
[0312] In summary, a combination of preservatives were evaluated
and shown to provide antimicrobial activity within Insulin Nasal
Spray formulations sufficient to pass US compendia antimicrobial
effectiveness testing requirements. In addition, these formulations
were shown to be stable for a least one month based upon physical
(i.e., appearance, pH, osmolality) and chemical (i.e., insulin
assay, A-21 desamido insulin, total other insulin related
substances, methylparaben assay, and propylparaben assay) stability
measurements.
[0313] All U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications,
non-patent publications, figures, tables, and websites referred to
in this specification are expressly incorporated herein by
reference, in their entirety.
[0314] Although the foregoing disclosure has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications are comprehended by the disclosure and may be
practiced without undue experimentation within the scope of the
appended claims, which are presented by way of illustration not
limitation.
* * * * *
References