Note: Descriptions are shown in the official language in which they were submitted.
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PHARMACEUTICAL SOLUBILIZED IN AEROSOL PROPELLANT
Technical Field
The present invention relates to an improved
delivery system for the administration of large-molecule
pharmaceuticals, e.g. peptidic drugs, vaccines and
hormones. In particular it relates to pharmaceuticals
which may be administered by means of an aerosol into
the mouth, for buccal or pulmonary application.
Background Art
Sub-optimal disease management for respiratory
illnesses, e.g. asthma, cystic fibrosis, and chronic
obstructive pulmonary disease (COPD) collectively
represents about billion dollar worldwide market for
biotechnology-derived proteins. The pulmonary delivered
protein, represent an enormous market opportunity for
pulmonary drug delivery. The delivery of drugs via
inhalation for local delivery to the upper lung (most
commonly in the form of metered-dose inhalers) and for
systemic delivery (into the bloodstream) via the deep
lung defines the scope of pulmonary drug delivery, is
the subject of intense research.
For more than a decade, companies have searched
extensively to find a drug delivery technology which is
patient-friendly, non-invasive, and an economically
viable alternative to injecting the large macromolecule
proteins. Some of the earliest efforts involved
transdermal delivery via electroporations but this has
mostly been abandoned as large molecules simply can't
pass through the skin. Oral delivery, which would
clearly be the preferable dosage form, has had some
success, but a major obstacle is the degradation and
denaturization of proteins in the gastrointestinal
tract.
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Drug delivery through the lung appears optimal for
two major reasons, i.e. the enormous surface area
available for delivery, and permeability to large
molecules. The lung has about a half billion alveoli.
The alveoli in an average adult lung have a surface area
the size of a tennis court, far exceeding the surface
area of most other delivery routes, e.g. GI tract, by
several orders of magnitude. The alveoli allow oxygen
and other molecules to readily pass into the circulatory
system. Conventional metered dose inhalers, primarily
used for asthma, deliver drugs into the upper branches
of the lung. In terms of permeability, the buccal
cavity and lung are ideal absorption areas for both
small and large molecules. Large proteins, including
antibodies, are readily absorbed through the alveoli
either directly into the circulatory system or, more
frequently, via the lymphatic system, which subsequently
releases the drug into the bloodstream.
The ability to deliver large molecule drugs orally,
e.g. buccally, and/or into the deep lung will represent
one of the most significant technical breakthroughs in
drug delivery.
New products that address these drug delivery needs
are sought, which simultaneously provide patients with a
convenient user friendly mechanism and physicians with a
tool to improve therapy, compliance, and to prevent or
reduce expensive hospital stays.
Oral delivery offers a variety of benefits for
systemic drug delivery. For example, it provides easy,
non-invasive access to a permeable mucosa, which
facilitates rapid drug absorption and a fast onset of
action of the drug. In comparison to the GI tract and
other organs, the buccal environment has lower enzymatic
activity and a neutral pH.
The absorption of proteins and peptides is believed
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to be enhanced by the diffusion of large molecules
entrapped in droplet form through the aqueous pores and
the cell structure perturbation of the tight
paracellular junctions. In order to further improve the
penetration and absorption of formulation it has now
been found that such formulations can be mixed with a
propellant (preferably a non-CFC) and delivered, e.g.
applied to the buccal mucosa, through metered dose
inhalers (NmIs) or similar. The present invention uses
novel formulations that are intended to improve the
quality (in terms of absorption), stability, and
performance of NmI-delivered pharmaceuticals. A novel
method is used to solubilize drugs in a propellant. The
formulation ingredients are selected specifically to
give enhancement in the penetration through the pores
and facilitate the absorption of the drugs to reach
therapeutic levels in the plasma.
With previous formulations, in order to administer
the pharmaceutical agent, it is necessary to shake the
vial in order to temporarily intimately mix the two
phases, so that a mixture of pharmaceutical formulation
and propellant are expelled from the vial upon opening a
dosing valve. The propellant and pharmaceutical phases
quickly separate after shaking. Separation of the
phases may lead to situations wherein the person
administering the drug does not shake the vial
sufficiently, forgets to shake the vial or waits too
long before opening the dosing valve. Such situations
lead to a lack of uniformity in the amount of
pharmaceutical being administered from one opening of
the valve to the next, i.e. from "shot" to "shot". This
is particularly problematic where the amount of
pharmaceutical agent to be administered is critical,
e.g. with insulin and some pain killing drugs and
narcotics. It is desirable, therefore, for the
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formulation and propellant to be evenly mixed, e.g. as a
solution, stable suspension or the like.
The present invention is directed to providing a
stable mixture of propellant and pharmaceutical agent.
The terms "comprising" and "comprises" when used in
this specification are taken to specify the presence of
the stated features, integers, steps or components but
do not preclude the presence or addition of one or more
other features, integers, steps, components or groups
thereof.
