Note: Descriptions are shown in the official language in which they were submitted.
CA 02229286 1998-02-10
MODI.P0020
MIXED MICELLAR DELIVERY SYSTEM AND METHOD OF PREPARATION
Field of the Invention
The present invention relates to an improved
5 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 through the oral and nasal
membranes.
Background to the Invention
In spite of significant efforts in academic and
commercial laboratories, major breakthroughs in oral
peptide and protein formulation have not been achieved.
Relatively little progress has been made in reaching the
15 target of safe and effeci~ive oral formulations for
peptides and proteins. The major barriers to developing
oral formulations for proteins and peptides include poor
intrinsic permeability, lumenal and cellular enzymatic
degradation, rapid clearance, and chemical stability in
20 the gastrointestinal (GI;~ tract. Pharmaceutical
approaches to address these barriers, which have been
successful with traditional small, organic drug
molecules, have not readily translated into effective
peptide and protein formulations. Although the
25 challenges are significant, the potential therapeutic
benefits remain high especially in the field of diabetes
treatment using insulin.
Scientists have explored various administration
routes other than the injection for proteins and
30 peptides. These routes include oral, intranasal, rectal,
vaginal cavities for the effective delivery of large
molecules. Out of the above four mentioned routes oral
and nasal cavities have been of greatest interest to
scientists. Both the oral and nasal membranes offer
35 advantages over other routes of administration. For
example, drugs administered through these membranes have
a rapid onset of action, provide therapeutic plasma
levels, avoid first pass effect of hepatic metabolism,
and avoid exposure of the drug to hostile GI
CA 02229286 1998-02-10
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environment. Additional advantages include easy access
to the membrane sites so that the drug can be applied,
localized and removed easily. Further, there is a good
potential for prolonged delivery of large molecules
through these membranes.
The oral routes have received far more attention
than has the other routes. The sublingual mucosa
includes the membrane of ventral surface of the tongue
and the floor of the mouth whereas the buccal mucosa
10 constitutes the lining of: the cheek. The sublingual
mucosa is relatively permeable thus giving rapid
absorption and acceptable bioavailability of many drugs.
Further, the sublingual mucosa is convenient, acceptable
and easily accessible. This route has been investigated
15 clinically for the delivery of a substantial number of
drugs.
The ability of molecules to permeate through the oral
mucosa appears to be related to molecular size, lipid
solubility and peptide protein ionization. Small
20 molecules, less than 1000 daltons appear to cross mucosa
rapidly. As molecular size increases, the permeability
decreases rapidly. Lipid soluble compounds are more
permeable than non-lipid soluble molecules. Maximum
absorption occurs when molecules are un-ionized or
25 neutral in electrical charges. Therefore charged
molecules present the biggest challenges to absorption
through the oral mucosae.
Most proteinic drug molecules are extremely large
molecules with molecular weight exceeding 6000 daltons.
30 These large molecules have very poor lipid solubility
and are practically impermeable. Substances that
facilitate the absorption or transport of large
molecules (>2000 daltons) across biological membranes
are known as the enhancers, (Lee et al., Critical
35 Reviews in Therapeutic drug Carrier Systems, 8, 91,
1991; Lee et al., Critical Reviews in Therapeutic drug
Carrier Systems, 8, 115, 1991, 1992). Enhancers may be
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characterized as chelators, bile salts, fatty acids,
synthetic hydrophillic and hydrophobic compounds, and
biodegradable polymeric compounds.
Various mechanisms of action of enhancers have been
proposed. These mechanic>ms of action, at least for
protein and peptidic drugs include (1) reducing
viscosity and/or elasticity of mucous layer, (2)
facilitating transcellular transport by increasing the
fluidity of the lipid bi7_ayer of membranes, and (3)
10 increasing the thermodynamic activity of drugs (Critical
Rev, 117-125, 1991, 1992).
Many enhancers have been tested so far and some
have found to be effective in facilitating mucosal
administration of large molecule drugs. However, hardly
15 any penetration enhancing products have reached the
market place. Reasons for this include lack of a
satisfactory safety profile respecting irritation,
lowering of the barrier function, and impairment of the
mucocilliary clearance protective mechanism. The main
20 factor to be considered in the use of enhancers
especially related to bile salts, and some protein
solubilizing agents is extremely bitter and unpleasant
taste. This makes their use almost impossible for human
consumption on a daily basis. Several approaches were
25 utilized to improve the taste of the bile salts based
delivery systems, but none one of them are commercially
acceptable for human consumption to date. Among the
approaches utilized includes patch for buccal mucosa,
bilayer tablets, controlled release tablets, use of
30 protease inhibitors, bucc:ally administered film patch
devices, and various polymer matrices.
The basic problem associated with the above
technologies is the use of large quantities of bile
acids and their salts to promote the transport of the
35 large molecules through membranes in the form of
localized delivery system using patches or tablets. In
spite of using protease inhibitors and polymer coatings
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the technologies failed to deliver proteinic drugs in
the required therapeutic concentrations. Further, the
problem is compounded because of the localized site
effect of the patch which resulted in severe tissue
5 damage in the mouth. Most: attempts were made to deliver
large molecules via the oral, nasal, rectal, and vaginal
routes using single bile acids or enhancing agents in
combination with protease inhibitors and biodegradable
polymeric materials. However, it is extremely difficult
10 to achieve therapeutic levels of proteinic drugs using
these formulations. As single enhancing agents fails to
loosen tight cellular junctions in the oral, nasal,
rectal and vaginal cavities for a required period of
time to allow passage of large molecules through the
15 mucosal membranes without: further degradation. This
problem makes it impractical to use the above mentioned
systems for a commercial purpose.
In order to overcome the above mentioned problem of the
bitter taste, irritation and the penetration of large
20 molecules through the sublingual, buccal and GI tract
mucosal lining, a system has now been designed where
protein drug was encapsulated in mixed micelles made up
of combination of enhancers, e.g. yolk proteins
(lecithins). This system allows opening of the
25 paracellular junctions (t:ight junctions) in oral as well
as in GI tract by gI moti_lity movement with high degree
of protease activity pre:cerved and protecting molecules
from premature degradation in the hostile acidic and
proteolytic GI environment.
