Note: Descriptions are shown in the official language in which they were submitted.
W094/~702 PCT~S94/04561
.
2160692
MODIFIED HYDROLYZED VEGETABLE PROTEIN MICROSPHERES
AND METHODS FOR PREPARATION AND USE THEREOF
This is a continuation-in-part of U.S. Patent applica-
tion Serial No. 08/051,739, filed April 22, 1993, which is a
continuation-in-part of U.S. Patent application Serial No.
07/995,508, filed December 21, 1992. This invention relates to
modified hydrolyzed vegetable proteins and microspheres made from
them. The microspheres releasably encapsulate active agents and
are suitable for oral ~mlnlstration to m~mm~ls. Methods for the
preparation of such microspheres are also disclosed.
Backqround of the Invention
The available means for delivering pharmaceutical and
therapeutic agents to m~mm~ls often are severely limited by
chemical or physical barriers or both, which are imposed by the
body. For example, oral delivery of many biologically-active
agents would be the route of choice if not for the presence of
chemical and physicochemical barriers such as extreme pH in the
gut, exposure to powerful digestive enzymes, and impermeability
of gastrointestinal membranes to the active ingredient. Among
the numerous pharmacological agents which are known to be
unsuitable for oral administration are biologically active
peptides and proteins, such as insulin. These agents are rapidly
destroyed in the gut by acid hydrolysis and/or by proteolytic
enzymes.
WO 94/23702 PCT/US94/04561
~,iGo692 2
Much research has been devoted to developing effective
oral drug delivery methods and systems for these vulnerable
pharmacological agents. The proposed solutions ha~re included:
(a) co-administration of adjuvants (such as resorci-
5 nols and non-ionic surfactants polyoxyethylene oleyl ether and n-
hexadecyl polyethylene ether to increase the permeability of the
intestinal walls; and ~
(b) co-administration of enzyr~tic inhibitors, such as
pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF)
10 and trasylol to avoid enzymatic degradation.
The use of such substances, in drug delivery systems,
is limited however either because of their:
(a) inherent toxicity when employed in effective
amounts; or
(b) failure to protect the activeingredient or
promote its absorption; or
(c) adverse interaction with the drug.
Liposomes as drug delivery systems have also been
described. They provide a layer of lipid around the encapsulated
20 pharmacological agent. The use of liposomes containing heparin
is disclosed in U.S. Patent No. 4,239,754 and several studies
have been directed to the use of liposomes containing insulin;
e.g., Patel et al. (1976) FEBS Letters Vol. 62, page 60 and
Hashimoto et al. (1979) Endocrinol. Japan, Vol. 26, page 337.
25 The use of liposomes, however, is still in the development stage
and there are continuing problems, including:
(a) poor stability;
(b) inadequate shelf life;
(c) limited to low MW (< 30,000) cargoes;
(d) difficulty in manufacturing;
(e) adverse interactions with cargoes.
More recently, artificial amino acid polymers or
proteinoids, forming microspheres, have been described for
encapsulating pharmaceuticals. For example, U.S. Patent No.
35 4,925,673 (the '673 patent), the disclosure of which is hereby
WO94/7170~ 216 0 6 9 Z PCT~594104561
incorporated by reference in its entirety, describes such
microsphere constructs as well as methods for their preparation
and use. The microspheres of the '673 patent are useful for
encapsulating a number of active agents, however there is a need
in the art for microsphere carriers that can encapsulate a
broader range of active agents such as lipophilic drugs.
Additionally, the method employed in the '673 patent
for preparing proteinoids results in a complex mixture of high
molecular weight (MW) (~ lO00 daltons) and low MW (~ lO00
daltons) peptide-like polymers which are difficult to separate.
Moreover, small amounts of the low MW microsphere-forming
proteinoids are obtained. Thus, an improved method of preparing
low molecular weight sphere-forming proteinoids is also desired.
Summary of the Invention
The present invention relates to a modified hydrolyzed
vegetable protein microsphere and to a method for preparation of
such microspheres. The invention provides stable microspheres
which are preparable from inexpensive hydrolyzed vegetable
protein, e.g. soybean protein, and a simple and economical method
for making such microsphere. Microspheres made according to the
invention display improved stability and performance in deliver-
ing biologically active materials to m~mm~l S.