The term "solubilized" is used in this
specification to refer to a stable intimate mixture of
ingredients. It has not been determined whether the
mixture is a solution, suspension or other form of
intimate mixture. Such a solubilized mixture is stable
for substantial periods of time, e.g. months, without
separation.
Disclosure of the Invention
Accordingly the present invention provides a
pressurized container containing a stable solubilized
mixture of propellant which is liquid under pressure and
an intermediate formulation which comprises a proteinic
pharmaceutical agent, water, first ingredient, second
ingredient and at least one third ingredient, wherein
the first ingredient is selected from glycerin and
polyglycerin and mixtures thereof in an amount of from
1-50 wt./wt.% of the intermediate formulation, the
second ingredient is selected from phenol, methyl phenol
and mixtures thereof in an amount of from 1-20 wt./wt.%
of the intermediate formulation, each third ingredient
is selected from the group consisting of alkali metal C8
to C22 alkyl sulphate, polidocanol C6 to C40 alkyl
ethers, trihydroxy oxo-cholanyl glycines and
pharmaceutically acceptable salts thereof,
polyoxyethylene ethers, alkyl-aryl polyether alcohols,
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hyaluronic acid and pharmaceutically suitable salts
thereof, monoolein, triolein, lysine, polylysine, oleic
acid, linoleic acid, linolenic acid, monooleates and
laurates, glycolic acid, lactic acid, chenodeoxycholate,
deoxycholate, chamomile extract, cucumber extract,
borage oil and evening of primrose oil and mixtures
thereof, in an amount of from 1-50 wt./wt% of the
intermediate formulation, and wherein the total
concentration of first, second and third ingredients is
less than 90 wt./wt% of the intermediate formulation.
In one embodiment, the alkali metal C8 to C22 alkyl
sulphate is in a concentration of from 2 to 20 wt./wt.%
of the intermediate formulation, especially 5 to
wt./wt.o.
15 In a further embodiment, the methyl phenol is
m-cresol.
In another embodiment, the alkali metal C8 to C22
alkyl sulphate is sodium lauryl sulphate.
In a further embodiment the polidocanol alkyl ether
is a polidocanol 10 or 20 lauryl ether.
In another embodiment, the polyoxyethylene ether is
polyoxyethylene sorbitan ether, and particularly
polyoxyethylene sorbitan 80 lauryl ether.
In yet another embodiment, the third ingredient is
present in a concentration of from about 1 to about
25 wt./wt.o.
In yet another embodiment, the propellant is
selected from the group consisting of tetrafluoroethane,
tetrafluoropropane, dimethylfluoropropane,
heptafluoropropane, dimethyl ether, n-butane and
isobutane.
In a further embodiment, the weight ratio of
intermediate formulation to propellant is from 5:95 to
25:75.
In one embodiment, the pharmaceutical agent, water,
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first, second and third ingredients and propellant. have
been solubilized by a process comprising the steps of:
a) dissolving the proteinic pharmaceutical agent in
water and adjusting the pH to a level suitable for
pharmaceutical use;
b) mixing with the first ingredient in an amount of from
1-50 wt./wt.% of the intermediate formulation;
c) then mixing with the second ingredient in an amount
of from 1-20 wt./wt.o of the intermediate formulation;
d) subsequently adding and mixing at least one third
ingredient to form the intermediate formulation;
e) charging the intermediate formulation to a
pressurizable container and subsequently charging the
container with the propellant.
The invention also provides a process for making a
stable aerosol pharmaceutical composition in which a
propellant and an intermediate formulation, which
comprises a pharmaceutical agent, water and first,
second and third ingredients, has been solubilized by a
process comprising the steps of:
a) dissolving the proteinic pharmaceutical agent in
water and adjusting the pH to a level suitable for
pharmaceutical use;
b) mixing with a first ingredient selected from
glycerin, polyglycerin and mixtures thereof in an amount
of from 1-50 wt./wt.% of the intermediate formulation;
c) then mixing with a second ingredient selected from
phenol, methyl phenol and mixtures thereof in an amount
of from 1-20 wt./wt.% of the intermediate formulation;
d) subsequently adding and mixing at least one third
ingredient to form the intermediate formulation, said
third ingredient being selected from the group
consisting of alkali metal C8 to C22 alkyl sulphate,
polidocanol C6 to C40 alkyl ethers, trihydroxy oxo-
cholanyl glycines and pharmaceutically acceptable salts
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thereof, polyoxyethylene ethers, alkyl-aryl polyether
alcohols, hyaluronic acid and pharmaceutically suitable
salts thereof, monoolein, triolein, lysine, polylysine,
oleic acid, linoleic acid, linolenic acid, monooleates
and laurates, glycolic acid, lactic acid,
chenodeoxycholate, deoxycholate, chamomile extract,
cucumber extract, borage oil and evening of primrose oil
and mixtures thereof, each of said third ingredients
being present in an amount of from 1-50 wt./wto of the
intermediate formulation, and wherein the total
concentration of first, second and third ingredients are
less than 90 wt./wtoof the intermediate formulation;
e) charging the intermediate formulation to a
pressurizable container and subsequently charging the
container with the propellant.