30 It is believed that the mixed micelles encapsulate
molecules with high degree of efficiency (>90~
encapsulation). These mixed micelles are extremely
small in the size (1 nm t:o 10 nm), and are smaller than
the pores of the membranes in the oral cavity or the GI
35 tract. It is therefore believed that the extremely
small size of mixed micelles helps encapsulated
molecules penetrate efficiently through the mucosal
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membranes of the oral cavity.
The absorption of proteins and peptides is believed
to be enhanced by the diffusion of large molecules
entrapped in the mixed micellar form through the aqueous
5 pores and the cell struct=ure perturbation of the tight
paracellular junctions.
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
10 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 a<:tive substance can never be
100, that is to say the administered dose of the active
15 drug is not completely absorbed, it is preferable to
incorporate slightly larger amount than the desired
dosage. Where the dosagf~ form is a spray (aerosol) or
the like which is repeatedly dispensed from the same
container, it is recommendably so arranged that the unit
20 dose will be slightly greater than the desired dose. It
should be understood that, dosage should vary with
species of warm blood animals such as man, domestic
animals, and their body weights. Although the
composition of this invention is prepared as the
25 microfine droplets (1 to 10 nm or less) by the virtue of
its preparation methods used and suitable combinations
of enhancer compound characteristics. The utilization
of atomizer or aerosol spray devices (metered dose
inhalers or nebulizers) rnay be useful to further a
30 sufficient reduction of particle size for effective
inhalation from the nasal or oral cavity so the drug may
successfully absorbed or reach to the specific site.
The therapeutic composition of the present
invention can be stored at room temperature or at cold
35 temperature. Storage of proteinic drugs is preferable at
the cold temperature to prevent the degradation of the
drugs and to extend their shelf life. While the mixed
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micellar therapeutic composition of the invention is applied to
the mucosal membranes, the sites of administration may be the
same as those used for the usual mucosal therapeutic
preparation. Generally, oral, transdermal and nasal are the
favourite sites of the administration but the composition can
be applied to the rectal and vaginal mucosa. According to the
physiologically active peptide or protein used, the dosage form
and the site of administration, a specific administration
method can be selected.
As used herein, the term "edetate" is used herein to
refer to pharmaceutically acceptable salts of
ethylenediaminetetraacetic acid.
Summary of the Invention
Accordingly the present invention provides a mixed
micellar pharmaceutical formulation having a pH of between 6.0
and 7.0 comprising a proteinic pharmaceutical agent in micellar
form, water, an alkali metal lauryl sulphate in a concentration
of from 1 to 10 wt./wt.% of the total formulation, a
pharmaceutically acceptable edetate in a concentration of from
1 to 10 wt./wt% of the total formulation, at least one alkali
metal salicylate in a concentration of from 1 to 10 wt./wt.o of
the total formulation, and at least one absorption enhancing
compound selected from the group consisting of lecithin,
hyaluronic acid, pharmaceutically acceptable salts of
hyaluronic acid, octylphenoxypolyethoxyethanol, glycolic acid,
lactic acid, chamomile extract, cucumber extract, oleic acid,
linolenic acid, borage oil, evening primrose oil, trihydroxy
oxo cholanylglycine, glycerin, polyglycerin, lysine,
polylysine, triolein and mixtures thereof, wherein each
absorption enhancing compound is present in a concentration of
from 1 to 10 wt./wt.% of the total formulation, and the total
concentration of absorption enhancing compounds is less than 50
wt./wt.% of the total formulation.
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In an embodiment, the alkali metal lauryl sulphate, the
edetate and the alkali metal salicylate are each in a
concentration of from 2 to 5 wt./wt.% of the total formulation.
In one embodiment, the edetate is an alkali metal
edetate. Preferably the alkali metal edetate is selected from
the group consisting of disodium edetate, dipotassium edetate,
and combinations thereof. In another embodiment, the alkali
metal lauryl sulphate is sodium lauryl sulphate.
In a further embodiment, the alkali metal salicylate is
sodium salicylate.
In another embodiment, the lecithin is selected from the
group consisting of Phospholipon-H (trade mark) saturated
phospholipid, Phospholipon-G (trade mark) unsaturated
phospholipid, phosphatidylcholine, phosphatidyl serine,
sphingomyelin, phosphatidylethanolamine, cephalin, lysolecithin
and mixtures thereof.
In one embodiment, one of the absorption enhancing
compounds is selected from the group consisting of hyaluronic
acid, pharmaceutically acceptable salts of hyaluronic acid and
mixtures thereof, the concentration of each such absorption
enhancing compound being from about 1 to about 5 wt./wt.% of
the total formulation.
In another embodiment, suitable for delivery through
nasal passages, the mixed micellar pharmaceutical formulation
is suitably diluted to avoid irritation of the nasal passages.
For insulin-containing and some other compositions, the
composition may also contain at least one inorganic salt which
opens channels in the gastrointestinal tract 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
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least one antioxidant to prevent degradation and oxidation of
the pharmaceutically active ingredients. It 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 and sorbitol.
In one embodiment the antioxidant is 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.% 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.% of the total
formulation. Soyabean trypsin and aprotinin may be used in
concentrations of about 1 to 2 wt./wt.% of the total
formulation.
The formulation suitable for delivery through oral
mucosal membranes may be in chewable form, in which case it
will be necessary to add ingredients suitable for such form.
Such ingredients include guar gum, powdered acacia,
carrageenin, beeswax and xanthan gum.
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 consisting of insulin, heparin, low molecular weight
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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 peptides (GLP), large molecule
antibiotics, protein-based thrombolytic compounds, platelet
inhibitors, DNA, RNA, gene therapeutics and antisense
oligonucleotides.