According to the invention, modified hydrolyzed
vegetable microspheres are prepared by dissolving hydrolyzed
vegetable protein in an aqueous alkaline solution and adding a
chemical modifier which reacts with free amine residues present
in the hydrolyzed protein. The pH of the reaction mixture is
then lowered until the modified vegetable protein precipitates
out from the mixture. The recovered protein readily forms micro-
spheres and can be used to encapsulate various cargoes such as
pharmaceutical agents. The microspheres are non-toxic and can be
orally or parenterally administered to m~mm~l S. Also
contemplated by the present invention are dosage unit forms that
include these compositions.
W094/~702 PCT~S94/04561
2l~O~92
Description of the Drawin~s
Figure 1 illustrates levels of glucose detected in rat
serum taken from rats orally administered microsphere encapsulat-
ed insulin or raw (unencapsulated) insulin as described in
Example 4. ~
Figure 2 illustrates rat seru~ calcium levels after
oral administration of calcitonin and càlcitonin encapsulated in
the vegetable protein microsphere of the present invention as
described in Example 5.
Figure 3 illustrates an an HPLC trace of the hydrolyzed
vegetable protein before modification.
Figure 4 illustrates the the change in the hydrolyzed
vegetable protein after modification with benzene sulfonyl
chloride in an HPLC trace.
Detailed Description of the In~ention
All patents, patent applications, and literatures cited
in the specification are hereby incorporated by reference in
their entirety. In the case of inconsistencies, the present
disclosure, including definitions, will prevail.
The modified vegetable protein microspheres of the
present invention may be prepared by reacting a hydrolyzed
vegetable protein with a chemical modifying agent which reacts
with free amino residues present in the protein. The modified
vegetable protein is then converted into microspheres which
encapsulate active ingredients, e.g. drugs. A number of
advantages are obt~;n~hle by the present invention which include
(a) the use of readily available and inexpensive starting
materials and (b) a cost-effective method for preparing and
isolating microsphere-producing modified proteins. The overall
modification process is simple to perform and is ~m~n~hle to
industrial scale-up production.
The compositions of the subject invention are useful
W094/~702 2 1 6 ~ ~ 9 ~ PCT~S94/04561
for ~ml n; stering biologically-active agents to any ~nlm~l s such
as birds; m~mm~ls such as primates and particularly humans; and
insects.
According to the method of the present invention, an
acid or enzyme hydrolyzed vegetable protein is useful in
practicing the invention. The vegetable protein generally
contains titratable carboxylic acid groups (COOH) ranging between
about 3 and about 8 milliequivalents/g, preferably between about
4 and about 6 milliequivalents/g, total free amino groups (NH2)
ranging between about 3 and about 9 milliequivalents/g, prefera-
bly ranging between about 4 and about 7 millie~uivalents/g NH2.
The molecular weight of the vegetable protein ranges between
about l00 D and about 2000 D, preferably between about 200 and
about 500 D.
Hydrolyzed vegetable protein is available from a
variety of commercial sources. Non-limiting examples of such
sources include Ajinomoto USA, Inc. (Teaneck, NJ 07666, USA);
Central Soya Co., Inc. (Fort Wayne, IN, USA); and Champlain
Industries, Inc. (Clifton, NJ, USA) and additional companies
listed in "Food Engineering Master", an annual publication of
Chilton Co., Radnor, PA 19089, USA. A particularly pre~erred
hydrolyzed vegetable protein in practicing this invention is
available from Ajinomoto USA under the tradename AJI-EKI. This
produc~ is an acid hydrolyzed li~uid soybean protein which is
derived from defatted soybean meal.
If desired, a dried protein extract of the hydrolyzed
vegetable protein solution may be used to prepare the modified
vegetable protein of the invention. The dried protein extract
is preparable by extracting the hydrolyzed vegetable solution
with a suitable solvent, e.g., methanol, followed by evaporating
the solvent extract.