In one embodiment, the alkali metal C8 to C22 alkyl
sulphate is in a concentration of from 2 to 25 wt./wt.o
of the intermediate formulation.
In a further embodiment, the methyl phenol is
m-cresol.
In another embodiment, the alkali metal C8 to C22
alkyl sulphate is sodium lauryl sulphate.
In a further embodiment the polidocanol alkyl ether
is a polidocanol 10 or 20 lauryl ether.
In another embodiment, the polyoxyethylene ether is
polyoxyethylene sorbitan ether, particularly
polyoxyethylene sorbitan 80 lauryl ether.
In yet another embodiment, the third ingredient is
present in a concentration of from about 1 to about
25 wt./wt.o.
In another embodiment, in step a) the pH is
adjusted to between 6.0 and 9.0, and preferably between
7Ø and 8Ø
In yet another embodiment, the propellant is
selected from the group consisting of tetrafluoroethane,
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tetrafluoropropane, dimethylfluoropropane,
heptafluoropropane, dimethyl ether, n-butane and
isobutane.
In a further embodiment, the weight ratio of
intermediate formulation to propellant is from 5:95 to
25:75.
In yet another embodiment, step d) is accomplished
with a high speed mixer or sonicator.
The present invention also provides a metered dose
aerosol dispenser with the stable aerosol pharmaceutical
composition of the present invention therein.
The present invention also provides a method for
administering stable aerosol pharmaceutical compositions
of the present invention, by spraying a predetermined
amount of the composition into the mouth with a metered
dose spray device.
The present invention also provides a method for
administration of a proteinic pharmaceutical agent in a
buccal cavity of a human being by spraying into the
cavity, without inhalation, from a metered dose spray
dispenser, a predetermined amount of stable solubilized
mixture of propellant which is liquid under pressure and
an intermediate formulation which comprises a proteinic
pharmaceutical agent, water, first ingredient, second
ingredient and at least one third ingredient, wherein
the first ingredient is selected from glycerin and
polyglycerin and mixtures thereof in an amount of from
1-50 wt./wt.% of the intermediate formulation, the
second ingredient is selected from phenol, methyl phenol
and mixtures thereof in an amount of from 1-20 wt./wt.%
of the intermediate formulation, each third ingredient
is selected from the group consisting of alkali metal C8
to C22 alkyl sulphate, polidocanol C6 to C40 alkyl
ethers, trihydroxy oxo-cholanyl glycines and
pharmaceutically acceptable salts thereof,
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polyoxyethylene ethers, alkyl-aryl polyether alcohols,
hyaluronic acid and pharmaceutically suitable salts
thereof, monoolein, triolein, lysine, poiylysine, oleic
acid, linoleic acid, linolenic acid, monooleates and
S laurates, glycolic acid, lactic acid, chenodeoxycholate,
deoxycholate, chamomile extract; cucumber extract,
borage oil and evening of primrose oil and mixtures
thereof, in an amount of from 1-50 wt./wt% of the
intermediate formulation, and wherein the total
concentration of first, second and third ingredients is
less than 90 wt./wt% of the intermediate formulation.
Modes For Carrying Out The Tnvention
The present invention provides an improved, stable
formulation. The formulation allows delivery of
macromolecular thigh molecular weighty pharmaceutical
agents, particularly through the membranes in the mouth
or lungs.
The pharmaceutical agents cover a wide spectrum of
agents, including proteins, peptides; hormones, vaccines
and drugs. The molecular weights of the macromolecular
pharmaceutical agents are preferably above 1000,
especially between 1000 and 2 000 000.
The proteinic pharmaceutical agent may be selected
from a wide variety of macromolecular agents, depending
on the disorder being treated, generally with molecular
weights greater than about 1000 and especially between
about 1000 and 2 000 000. Preferred pharmaceutical
agents are selected from the group Gansisting of
insulin, heparin, low molecular weight heparin, hirulog,
hirugen, huridine, interferons, interleukins, cytokins,
mono .and polyclonal antibodies, immunoglobins,
chemotherapeutic agents, vaccines, glycoproteins,
bacterial toxoids, hormones, calcitonins, insulin like
growth factors (IGF?, glucagon-like peptide 1 (GLP-1),
large molecule antibiotics, protein based thrombolytic
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compounds, platelet inhibitors, DNA, RNA, gene
therapeutics and antisense oligonucleotides, opioids,
narcotics, hypnotics, steroids and pain killers (non-
steroidal anti-inflammatory drugs?.