The present invention also provides a process for making
a pharmaceutical formulation suitable for delivery through
transdermal membranes comprising:
a) preparing a micellar proteinic pharmaceutical agent
composition in an aqueous medium having a proteinic
pharmaceutical agent, an alkali metal salicylate in a
concentration of from 1 to 10 wt./wt.% of the total aqueous
micellar pharmaceutical agent composition, an alkali metal
lauryl sulphate in a concentration of from 1 to 10 wt./wt.% of
the total aqueous micellar pharmaceutical agent composition and
a pharmaceutically acceptable edetate in a concentration of
from 1 to 10 wt./wt.% of the total aqueous micellar
pharmaceutical agent composition;
b) slowly adding the micellar proteinic pharmaceutical
agent composition to at least one of the absorption enhancing
compounds selected from the group consisting of lecithin,
hyaluronic acid, pharmaceutically acceptable salts of
hyaluronic acid, octylphenoxypolyethoxyethanol, glycolic acid,
lactic acid, chamomile extract, cucumber extract, oleic acid,
linolenic acid, borage oil, evening primrose oil, trihydroxy
oxo cholanylglycine, glycerin, polyglycerin, lysine,
polylysine, triolein and mixtures thereof, while mixing
vigorously, to form a mixed micellar proteinic pharmaceutical
composition; wherein the absorption enhancing compounds are
each present in a concentration of from 1 to 10 wt./wt.% of the
total formulation, and the total concentration of alkali metal
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salicylate, alkali metal lauryl sulphate, edetate and
absorption enhancing compounds is less than 50 wt./wt.o of the
total formulation.
In one embodiment, the process provides an additional
S step of adding, while continuing vigorous mixing, at least one
absorption enhancing compound different from that added in step
b), selected from the group consisting of lecithin, hyaluronic
acid, pharmaceutically acceptable salts of hyaluronic acid,
octylphenoxypolyethoxyethanol, glycolic acid, lactic acid,
chamomile extract, cucumber extract, oleic acid, linolenic
acid, borage oil, evening primrose oil, trihydroxy oxo
cholanylglycine, glycerin, polyglycerin, lysine, polylysine,
triolein and mixtures thereof.
In one embodiment the alkali metal lauryl sulphate is
sodium lauryl sulphate.
In another embodiment the alkali metal salicylate is
sodium salicylate.
In a further embodiment the alkali metal edetate may be
selected from the group consisting of disodium edetate and
dipotassium edetate.
In yet another embodiment, the formulation has a
combination selected from the group consisting of i) sodium
hyaluronate and Phospholipon-H, ii) Phospholipon-H and glycolic
acid, and iii) sodium hyaluronate and lecithin.
The vigorous mixing may be accomplished by using high
speed stirrers, e.g. magnetic stirrers or propellor stirrers,
or by sonication.
In one embodiment, the mixed micellar formulation is
formed by sonication of the aqueous micellar pharmaceutical
agent composition in the presence of lecithin.
Detailed Description of Preferred Embodiments
The present invention provides an improved method for
delivery of macromolecular (high molecular weight)
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pharmaceutical agents, particularly through the
membranes in the nose, mouth, vagina or rectum. The
preferred delivery is through oral and nasal cavities.
The pharmaceutical agents cover a wide spectrum of
5 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.
For example, hormones which may be administered
with the present invention include thyroids, androgens,
estrogens, prostaglandin;s, somatotropins, gonadotropins,
erythropoetin, interferons, interleukins, steroids and
cytokins. Vaccines which may be administered with the
present invention include bacterial and viral vaccines
15 such as vaccines for hepatitis, influenza, tuberculosis,
canary pox, chicken pox, measles, mumps, rubella,
pneumonia, BCG, HIV and AIDS. Bacterial toxoids which
may be administered using the present invention include
diphtheria, tetanus, pseudonomas and mycobactrium
20 tuberculosis. Examples of specific cardiovascular or
thromobolytic agents inc:Lude heparin, hirugen, hirulos
and hirudine. Large molecules usefully administered
with the present invention include monoclonal
antibodies, polyclonal antibodies and immunoglobins.
25 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
30 agent administered will depend on the parameters
determined for the agent and the method of
administration, e.g. oral, nasal. For example, nasal
formulations tend to require much lower concentrations
of some ingredients in order to avoid irritation or
35 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.
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The mixed micellar formulation is prepared by first
preparing a first micella.r composition which contains
the pharmaceutically active agents, akali metal lauryl
sulphate, edetate and alkali metal salicylate. For
5 those compositions intended for administration through
the nasal, oral, vaginal or rectal cavities, the first
micellar composition is then added to at least one of
the absorption enhancing compounds to form a mixed
micellar composition. At. least one other absorption
10 enhancing compound may also be added subsequently.
Preferably the first absorption enhancing compound is
lecithin.
Although the present. invention has such wide
applicability, the invention is described hereinafter
15 with particular reference to insulin and its analogues,
which are used for the treatment of diabetes.
As indicated hereinbefore, the compositions of the
present invention require: that the pharmaceutical
formulation be in mixed micellar form.
20 In the case of insulin, which is intended for
administration through nasal or oral cavities, the first
micellar solution may be made by adding a buffer
solution to powdered insulin, and then stirring until
the powder is dissolved and a clear solution is
25 obtained. A typical buffer solution is an aqueous
solution of sodium salicylate and sodium lauryl sulphate
and disodium edetate. Typical concentration of sodium
salicylate and sodium lauryl sulphate in the aqueous
solution are about 3 to 20 wt./wt.~ of each compound in
30 the solution. Typically, insulin is present in the
micellar solution in an amount which will give a
concentration of about 2 to 4 wt./wt.$ of the final
formulation. Typically t:he concentration may be about
wt./wt.~ of the first micellar composition.