The vegetable protein is then dissolved in aqueous
alkaline solution of a metal hydroxide, e.g., sodium or potassium
hydroxide, and heated at a temperature ranging between about 50C
W094/~702 PCT~S94/04561
2~6~ ~ 6
and about 70C, preferably between about 50C and about 60C, for
a period ranging between about 10 minutes and about 40 minutes,
preferably about 15 minutes. The amount of alkali employed per
mmole of titratable NH2 in the vegetable protein generally ranges
between about 2 and about 3 mmole, preferably between about 2.2
and about 2.5 mmole. The pH of the solution generally ranges
between about 8 and about 13, preferably;~ranging between about 9
and about 10.
Thereafter, an amine modifying agent is then added to
the reaction mixture while stirring. The amine modifying agents
are compositions that can react with the free amino (NH2)
residues present in the protein. Some non-limiting examples of
amine modifying agents useful in practicing the present invention
include sulfonating agents such as benzene sulfonyl chloride and
acylating agents such as benzoyl chloride.
The amount of amine modifying agent in relation to the
quantity of hydrolyzed vegetable protein employed is based on the
equivalents of total free NH2 in the vegetable protein. Thus,
between about 0.3 and about 1.2 equivalents of modifying agent
are used for each molar equivalent of total NH2 groups in
vegetable protein, and preferably between about 0.6 and about 1.0
equivalents of the modifying agent for each molar equivalent of
total NH2 groups in the hydrolyzed vegetable protein.
In practicing the invention, the mixture of vegetable
protein and modifying agent is maintained at a temperature
generally ranging between about 50C and about 70C, preferably
between about 60C and about 65C for a period ranging between
about 2 and about 5 hours.
The reaction is quenched by adjusting the pH of the
mixture with a suitable acid, e.g., concentrated hydrochloric
acid, until the pH reaches between about 2 and about 3. The
mixture separates on standing at room temperature to form an
opaque upper layer and a dark viscous lower layer. The upper
layer is discarded and modified vegetable protein is collected
W094/~702 21 6 0 69 2 PCT~S94/04561
from the lower layer by filtration. The crude modified vegetable
protein is then dissolved in water at a pH ranging between about
9 and about 13, preferably between about 11 and about 13.
Insoluble materials are removed by filtration and the filtrate is
dried in vacuo. The yield of modified protein generally ranges
between about 30 and about 60~, usually about 45~.
The modified vegetable protein of the present invention
is soluble in alkaline aqueous solution (pH> 9.0); partially
soluble in ethanol, n-butanol and 1:1 (v/v) toluene/ethanol
solution and insoluble in neutral water. The titratable function-
al groups remaining in the vegetable protein after modification
are as follows: carboxylic acid groups (COOH) ranging between
about 1.5 and about 3.5 milliequivalents/g, preferably about 2.3
milliequivalents/g, amino groups (NH2) ranging between about 0.3
and about 0.9 milliequivalents/g, preferably about 0.5 milliequi-
valents/g. The molecular weight of the modified vegetable
protein ranges between about 200 D and about 2000 D, preferably
between about 200 D and about 500 D.
The modified vegetable protein of the present invention
can be used immediately to microencapsulate an active pharmaco-
logical agent or the protein can be concentrated or dried by
conventional means and stored for future use.
The modified vegetable protein may be purified by
fractionation on solid column supports such as alumina, using
methanol/n-propanol mixtures as the mobile phase; reverse phase
column supports using trifluoroacetic acid/acetonitrile mixtures
as the mobile phase; and ion exchange chromatography using water
as the mobile phase. When anion exchange chromatography is
performed, a subsequent 0-500 mM sodium chloride gradient is
employed. The modified vegetable protein may also be purified by
extraction with a lower alcohol such as methanol, butanol, or
isopropanol to remove low molecular weight cont~m~n~nts.
The following procedure may be employed to make
microspheres from purified modified vegetable protein. Modified
W094/~702 PCT~S94tO4561
6 9 ~
vegetable protein is dissolved in deionized water at a concentra-
tion ranging between about 75 and about 200 mg/ml, preferably
about 100 mg/ml at a temperature between about 25~C and about
600C, preferably about 400C. Particulate matter rPm~;n;ng in
the solution may be removed by conventional means such as gravity
filtration over filter paper.