As will be understood, the concentration of the
pharmaceutical agent is an amount sufficient to be
effective in treating or preventing a disorder or to
regulate a physiological condition in an animal or
human. The concentration or amount of pharmaceutical
agent administered will depend on the parameters
determined for the agent and the method of ,
administration, e.g. nasal, buccal, pulmonary. For
example, nasal formulations tend to require much lower
concentrations of some ingredients in order to avoid
irritation or burning of the nasal passages. It is
sometimes desirable to dilute an oral formulation up to
10-100 times in order to provide a suitable nasal
formulation.
For insulin-containing and some other compositions,
the composition may also contain at least one inorganic
salt which helps to open channels in the membranes of
the mouth or lungs, and may provide additional
stimulation to release insulin. Non-limiting examples
of inorganic salts are sodium, potassium, calcium and
zinc salts, especially sodium chloride, potassium
chloride, calcium chloride, zinc chloride and sodium
bicarbonate.
It will be recognized by those skilled in the art
that for many pharmaceutical compositions it is usual to
add at least one antioxidant to prevent degradation and
oxidation of the pharmaceutically active ingredients.
2t will also be understood by those skilled in the art
that colorants, flavouring agents and non-therapeutic
amounts of other compounds may be included in the
formulation. Typical flavouring agents are menthol,
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sorbitol and fruit flavours.
The antioxidant may be selected from the group
consisting of tocopherol, deteroxime mesylate, methyl
paraben, ethyl paraben and ascorbic acid and mixtures
thereof. A preferred antioxidant is tocopherol.
In a preferred embodiment at least one protease
inhibitor is added to the formulation to inhibit
degradation of the pharmaceutical agent by the action of
proteolytic enzymes. Of the known protease inhibitors,
most are effective at concentrations of from 1 to
3 wt./wt. o of the formulation.
Non-limiting examples of effective protease
inhibitors are bacitracin, soyabean trypsin, aprotinin
and bacitracin derivatives, e.g. bacitracin methylene
disalicylate. Bacitracin is the most effective of those
named when used in concentrations of from 1.5 to
2 wt./wt.%. Soyabean trypsin and aprotinin two may be
used in concentrations of about 1 to 2 wt./wt. o of the
formulation.
The amount of the first ingredient is present in a
concentration of from 1 to 50 wt/wt% of the intermediate
formulation. The amount of the second ingredient is
present in a concentration of from 1 to 20 wt/wt% of the
intermediate formulation and the third ingredient is
present in a concentration of from 1 to 50 wt/wt% of the
intermediate formulation, and total concentration of
such ingredients is less than 90 wt./wt% of the
formulation. It is believed that the phenolic compounds
act mainly as preservatives and complexing agents to
stabilize drugs, e.g. insulin. Besides their function
as a stabilizer and preservative, they may also act as
antiseptic agents and furthermore may help in
absorption. The methyl phenol may be o-cresol, m-cresol
or p-cresol, but m-cresol is preferred.
The order of addition of the ingredients in the
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formulation are important in order to obtain a stable
mixture. First, the pharmaceutical agent is dissolved
in water. Preferably, the pH is adjusted to between
about 6.0 and 9.0, and even more preferably to between
about 7.0 and 8Ø Secondly, the aqueous pharmaceutical
agent mixture is mixed first with glycerin, polyglycerin
or mixtures thereof (the first ingredient), and then
with phenol, methyl phenol or mixtures thereof (the
second ingredient). Subsequently the third ingredient
is added and mixed to form the intermediate formulation.
The third ingredient is at least one of the following
compounds: alkali metal C8 to C22 alkyl sulphate,
polidocanol C6 to C40 alkyl ethers, trihydroxy oxo-
cholanyl glycines and pharmaceutically acceptable salts
thereof, polyoxyethylene ethers, alkyl-aryl polyether
alcohols, hyaluronic acid and pharmaceutically suitable
salts thereof, monoolein, triolein, lysine, polylysine,
oleic acid, linoleic acid, linolenic acid, monooleates
and laurates, glycolic acid, lactic acid,
chenodeoxycholate, deoxycholate, chamomile extract,
cucumber extract, borage oil and evening of primrose
oil. The ingredients are mixed together with a mixer.
When the third ingredient is added, a high speed mixer
or sonicator is preferred. The resulting mixture is
referred to herein as the intermediate formulation.
Each of the non-pharmaceutical substances referred
to in the previous paragraph may be added in
concentrations previously indicated, provided that the
total amount of such substances does not exceed
90 wt./wt.o of the intermediate formulation.
After formation of the intermediate formulation,
the formulation is charged to a pressurizable container.