35 The micellar solution is then added slowly to the
first absorption enhancing compound, e.g. lecithin while
mixing vigorously, e.g. ~~onicating, to form a mixed
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micelle liposomal solution. At least one other
absorption enhancing compounds selected from the group
consisting of lecithin, hyaluronic acid,
pharmaceutically acceptable salts of hyaluronic acid,
5 octylphenoxypolyethoxyetlzanol, glycolic acid, lactic
acid, chamomile extract, cucumber extract, oleic acid,
linolenic acid, borage o:il, evening of primrose oil,
trihydroxy oxo cholanylg:Lycine, glycerin, polyglycerin,
lysine, polylysine, trio:Lein is then added. The mixing
10 may be done with a high speed mixer or sonicator to
ensure uniform micelle p<~rticle size distribution within
the formulation.
Each of the absorpt_~on enhancing compounds, when
present, is in a concentration of from 1 to 10 wt./wt.~
15 of the total formulation.
Preferred salts of hyaluronic acid are alkali metal
hyaluronates, alkaline earth hyaluronates and aluminium
hyaluronate. The preferred salt is sodium hyaluronate.
The preferred concentrat_Lon of hyaluronic acid or
20 pharmaceutically acceptable salts of hyaluronic acid is
from 1 to 5 wt./wt.$ of i:he total formulation. An even
more preferred range is from 1.5 to 3.5 wt./wt.~ of the
total formulation.
Other ingredients may be added to the mixed
25 micellar solution. For example, flavouring agents,
antioxidants, salts, proi;ease inhibitors or other
pharmaceutically acceptable compound may be added.
In general the size of the micelle particles in the
solution is about 1 to 10 nm, and preferably from 1 to
30 5 nm. Such a size distr_Lbution ensures effective
absorption of the formulation, and therefore the
pharmaceutical agent, through the membranes, for example
the membranes in the oral and nasal cavities.
The specific concentrations of the essential
35 ingredients can be determined by relatively
straightforward experimentation. For absorption through
the nasal and oral cavities, it is often desirable to
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increase, e.g. double or triple, the dosage which is
normally required through injection of 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.
Preferred formulations oral or nasal application have
the following combinations: i) sodium lauryl sulphate,
sodium salicylate, disodi.um edetate, Phospholipon-H and
10 sodium hyaluronate; ii) sodium lauryl sulphate, sodium
salicylate, disodium edet.ate, lecithin and sodium
hyaluronate; iii) sodium lauryl sulphate, sodium
salicylate, disodium edet.ate, sodium hyaluronate and
evening of primrose oil; iv) sodium lauryl sulphate,
15 sodium salicylate, disodi.um edetate, Phospholipon-H and
bacitracin; v) sodium lauryl sulphate, sodium
salicylate, disodium edet.ate, Phospholipon-H, sodium
hyaluronate and bacitraci.n; and vi) sodium lauryl
sulphate, sodium salicylate, disodium edetate, sodium
20 hyaluronate, oleic acid and gamma linoleic acid.
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
25 drugs and to extend their shelf life.
As indicated hereinbefore, generally, oral and
nasal are the favourite ~;ites of the administration but
the composition can be applied to the rectal and vaginal
mucosa. According to the: physiologically active peptide
30 or protein used, the dosage form and the site of
administration a specific: administration method can be
selected.
The composition of this invention is generally
prepared as microfine mixed micellar particles (1 to 10
35 nm or less) by the virtue of its preparation methods
used and suitable combinations of absorption enhancer
characteristics.
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Administration of the formulation is by methods
generally known in the ant. For oral and nasal
application, sprays are preferable, but also drops,
chewable tablets, chewable gum and other suitable forms
5 may be used. Utilization of atomizer or aerosol spray
devices (metered dose inhalers or nebulizers) can be
used to further reduce the particle size for effective
inhalation from the nasal or oral cavity so the drug may
successfully reach to the specific site and be absorbed.
10 It is also possible to utilize a drug delivery system
such that an enteric coating is applied to the gelatin
capsule to cause the micelles to be released only in the
duodenum or in the proximity of the large intestine and
not in the stomach.
15 The invention is illustrated by reference to the
following examples.
Example 1
A first experiment was conducted to provide data
for comparative purposes. This example does not fall
20 within the scope of the present invention.
A solution was prepared using 0.5 g sodium lauryl
sulphate, 0.5 g sodium salicylate and 0.25 g disodium
edetate dissolved in 10 mL of water. To this solution
40 mg (1000 units) of in:~ulin was added and dissolved
25 completely while stirring, to give about 100 units/mL
insulin solution.
In one set of tests, five healthy non-diabetic
human volunteers were te:~ted with insulin, by injection.
In another set of tests t:he volunteers were tested with
30 insulin, taken orally. The volunteers fasted from
midnight prior to the te:~t, with no food being taken
during the 4 hour study.
On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
35 available from Eli Lilly). On the second day, the
volunteers received 100 units (1 mL volume per drop,
approximately 20 drops) of the above-prepared oral
CA 02229286 1998-02-10
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insulin (10 times the injection dose). In both tests,
blood glucose levels werE: monitored every 15 minutes by
Bayer's Glucometer Elite.
The average results for the five volunteers, of the
first day's trial (sub-cutaneous injection with 10
units) were as follows:
Table I
Time*: 0 15 30 60 75 90 120 150 180
Avg: 5.8 5.8 5.4 5.0 4.6 4.3 3.8 3.6 3.4
10 Time*: 210 240
Avg: 4.2 4.5
* time in minutes
The results for each of the five volunteers, of the
second day's trial (oral drops with 100 units) were as
follows:
'fable II
Time*: 0 15 30 60 75 90 120 150 180
Subject Nos:
1 6.2 5.8 5.2 5.0 4.9 5.0 5.0 4.8 4.7
2 5.8 5.4 5.0 4.7 4.9 4.3 5.0 5.5 5.2
3 4.8 4.6 4.3 4.3 4.4 4.6 4.8 4.7 5.2
4 6.6 6.1 5.8 5.5 5.1 4.9 5.0 5.0 5.9
5 6.0 5.8 5.7 5.5 5.1 4.8 4.7 4.9 5.0
Time*: 210 240
Subject Nos:
1 5.5 6.0
2 5.8 6.1
3 5.5 5.1
4 6.2 6.8
5 5.9 6.7
* time in minutes
Th ese ts indicatethat compared to e injection
tes th
method, oral nsulin givE:s a faster onset action and
i of
lowers blood lucose levels without creating
g
hypogly caemic condition. Due to the hepatic glucose
product ion, ere was ebound effect. This is
th a r
believe d to due to the incomplete absorpti on of
be
CA 02229286 1998-02-10
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insulin.