Thereafter, the protein solution, maintained at a
temperature of about 400C, is mixed 1.1 (V/V) with an aqueous
acid solution (also at about 400C) having an acid concentration
ranging between about 0.05 N and about 2 N, preferably about 1.7
N. The resulting mixture is further incubated at 400C for a
period of time effective for microsphere formation as observed by
light microscopy. In practicing this invention, the preferred
order o~ addition is to add the protein solution to the agueous
acid solutlon.
Suitable acids include any acid which does not (a)
adversely effect the protein, e.g., chemical decomposition; (b)
interfere with microsphere formation; (c) interfere with
microsphere encapsulation of cargo; and (d) adversely interact
with the cargo. Preferred acids for use in this invention include
acetic acid, citric acid, hydrochloric acid, phosphoric acid,
malic acid and maleic acid.
In practicing the invention, a microsphere stabilizing
additive preferably incorporated into the aqueous acid solution
or into the protein solution, prior to the microsphere formation
process. The presence of such additives promotes the stability
and dispersibility of the microspheres in solution.
The additives may be employed at a concentration
ranging between about 0.1 and 5 ~ (W/V), preferably about 0.5 ~
(W/V). Suitable, but non-limiting, examples of microsphere
stabilizing additives include gum acacia, gelatin, polyethylene
glycol, and polylysine.
Under the above conditions, the carrier forms hollow or
solid matrix type microspheres wherein the cargo is distributed
in a carrier matrix or capsule type microspheres encapsulating
W094l~702 21 6 0 6 9 2 PCT~S94/04561
liquid or solid cargo. If the carrier microspheres are formed in
the presence of a soluble material, e.g., a pharmaceutical agent
in the aforementioned aqueous acid solution, this material will
be incorporated in the microspheres. In this way, one can
incorporate pharmacologically active materials such as peptides,
proteins, and polysaccharides as well as charged organic
molecules, e.g., antimicrobial agents, which normally have poor
bioavailability by the oral route. The amount of ph~rm~ceutical
agent which may be incorporated in the microsphere is dependent
on a number of factors which include the concentration of agent
in the microsphere forming solution, as well as the affinity of
the cargo for the carrier.
Under these conditions, the modified vegetable protein
molecules form hollow microspheres of less than lO microns in
diameter. If the protein microspheres are formed in the presence
of a soluble material, e.g., a pharmaceutical agent in the
aforementioned aqueous acid solution, this material will be
encapsulated in the hollows of the microspheres and confined
within the protein wall defined by the spherical structure. In
this way, one can encapsulate pharmacologically active materials
such as peptides, proteins, and polysaccharides as well as
charged organic molecules, e.g., quinolones or antimicrobial
agents, having poor bioavailability by the oral route. The
amount of pharmaceutical agent which may be encapsulated by the
microsphere is dependent on a number of factors which include the
concentration of agent in the encapsulating solution, as well as
the affinity of the cargo for the carrier.
Biologically-active agents suitable for use with
carriers disclosed herein include, but are not limited to,
peptides, and particularly small peptide hormones, which by
themselves do not pass or only pass slowly through the gastro-
intestinal mucosa and/or are susceptible to chemical cleavage by
acids and enzymes in the gastro-intestinal tract; polysaccharides
and particularly mixtures of mucopolysaccharides; carbohydrates;
lipids; or any combination thereof. Examples include, but are
W094/~702 21~ ~ 6 9 ~ PCT~S94/04561 ~
not limited to, human growth hormone; bovine growth hormone;
growth hormone releasing hormone; interferons; interleukin-I;
insulin; heparin, and particularly low molecular weight heparin;
calcitonin; erythropoietin; atrial naturetic factor; antigens;
monoclonal antibodies; somatostati~i adrenocorticotropin;
gonadotropin releasing hormone; oxytocin; vasopressin; cromolyn
sodium (sodium or disodium cromoglycate); vancomycin;
desferrioxamine (DFO); or any combination thereof.
Additionally the carriers of the present invention can
be used to deliver other active agents such as pesticides and the
like.
The amount of active agent in the composition typically
is a pharmacologically or biologically effective amount.