Preferably the container is a vial suitable for use with
a metered dose inhaler or applicator. Then the vial is
charged with propellant. As the propellant is
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introduced into the vial, there is great turbulence in
the vial and the propellant and pharmaceutical
formulation become intimately mixed and do not separate
on standing. It is believed that the propellant and
pharmaceutical mixture so formed would be stable for
several months. As a result, it is not necessary to
shake the vial before use, although, through habit with
other formulations, many users may shake the vial. The
advantage of the solubilized formulation will be
immediately apparent to those skilled in the art. For
example, the relative homogeneity of the mixture
provides good accuracy of pharmaceutical dispensing from
"shot" to "shot" and from the first shot to the last
from the container. As is known, in order to deliver
the pharmaceutical agent to the lung, it is necessary
for the user to breathe deeply when the aerosol spray
from the pressurized container is released. Without
breathing in, the pharmaceutical agent is delivered to
the buccal cavity. The method chosen will depend on a
number of factors, including the type of pharmaceutical
agent, the concentration in the aerosol, the desired
rate of absorption required and the like.
A particular advantage with the use of metered dose
applicators or inhalers is that the formulation can be
delivered in a relatively precise dose, e.g. titratable
to injection within 1 unit of insulin dose. The droplet
size of the formulation preferably falls between 1-5 ~.m
in order for droplets to penetrate buccal mucosa or to
reach to the deep lung surface. Thus, the present
invention is suitable for delivery of proteinic drugs
such as insulin for the treatment of diabetes.
The pressurized inhalers also offer a wide dosing
range and consistent dosing efficiency. With such a
delivery, greater than about 950 of the dose may reach
the target area. The smaller particle size (1-5 Vim)
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obtained using pressurized inhalers also enhances dosing
due to broader coverage within the lung cavity. In this
situation, increased coverage can help more absorption
of a drug like insulin. Furthermore, because these
devices are self-contained, potential contamination is
avoided.
The amount of physiologically peptide or protein in
the compositions of this invention is typically a
quantity that provides an effective amount of the drug
to produce the physiological activity (therapeutic
plasma level) for which peptide or protein is being
administered. In consideration of the fact that the
bioavailability of any active substance can never be
1000, that is to say the administered dose of the active
drug is not completely absorbed, it is preferable to
incorporate slightly larger amount than the desired
dosage. Where the dosage form is a spray (aerosol) or
the like which is repeatedly dispensed from the same
container, it is preferably so arranged that the unit
dose will be slightly greater than the desired dose. It
should be understood that dosage will vary with species
of warm blooded animals such as man, domestic animals,
and their body weights. The utilization of atomizer or
aerosol spray devices (metered dose inhalers or
nebulizers) is important to provide particle sizes for
effective absorption from the nasal or lung cavity, or
in the mouth, e.g. in the buccal cavity, so the drug may
successfully absorbed or reach to the specific site. It
is believed that a variety of proteins retain their
biological activity even after prolonged exposure to
propellants commonly used in metered dose inhalers.
The specific concentrations of the essential
ingredients can be determined by relatively
straightforward experimentation. It will be understood
that the amounts of certain ingredients may need to be
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limited in order to avoid compositions which produce
foam when sprayed rather than forming a fine spray. For
absorption through the oral cavities, it is often
desirable to increase, e.g. double or triple, the dosage
which is normally required through injection or
administration through the gastrointestinal tract.
As will be understood, the amount of each component
of the formulation will vary depending on the
pharmaceutical agent and the site of application.
The therapeutic compositions of the present
invention may be stored at room temperature or at cold
temperature. Storage of proteinic drugs is preferable
at a cold temperature to prevent degradation of the
drugs and to extend their shelf life.
The desired size of aerosol droplets which are
sprayed from the aerosol dispenser will depend, in part,
on where the pharmaceutical is to be deposited. For
example, for deposition in the lungs, particle sizes of
less than about 5 ~.m are preferred whereas for
absorption in the buccal cavity of the mouth, particle
sizes of about 6-10 ~m are preferred.
The amount of physiologically peptide or protein in
the compositions of this invention is typically a
quantity that provides an effective amount of the
pharmaceutical or drug to produce the physiological
activity (therapeutic plasma level) for which peptide or
protein is being administered. In consideration of the,
fact that the bioavailability of any active substance
can never be 100%, that is to say the administered dose
of the active drug is not completely absorbed, it is
preferable to incorporate slightly larger amount than
the desired dosage.
Administration of the formulation into the buccal
cavity is by spraying the formulation into the mouth,
substantially without inhalation, so that the droplets
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stay in the mouth rather than be drawn into the lungs.
The advantages of the present invention are
illustrated by the following non-limiting examples in
which insulin is the pharmaceutical agent.