Example 2
Another experiment, not within the scope of the
present invention, was performed for comparative
purposes.
Oral insulin (100 units) was formulated in
(Phospholipon-H, 10 mg) without any sodium lauryl
sulphate, sodium salicylate, edetate or absorption
enhancers, to evaluate its efficacy of blood glucose
10 lowering in a fasted stage, for healthy volunteers.
Volunteers were asked to fast overnight and not
have any breakfast prior to dosing. Volunteers were
asked to take this oral insulin formulation in their
mouth and swallow it. B7_ood glucose levels were
15 monitored every 15 minutes using Bayer's glucometer
Elite for 3 hours, and the average results for 5
volunteers are shown in Table III.
Table III
Time*: 0 15 30 45 60 75 90 120 150
20 180
Avg: 5.6 5.8 5.8 5.7 5.7 5.8 5.7 5.7 5.8
5.7
* time in minutes
This indicates that orally administered insulin
25 with lecithin alone has no effect on blood glucose
lowering.
Exam~l a 3
A further experiment:, not within the scope of the
present invention, was performed for comparative
30 purposes.
Oral insulin (100 units) was formulated with sodium
salicylate and alkali metal edetate (both 5$ by wt.) to
evaluate its efficacy of blood glucose lowering in
fasted state in healthy volunteers.
35 Volunteers were asked to fast overnight and not
have any breakfast prior to dosing. Volunteers were
asked to take this oral insulin formulation in their
CA 02229286 1998-02-10
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mouth and swallow it. Blood glucose levels were
monitored every 15 minutes using Bayer's glucometer
Elite for 3 hours and the: average results for 5
volunteers are shown in 'fable IV.
Table IV
Time*: 0 15 30 45 60 75 90 120 150
180
Avg: 5.8 5.8 5.8 5.9 5.8 5.9 5.7 5.9 6.2
6.0
* time in minutes
This indicates that orally administered insulin
with sodium salicylate and alkali metal edetate alone
has no effect on blood glucose lowering. In addition,
this formulation caused irritation and burning
15 sensation, which lasted f:or several hours.
Example 4
A further experiment:, not within the scope of the
present invention, was performed for comparative
purposes.
20 Oral insulin (100 units) was formulated using
sodium salicylate and alkali metal edetate (both 5~ by
wt.) with Phospholipon-H (10 mg) and tested on healthy
subjects. Blood glucose levels were monitored every
minutes using Bayer's glucometer Elite for 3 hours
25 and the results are shown in Table V.
Table V
Time*: 0 15 30 45 60 90 120 180
Avg: 5.3 5.3 5.3 5.4 5.6 5.7 5.7 5.8
* time in minutes
30 This indicates that orally administered insulin
with sodium salicylate, alkali metal edetate and
Phospholipon-H has no effect on blood glucose lowering.
Example 5
Another experiment, not within the scope of the
35 present invention, was performed for comparative
purposes.
Oral insulin (50 units) was formulated using only
CA 02229286 1998-02-10
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alkali metal lauryl sulphate (5~ by wt). Blood glucose
levels were monitored every 15 minutes using Bayer's
glucometer Elite for 3 hours and the average results for
four volunteers are shown in Table VI.
Table VI
Time*: 0 15 30 60 90 120 180
Avg: 5.8 5.6 5.4 5.3 5.4 5.4 5.6
* time in minutes
This data shows that orally administered insulin
with only alkali metal lauryl sulphate has little
metabolic effect on the blood glucose lowering in
healthy subjects. This formulation caused substantial
burning sensation and irritation in the subjects and
lasted for two days.
Example 6
Yet another experiment, within the scope of the
present invention, was performed.
Mixed micellar oral insulin (50 units) was
formulated using alkali metal lauryl sulphate and sodium
salicylate (both 4.4~ by wt.) and alkali metal edetate
(2.2~ by wt) with Phospholipon-H (10 mg) and tested on
healthy volunteers.
The method involved mixing the sodium lauryl
sulphate, sodium salicylate and alkali metal edetate
25 with water in a beaker with a magnetic stirrer at medium
speed until the ingredients were dissolved, to form
buffer solution. Insulin powder was placed in a beaker
and to this powder was added the buffer solution. The
solution was continuously stirred using a magnetic stir
30 bar until all of the insulin powder was dissolved and a
clear solution obtained. The micellar solution so
formed was stored in clean glass bottles and
refrigerated.
Mixed micellar liposomal insulin was then prepared
35 in a glass beaker, in which was placed the Phospholipon-
H and a small amount of isopropyl alcohol. The mixture
was stirred at a high speed (1000 rpm) for about 10
CA 02229286 1998-02-10
- 20 -
minutes to ensure complete dissolution of the
Phospholipon-H. To this solution was added the micellar
insulin solution very slowly, drop wise, using glass
dropper, with continuous stirring at a high speed. The
5 solution was stirred continuously for another 30 minutes
at a high speed to ensure: uniform micellar particle size
distribution.
Samples of the mixed micellar solution were taken
orally by the volunteers.
10 Blood glucose levels were monitored every 15
minutes using Bayer's glucometer Elite for 3 hours and
the average results for 5 volunteers are shown in Table
VII.
Table VII
15 Time*: 0 15 30 45 60 90 120 150 180
Avg: 6.5 6.1 5.5 5.3 5.3 5.4 5.5 5.5 5.5
* time in minutes
This data shows that. orally administered insulin
with alkali metal lauryl sulphate combined with the
20 sodium salicylate and alkali metal edetate with
Phospholipon-H has a small metabolic effect on blood
glucose levels in healthy volunteers.
Example 7
An experiment, within the scope of the present
25 invention, was performed. In this example, the
formulation was for oral administration.
Oral insulin (50 units) was formulated using alkali
metal lauryl sulphate and sodium salicylate (both 4.4~
by wt.) and alkali metal edetate (2.2~ by wt.) with
30 Phospholipon-H (10 mg) and sodium hyaluronate (1.1~ by
wt). This formulation was tested on healthy subjects
under fasting condition.
The method involved mixing the sodium lauryl
sulphate, sodium salicylate and alkali metal edetate
35 with water in a beaker with a magnetic stirrer at medium
speed until the ingredients were dissolved, to form
buffer solution. Insulin powder was placed in a beaker
CA 02229286 1998-02-10
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and to this powder was added the buffer solution. The
solution was continuously stirred using a magnetic stir
bar until all of the insulin powder was dissolved and a
clear solution obtained. The micellar solution so
5 formed was stored in clean glass bottles and
refrigerated .
Mixed micellar liposomal insulin was then prepared
in a glass beaker, in which was placed the Phospholipon-
H and a small amount of :isopropyl alcohol. The mixture
10 was stirred at a high speed (1000 rpm) for about 10
minutes to ensure complete dissolution of the
Phospholipon-H. To this solution was added the micellar
insulin solution very slowly, drop wise, using glass
dropper, with continuous stirring at a high speed. The
15 solution was stirred continuously for another 30 minutes
at a high speed to ensure uniform micellar particle size
distribution. The hyaluronate and small amounts of
menthol and sorbitol were then added, with continuous
stirring.
20 Samples of the mixed micellar solution were taken
orally by the volunteers.
Blood glucose levels were monitored every 15
minutes using Bayer's glucometer Elite for 3 hours and
the average results for 5 volunteers are shown in Table
25 VIII.
Table VIII
Time*: 0 15 30 45 60 90 120 150 180
Avg: 6.5 5.9 5.6 5.4 4.9 5.0 4.9 5.2 5.4
* time in minutes
30 This data shows that: orally administered insulin
with alkali metal lauryl sulphate, sodium salicylate,
alkali metal edetate, Phospholipon-H and sodium
hyaluronate has resulted in lowering of blood glucose
levels in healthy subjects better than the above
35 mentioned formulations.
Example 8
A further experiment., within the scope of the
CA 02229286 1998-02-10
- 22 -
present invention, was performed. In this example, the
formulation was for oral administration.
A buffer solution was prepared using 0.5 g sodium
lauryl sulphate, 0.5 g sodium salicylate and 0.25 g
5 disodium edetate dissolved in 10 mL of water. The
solution was added to insulin and mixed, to form
micellar insulin.
Separately, 100 mg of powdered Phosphatidylcholin-H
was added to a glass beaker and to this powder was added
10 10 mL 50~ ethanol. The powder was dissolved completely.
To this solution 16 mg (900 units) of micellar insulin
solution dissolved in 3 mL of the buffer solution to
(give 30 units/mL insulin solution) was added slowly
with vigorous mixing, to form a mixed micellar solution.
15 To this was added 0.6 mL of sodium hyaluronate and 0.2
ml of 2~ menthol solution containing 3~ sorbitol.
In one set of tests, ten Type II diabetic human
volunteers who took insulin, by injection three times a
day, were studied. In another set of tests the
20 volunteers were tested with insulin, taken orally. The
volunteers fasted from midnight prior to the test, with
no food being taken during the 4 hour study.
On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
25 available from Eli Lilly). On the second day, the
volunteers received 30 units (1 mL volume per drop,
approximately 20 drops) of the above-prepared oral
insulin (3 times the injection dose). In both tests,
blood glucose levels were: monitored every 15 minutes by
30 Bayer's Glucometer Elite.
The results, showing the average for the ten
volunteers, were as shown on the following page:
CA 02229286 1998-02-10
- 23 -
Table IX
Blood glucose levels (mmol/L)
Time (minutes) Oral Dose Injection
(30 units) (10 units)
0 6.4 6.8
15 5.8 6.9
30 5.4 6.1
45 5.3 5.8
60 5.3 5.8
75 5.2 5.8
90 5.2 5.4
105 5.2 5.4
120 5.1 5.2
135 5.1 5.1
150 5.2 4.9
165 5.3 4.9
180 5.3 4.8
195 5.4 4.8
210 5.4 5.2
225 5.6 5.2
240 5.6 5.4
The results show that the oral insulin formulation
of the present invention, at a dosage of three times
25 higher than the injected level, is comparable to the
injected insulin.
Example 9
This example illustrates a method for making a
mixed micellar formulation according to the present
invention.
In a 250 mL capacity glass beaker was added 5 g
sodium lauryl sulphate, 5 g sodium salicylate and 2.5 g
edetate. The beaker was placed on the hot plate with a
magnetic stirrer. To this dry powder mixture was added
35 100 mL distilled water and the mixture was stirred,
using the magnetic stir bar, at a medium speed until all
the powder was dissolved. The buffer solution was
CA 02229286 1998-02-10
- 24 -
stored in a clean glass bottle at room temperature (pH
6.5).
A micellar insulin solution was then prepared in a
50 mL capacity glass beaker, into which was placed
5 11.54 mg insulin powder. To this powder was added 10 mL
of the buffer solution. The solution was continuously
stirred using a magnetic stir bar until all of the
insulin powder was dissolved and a clear solution
obtained. The micellar solution so formed was stored in
clean glass bottles and refrigerated.
A 2$ menthol solution was then prepared from 100 mg
menthol crystals, dissolved in 5 mL ethanol. To this
solution was added 5 mg F'D & C blue dye. The solution
was stirred for 10 minutes and stored in a glass bottle
at room temperature.
Mixed micellar liposomal insulin was then prepared
in a 50 mL glass beaker, in which was placed 100 mg of
phosphatidylcholine (Sigma, type I=EH, hydrogenated). To
this powder was added 10 mL of isopropyl alcohol. The
20 mixture was stirred at a high speed (1000 rpm) for about
10 minutes to ensure complete dissolution of the
phosphatidylcholine. To this solution was added the
micellar insulin solution very slowly, drop wise, using
glass dropper, with continuous stirring at a high speed.