Example 1
Method of Insulin Solution Preparation: (U200, 400,
600, 800 and 1000 per mL)
Appropriate quantities of insulin powder (in order
to make 200 units, 400 units or 600 units 800 units or
1000 units per mL, depending on the activity (27.5-28.3
units/mg) were weighed accurately on an analytical
balance. The powders were transferred to glass beakers
equipped with stirrer. Distilled water was added to the
beakers and the solution was stirred at low speed. To
each beaker was added 5M HCl (pH 2) solution dropwise
until the insulin powder therein was solubilized
completely. These solutions were then neutralized with
5M NaOH dropwise to pH 7-8. The solution was stirred
continuously at low speed. The solution was stirred
further for 30 minutes and stored at 10°C or at room
temperature. This gave solutions containing insulin
with 200U, 400U, 600U, 800U and 1000U/mL.
Glycerin was added to each of these solutions, with
stirring, in an amount of 20 wt./wt.o glycerin in the
intermediate formulation. After this, phenol was added,
with stirring, in an amount of 10 wt./wt.% phenol in the
intermediate formulation. Then 15 wt./wto sodium
lauryl sulphate, 10 wt./wt.% trihydroxy oxo cholanyl
glycine and 20 wt./wt.o polidocanol 20 lauryl ether was
added and mixed with a high speed stirrer.
One millilitre portions of the solutions of insulin
(U200, U400, U600, U800 or U1000/mL) were pipetted into
special glass vials coated on the outside with a plastic
coating, for protection in the event of mechanical
failure of the glass. The vials were then charged with
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a non-CFC tetrafluoroethane (134a) propellant with the
aid of a Pamasol 2008 (trade mark) semi-automatic gas
filling equipment. The amount of 134a propellant in
each vial was adjusted to 9 mL shot size in order to
deliver amounts of insulin equivalent to 2, 4, 6, 8 or
units/actuation when actuated through the valve of
the vial. For example, the shot size of 2 units per
actuation refers to the U200 insulin solution in a vial.
The valves were specially designed to deliver exactly
10 100 ~,L spray per actuation.
Aerodynamic Particle Size: The aerodynamic particle
sizes of formulations sprayed from the vials were then
determined by 8-stage USP Anderson Multistage Cascade
Impactor-Mark-II (trade mark). The Multistage Cascade
Impactor was cleaned with methanol and air-dried at
30°C. Glass fibre filters were placed on the collection
plates. Seals were aligned properly and the actuator
was attached to a mouthpiece and assembled onto the USP
induction port and jet stages. A vacuum pump was
connected and air flow rate is set to 28.3 litres/min.
Each vial was actuated twice to waste. The shots were
then delivered by discharging the actuator into the
mouthpiece and repeated for 25 times. The deposited
insulin was collected by rinsing the mouthpiece with
0.6 mg/mL EDTA in 10 mL water at pH 8.7. The filters
were carefully removed and placed in scintillation vials
and the vials sonicated for 15 minutes. The quantity of
the insulin was then analysed using RP-HPLC. The
results are shown in Tables I, II and III for U400, U600
and U800 solutions.
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Table I (U400 , units/actuation)
4
Stage vol. mg units actuationunits/ Particle
size
# mL Actuation ~m
0 10
1 10
2 10
3 10 0.77 20.1 5 4.0 4.0
4 10 0.78 20.1 5 4.0 3.8
5 10 0.81 20.0 5 4.0 3.0
106 10 0.80 20.3 5 4.0 2.1
7 10 0.80 20.1 5 4.0 1.0
8 10 0.79 20.1 5 4.0 0.7
Table II (U600, units/actuation)
6
Stage vol. mg units actuation units/ Particle
size
15# mL Actuation ~.m
0 10 n/d
1 10 n/d
2 10 n/d
3 10 0.77 30.1 5 6.0 4.0
204 10 0.78 30.1 5 6.0 3.8
5 10 0.81 30.0 5 6.0 3.0
6 10 0.80 30.3 5 6.0 2.1
7 10 0.80 30.1 5 6.0 1.0
8 10 0.79 30.1 5 6.0 0.7
25 Table III (U800, units/actuation )
8
Stage vol. mg units actuation units/ Particle
size
# mL Actuation /gym
0 10 n/d
1 10 n/d
302 10 n/d
3 10 0.77 40.1 5 8.0 3.8
4 10 0.78 40.1 5 8.0 3.3
5 10 0.81 40.0 5 8.0 3.0
6 10 0.80 40.3 5 8.0 2.0
357 10 0.80 40.1 5 8.0 1.0
8 10 0.79 40.1 5 8.0 0.6
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Conclusion: The particle sizes were determined to
be around 3 ~.m and stages 0-2 showed no insulin
deposition indicating that most particles were smaller
than 6 ~.m. Thus, this analysis suggests a strong
likelihood of deep lung deposition, as the droplet sizes
were generally smaller than 4 ~,m.
Shot size accuracy: The shot size accuracy was
determined by firing shots in specially designed glass
thiel tubes and weighing the tubes before and after the
sample collection. Each vial had a capacity of 100
shots. The number of units per actuation are shown in
Table IV.