25 The solution was stirred continuously for another 30
minutes at a high speed t.o ensure uniform micellar
particle size distribution. To this solution was added
1 mL of the 2~ menthol solution and 50 mg sodium
hyaluronate. The semi-clear, translucent, light blue
30 colour, liposomal insulin mixed micellar solution {final
volume 15 mL) was stored in a clean glass bottle and
refrigerated. The solution had a pH of 6.5.
If the phosphatidylcholine powder does not dissolve
completely, then heating up to about 45°C may be
35 required, e.g. using a water bath.
It has been found that if the micellar insulin
composition is not added slowly, then the mixed micellar
CA 02229286 1998-02-10
- 25 -
formulation will not be formed and the formulation will
be gelatinous and sticky.
Example 10
The formulation of Example 9 was tested in a manner
similar to that indicated in Example 8 except that the
formulation of the present invention was administered
nasally.
On the first day, the ten volunteers each received
units insulin injection (regular fast acting, Eli
10 Lilly). On the second day, the volunteers received 20
units of the "oral" insulin of Example 9 (2 times the
injection dose). The "oral" insulin was administered as
drops (0.4 mL volume per drop, approximately 4 large
drops in total, i.e. two drops in each nostril).
15 The results, showing the average for the ten
volunteers, were as follows:
m -. t-. ~ .. v
Blood glucose levels (mmol/L)
Time (minutes) Nasal Dose Injection
(20 units) (10 units)
0 7.4 6.8
15 6.7 7.0
30 5.9 6.8
45 5.3 6.3
60 5.0 6.3
75 5.2 5.8
90 5.1 5.2
105 5.0 5.0
120 4.6 5.2
135 4.5 4.2
150 4.3 4.6
165 4.3 4.0
180 4.8 4.1
195 5.3 4.3
210 5.4 4.5
225 5.7 4.7
240 5.6 5.0
CA 02229286 1998-02-10
- 26 -
The results show that the nasal insulin formulation
of the present invention,. at a dosage of twice the
injected level, is comparable to the injected insulin.
Example 11
5 The formula of Example 9 was taken and tests
performed to determine the insulin action on meal
glucose on healthy volunteers.
Usually, diabetic patients take an insulin
injection 30 minutes prior to a meal, because injected
insulin takes a long timE: to take effect. Injected
insulin is slowly absorbed into bloodstream within 60
minutes and has metabolic: effect on meal glucose levels.
The mixed micellar formulation of Example 9 was
tested in healthy voluntE:ers under controlled conditions
to determine the oral insulin effect on meal glucose
when compared to injected insulin.
In one set of tests, ten healthy non-diabetic human
volunteers were tested with insulin, by injection. In
another set of tests the volunteers were tested with
20 insulin, taken orally. The volunteers fasted from
midnight prior to the tests, with food being taken 30
minutes after dosing. The meals were standard Sastacal
240 mL liquid diet approved by the Diabetic Society,
containing 400 calories.
25 On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
available from Eli Lilly). On the second day, the
volunteers received 30 units of the above-prepared oral
insulin (3 times the injE:ction dose). In both tests,
30 blood glucose levels werE: monitored every 15 minutes by
Bayer's Glucometer Elite. The results are shown on the
following page:
CA 02229286 1998-02-10
- 27 -
Table XI
Blood glucose levels (mmol/L)
Time (minutes) Oral Dose Injection
(30 units) (10 units)
0 5.7 5.5
5.2 5.6
30 5.0 5.4
45 5.3 5.4
10 60 5.4 5.6
75 6.3 6.6
90 6.9 7.0
105 6.0 5.9
120 5.8 5.6
15 135 5.5 5.1
150 5.1 4.8
165 4.9 4.6
180 4.8 4.3
The results indicate that the oral insulin helps
control meal glucose levels in healthy volunteers when
compared to injected insulin.
Example 12
The mixed micellar formulation of Example 9 was
tested in diabetic volunteers under controlled
25 conditions to determine the oral insulin effect on meal
glucose when compared to injected insulin.
In one set of tests, ten Type II diabetic human
volunteers who took insulin, by injection three times a
day, were studied. In another set of tests the
30 volunteers were tested with insulin, taken orally. The
volunteers fasted from midnight prior to the tests, with
food being taken 30 minutes after dosing. The meals
were standard Sastacal 290 mL liquid diet approved by
the Diabetic Society, containing 400 calories.
35 On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
available from Eli Lilly). On the second day, the
CA 02229286 1998-02-10
- 28 -
volunteers received 30 units of the above-prepared oral
insulin (3 times the injection dose). In both tests,
blood glucose levels were monitored every 15 minutes by
Bayer's Glucometer Elite.
The average results for the 10 volunteers were as
follows:
Table XII
Blood glucose levels
(mmol/L)
Time (minutes) Oral Dose Injection
(30 units) (10 units)
0 8.8 8.7
15 8.1 8.8
30 8.0 8.9
45 8.4 10.1
60 10.2 11.8
75 11.8 11.8
90 12.3 12.2
105 10.8 11.2
120 9.6 10.4
135 8.1 8.4
150 6.9 7.3
165 6.2 6.5
180 4.8 4.3
The results ind icate that oral insulin helps to
control meal glucoselevels in diabetic pati ents when
compared to injectedinsulin.
Example 13
A chewable gum insulin formulation was prepared
by
vigorously stirring the l.iposomal insulin xed micellar
mi
solution of Example 9 while adding guar gum,beeswax,
powdered acacia, ic acid, gamma-linoleic acid and
ole
sorbitol. For each 30 units of insulin, mixture
the
contained 100 mg r gum, 50 mg beeswax, mg powdered
gua 50
acacia, 100 mg oleicacid, 100 mg gamma-lino leic acid
and 1 mL 3~ sorbitolin ethanol solution. he mixture
T
was then poured intoa flat tray coated with
polytetrafluoroethylene until the mixture s about
wa
CA 02229286 1998-02-10
- 29 -
mm deep. The mixture then solidified and after
solidification was cut into sticks about 1 cm by 3 cm.