Table IV (U400
Shot Number Shot 4~leight ( g)
4 units/act. 6 units /act 8 units/act
10 0.076 0.090 0.179
15 0.073 0.093 0.180
0.076 0.096 -
0.074 0.094 -
20 30 0.070 0.090 0.178
40 - - 0.176
70 - - 0.177
Conclusion: The analysis indicates the uniformity
of the shot size delivered through the valves.
25 Insulin dose: The volume of insulin dose delivered, in
terms of units/actuation was then determined by HPLC
analysis.
The vials were actuated twice to waste. Shots were
delivered by discharging the actuator into the
mouthpiece and repeated for 25 times. The deposited
insulin was collected by rinsing the mouthpiece with
0.6mg/mL EDTA in 10 mL water at pH 8.7, carefully remove
the filters and place them in scintillation vials and
sonicate the vials for 15 minutes. The quantity of the
insulin was then analysed using RP-HPLC. The results for
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6 and 8 units/actuation formulations are shown in Tables
V and VI. Each vial had a capacity of 100 shots. Shot
numbers 5-10 were at the beginning of the vial's
discharge, 45-50 were in the middle and 85-90 were at
the end.
Table V (6 units/actuation)
Shot Nos. Dose delivered Dose delivered
~.g unit s
5-10 118 6.2
45-50 110 6.0
85-90 105 5.8
Table VI (8 units/actuation)
Shot Nos. Dose delivered Dose delivered
~g units
5-10 173.3 8.1
45-50 171.1 7.9
85-90 172.7 8.0
Conclusion: The analysis indicates the uniformity
of the dose delivered per actuation through the valves.
Clinical Results: 15 healthy volunteers were given the
following doses of insulin for three days.
Day-1: 5 puffs of 4 units each (total 20 units)
Day-2: 5 puffs of 6 units each (total 30 units)
Day-l: 5 puffs of 8 units each (total 40 units)
Plasma insulin levels, in pmol/L, were measured
every 15 minutes for first 90 minutes and then every 30
minutes for 2 hours. The results are shown in Table VII
on the following page.
35
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Table VII
Day-1 Day-2 Day-3
Time
20 units 30 units 40 units
0 35 38 42
56 62 72
30 89 97 112
45 119 138 178
60 160 178 202
10 75 160 175 190
90 142 157 173
120 78 112 141
150 62 87 92
180 37 49 67
15 These data shows significant absorption of insulin
through buccal mucosa, oropharynx, and lungs regions.
Example 2
As a comparison, i.e. outside the scope of the
invention, tests were conducted with an insulin
formulation which did not have any of the solubilizing
ingredients.
Appropriate quantities of insulin powder (in order
to make 200 units, 400 units or 600 units 800 units or
1000 units per mL, depending on the activity (27.5-28.3
units/mg) was weighed accurately on an analytical
balance. The powders were transferred to glass beakers
equipped with stirrers. Distilled water was added and
the solution was stirred at low speed. To this was
added 5M HCl (pH 2) solution dropwise till insulin
powder was solubilized completely. This solution was
then neutralized with 5M NaOH dropwise to pH 7-8. The
solution was stirred continuously at low speed. The
solution was stirred further for 30 minutes and stored
at 10°C. This gave solutions containing insulin (200U,
400U, 600U, 800U or 1000U/mL).
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Shot Size: Shot size accuracy was determined by firing
shots in thief tubes and weighing the tubes before and
after the sample collection. Each vial had a capacity
of 100 shots. The average shot weights for 5 sequential
shots were determined, as shown in Tables VIII, IX and
X.
Table VIII (400U/mL)
Shot # # of Shots Shot Weight (g)
10-15 5 0.065
20-25 5 0.087
30-35 5 0.077
40-45 5 0.063
70-75 5 0.051
Table IX (600U/mL)
Shot # # of Shots Shot Weight (g)
10-15 5 0.077
20-25 5 0.064
30-35 5 0.091
40-45 5 0.051
70-75 5 0.083
Table X (800U/mL)
Shot # # of Shots Shot Weight (g)
10-15 5 0.049
20-25 5 0.071
30-35 5 0.065
40-45 5 0.088
70-75 5 0.102
Highly irregular shot weight distribution was
observed due to the insolubility of insulin in the
propellant and the inability to facilitate formation of
small droplets
Aerodynamic particle size: The aerodynamic particle
sizes of formulations sprayed from the vials were
determined by 8-stage USP Anderson Multistage Cascade
Impactor-Mark-II (trade mark) by the same procedure
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outlined in Example 1. The results are shown in Tables
XI and XII.