Each stick contained about 30 units insulin.
The mixed micellar :Formulation in chewable stick
5 form was tested in diabetic volunteers under controlled
conditions to determine the oral insulin effect on meal
glucose when compared to injected insulin.
In one set of tests, five Type II diabetic human
volunteers who took insu:Lin, by injection three times a
10 day, were studied. In another set of tests the
volunteers were tested with the chewable gum insulin,
taken orally. The volunteers fasted from midnight prior
to the tests, with food being taken 30 minutes after
dosing. The meals were standard Sastacal 240 mL liquid
15 diet approved by the Diabetic Society, containing 400
calories.
On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
available from Eli Lilly). On the second day, the
20 volunteers received 30 units of the above-prepared
chewable gum oral insulin (3 times the injection dose).
In both tests, blood glucose levels were monitored every
minutes by Bayer's Glucometer Elite.
The average results for the five volunteers were as
follows:
Table XIII
Blood glucose levels (mmol/L)
Time {minutes) Oral Dose Injection
(30 units) (10 units)
0 9.1 8.8
15 9.3 8.2
30 9.3 8.0
45 10.2 8.4
60 11.2 9.2
75 12.1 10.3
90 12.9 11.8
105 13.2 11.6
CA 02229286 1998-02-10
- 30 -
Table XIII (continued)
Blood glucose levels (mmol/L)
Time (minutes) Oral Dose Injection
(30 units) (10 units)
120 12.8 11.0
135 12.2 10.2
150 11.6 9.6
165 11.0 9.5
180 10.6 9.1
195 10.0 8.7
210 9.5 8.2
225 8.8 8.0
240 8.2 7.5
Example 14
15 Another experiment, within the scope of the present
invention, was performed.. In this example, the
formulation was for oral administration.
A buffer solution was prepared using 0.5 g sodium
lauryl sulphate, 0.5 g sodium salicylate and 0.25 g
20 disodium edetate dissolved in 10 mL of water. The
solution was added to 8 mg (200 units) insulin and
mixed, to form micellar insulin.
To this micellar so7_ution were added 0.2 g
bacitracin and 0.5 g evening of primrose oil and the
25 solution was mixed vigorously to form a mixed micellar
insulin solution (about 2.0 units/mL).
Six human volunteers were studied. The volunteers
fasted from midnight prior to the test, with no food
being taken during the 4 hour study.
30 On the first day, the volunteers received 10 units
insulin by injection (regular fast acting insulin,
available from Eli Lilly). On the second day, the
volunteers received 20 units of the above-prepared oral
insulin (twice the injection dose). In both tests,
35 blood glucose levels were monitored at intervals by
Bayer's Glucometer Elite.
The results, showing the average for the six
CA 02229286 1998-02-10
- 31 -
volunteers, were as follows:
Table XIV
Blood glucose levels (mmol/L)
Time (minutes) Oral Dose Injection
(20 units) (10 units)
0 8.8 7.9
8.4 7.9
30 8.1 8.2
45 7.4 8.3
10 60 6.3 7.6
90 5.1 6.2
120 5.0 5.2
150 4.8 4.6
180 5.1 3.9
15 210 5.3 4.4
240 5.6 5.2
The results show that the oral insulin formulation
of the present invention, at a dosage of twice the
injected level, is comparable to the injected insulin.
Example 15
A further experiment: was performed to show another
method of making the mixE:d micellar formulation of the
present invention.
In a 250 mL round bottom flask was added 100 mg of
saturated lecithin powder (Phospholipon-90H) purchased
from the American Lecithin Co. To this powder was added
5 mL of absolute ethanol (USP grade). The flask was
then attached to a rotary evaporator equipped with the
vacuum pump and nitrogen inlet for inert atmosphere
30 condition to minimize oxidation of the lecithin. The
flask was rotated at 100--150 rpm under vacuum. The
solution in the flask was heated to 60°C by means of
water bath to dissolve the powder completely. After
complete dissolution of t:he powder, heating was stopped
35 and the rotation speed was increased to 300 rpm, under
vacuum in nitrogen atmosphere until the alcohol
evaporated completely, leaving a uniform film on the
CA 02229286 1998-02-10
- 32 -
side of the flask. The rotation was continued for at
least 30 minutes to ensure uniform coating of film on
the wall and complete so:Lvent removal. After 30
minutes the rotation was stopped and the vacuum was
released.
To this flask was added micellar insulin solution
which had been prepared :From an aqueous solution of
insulin, sodium lauryl sulphate, sodium salicylate and
disodium edetate. The f:Lask was shaken with the help of
10 shaker plate. Shaking was continued for at least 30
minutes and then the solution was sonicated with a high
frequency sonicating probe for another 60 minutes in
order to form small uniform mixed micelles. The mixed
micelles so obtained were analyzed by Malvern Zeta
15 (trade mark) particle sire distribution measurement
equipment equipped with i~he laser light scattering
device. The mixed micelles particle size distribution
obtained by this method was between 2 and 9nm.
To this solution was added 1 mL of 2~ menthol solution
20 and 50 mg sodium hyaluronate. The semi-clear,
translucent, light blue colour solution (final volume
mL) was stored in a clean glass bottle and
refrigerated. The solution had a pH of 6.5.
Example 16
25 Another experiment, within the scope of the present
invention, was performed..
A buffer solution was prepared using 0.5 g sodium
lauryl sulphate, 0.5 g sodium salicylate and 0.25 g
disodium edetate dissolved in 10 mL of water. The
30 solution was added to 8 rng (200 units) insulin and
mixed, to form micellar insulin.
To this micellar solution were added 0.5 g borage
oil and the solution was mixed vigorously to form a
mixed micellar insulin solution (about 20 units/mL).