Table XI (U600, 6 units/actuation)
Stage vol. mg units actuation units/ Particle size
# mL Actuation ~Cm
0 10 0.91 56.5 5 11.3 >9
1 10 0.60 46.7 5 9.3 >7
2 10 0.42 33.3 5 6.7 > 5
3 10 not detected
Table XII (U800, 8 units/actuation)
Stage vol. mg units actuation units/ Particle size
# mL Actuation ~.m
0 10 0.97 77.7 5 15.5 >9
1 10 0.88 66.9 5 13.4 >7
2 10 0.42 55.6 5 11.1 > 5
3 10 not detected
This demonstrates highly irregular droplet sizes
and number of units delivered through the aerosol
valves.
Example 3
Another experiment was conducted to provide data
for comparative purposes. This example does not fall
within the scope of the present invention.
Powdered insulin was placed in a glass beaker
equipped with a stirrer. Distilled water was added and
the solution was stirred at low speed. To this solution
was added 5M HCl (pH 2) solution dropwise until the
insulin was solubilized completely. This solution was
then neutralized with 5M NaOH solution dropwise until
the pH was between 7 and 8. Seven mg phenol and 7 mg
m-cresol were added to the solution and mixed
thoroughly. The solution was diluted with distilled
water until there were 200 units insulin per millilitre
of solution. One millilitre portions were then
transferred to glass vials, which were then charged with
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10.8 g HFA 134a propellant using a Pamasol (trade mark)
2008 semi-automatic gas filling apparatus.
The propellant and insulin solution remained as
separate phases.
Example 4
A further comparative experiment was conducted.
Powdered insulin was placed in a glass beaker equipped
with a stirrer. Distilled water was added and the
solution was stirred at low speed. To this solution was
added 5M HC1 (pH 2) solution dropwise until the insulin
was solubilized completely. This solution was then
neutralized, while stirring slowly, with 5M NaOH
solution dropwise until the pH was between 7 and 8. To
this solution was added 7 mg sodium lauryl sulphate,
7 mg polyoxyethylene ether (10 lauryl) and 7 mg
trihydroxy oxo cholanyl glycine and dissolved
completely. Seven mg lecithin, solubilized in a water
alcohol solution (7 mg/mL) was then added while stirring
at high speed, i.e. 2000 rpm. The solution was stirred
for 30 minutes and then stored at 10°C. The resulting
mixed micellar solution had about 200 units insulin. To
this mixture 5 mg phenol, 5 mg m-cresol and 10 mg
glycerin were added.
The solution was pipetted (1mL/vial) into 10 mL
capacity glass vials. The vials were then charged with
HFA 134a propellant with a Pamasol 2008 automatic gas
filling apparatus. The amount of propellant was
adjusted to 9 mL shot size in order to deliver 2 units
insulin per actuation of the aerosol vial. The valves
of the vials were designed to deliver 100 ~,L spray per
actuation, containing 2 units insulin. The aqueous
pharmaceutical composition and the propellant remained
as separate phases. Prior to discharging shots of the
formulation, shaking of the vial was necessary in order
to entrain the pharmaceutical in the propellant phase.
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After discharging a shot, the propellant separated from
the aqueous pharmaceutical composition within about 20
seconds.
The aerodynamic particle size was determined by an
8-stage USP Anderson (trade mark) Cascade Impactor Mark
II. The impactor was cleaned with methanol and air
dried at 30°C. Glass fibre filters were placed on the
collection plates. The actuator was attached to the
mouthpiece of the impactor and assembled onto the USP
induction port and jet stages. A vacuum pump was
connected and the air flow rate set to 28.3 litres per
minute. The vial was primed by shaking for 10 seconds
and actuating twice to waste. The shot was delivered by
discharging the actuator into the mouthpiece and
repeating 25 times. The deposited insulin was collected
by rinsing the mouthpiece with 0.6 mL EDTA in 10 mL
water at pH 8.7. The filters were removed and placed in
scintillation vials and sonicated for 15 minutes. The
quantity of insulin was then analysed using RP-HPLC.
The results are shown in Table XIII (2 units per
actuation) and XIV (4 units per actuation).
Table XIII
Stage No. 0 1 2 3
Volume (mL) 10 10 10 10
Mass (mg) 0.79 0.81 0.78
Units 10.4 10.0 10.0
Actuation 5 5 5
Units per
actuation 2.0 2.0 2.1
Particle size(,um) 8.8 5.8 5.7
* not determined/detected
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Table XIV
Stage No. 0 1 2 3
Volume (mL) 10 10 10 10
Mass (mg) 0.79 0.81 0.78 **
Units 20.7 21.0 20.1
Actuation 5 5 5
Units per
actuation 4.15 4.18 4.01
Particle size (,um) 9 5.8 4.7
** not determined
Based on these tests, the particle size was
determined to be about 7 ,um, and stages 3-8 showed no
insulin deposition, indicating that most particles were
larger than about 6 ,um. This suggests that there would
be no deep lung deposition formulation and that most of
the formulation would be deposited in the buccal cavity.