However, the amount can be less than a ph~rm~cologically or
biologically effective amount when the composition is used in a
dosage unit form, such as a capsule, a tablet or a liquid,
because the dosage unit form may contain a multiplicity of
carrier/biologically-active agent compositions or may contain a
divided pharmacologically or biologically effective amount. The
total effective amounts will be administered by cumulative units
containing in total pharmacologically or biologically active
amounts of biologically-active agent.
Dosage unit forms can also include any of excipients;
diluents; disintegrants; lubricants; plasticizers; colorants; and
dosing vehicles, including, but not limited to water, 1,2-propane
diol, ethanol, olive oil, or any combination thereof.
The modified vegetable protein microspheres of the
invention are pharmacologically harmless and do not alter the
physiological and biological properties of the active agent.
Furthermore, the encapsulation process does not alter the
pharmacological properties of the active agent. While any
pharmacological agent can be encapsulated within the protein
microspheres, it is particularly valuable for delivering chemical
or biological agents which otherwise would be destroyed or
rendered less effective by conditions encountered within the body
W094/~702 2 ~ G 0 6 ~ 2 PCT~S94/04561
11
o~ the m~mm~l to which it is administered, before the microsphere
reaches its target zone (i.e., the area in which the contents of
the microsphere are to be released) and which are poorly absorbed
in the gastrointestinal tract.
The protein microspheres of the invention are particu-
larly useful for the oral administration of certain pharmacologi-
cal agents, e.g., small peptide hormones, which, by themselves,
pass slowly or not at all through the gastro-intestinal mucosa
and/or are susceptible to chemical cleavage by acids and enzymes
in the gastrointestinal tract. Non-limiting examples of such
agents include human or bovine growth hormone, interferon and
interleukin-II, calcitonin, atrial naturetic factor, antigens and
monoclonal antibodies.
The particle size of the microsphere plays an important
role in determ;nlng release of the active agent in the targeted
area o~ the gastrointestinal tract. Microspheres having
diameters between about ~ O.l microns and about lO microns,
preferably between about 5.0 microns and about O.l microns, and
encapsulating active agents are sufficiently small to effectively
release the active agent at the targeted area within the
gastrointestinal tract. Small microspheres can also be ~m;n;s-
tered parenterally by being suspended in an appropriate carrier
fluid (e.g., isotonic saline) and injected into the circulatory
system or subcutaneously. The mode of administration selected
will, of course, vary, depending upon the requirement o~ the
active agent being administered. Large protein microspheres (>lO
microns) tend to be less effective as oral delivery systems.
The size of the microspheres formed by contacting
modified vegetable protein with water or an aqueous solution
containing active agents can be controlled by manipulating a
variety of physical or chemical parameters, such as the pH,
osmolarity or ionic strength of the encapsulating solution, and
by the choice of acid used in the encapsulating process.
The vegetable protein-derived microspheres of the
present invention are suitable for oral administration of peptide
W094/~702 PCT~S94104561
21~0~92 ~
~ 12
hormones, e.g., insulin, and polysaccharides, e.y., heparin,
which otherwise would be quickly destroyed in the stomach. They
also are suitable ~or protecting the stomach ~rom gastric
irritants, such as aspirin and NSAID~S~. When such aspirin
containing microspheres are orally ~m; n~ stered, they pass
through the gastrointestinal mucosa and release the aspirin far
more rapidly than conventional enterically coated aspirin, which
first must traverse the stomach and then must enter the blood-
stream from the intestine after the enteric coating has dis-
solved.
The microspheres of the invention may be orallyadministered alone as solids in the form of tablets, pellets,
capsules, and granulates suitable for suspension in liquids such
as water or edible oils. Similarly, the microspheres can be
formulated into a composition containing one or more physiologi-
cally compatible carriers or excipients, and which can be
administered via the oral route. These compositions may contain
conventional ingredients such as gelatin, polyvinylpyrrolidone
and fillers such as starch and methyl cellulose. Alternatively,
small microspheres (size less than lO ~m) can be administered via
the parenteral route.
The following examples are illustrative of the
invention but are not intended to limit the scope of the
lnvention.
Exam~le l: Modification of Soybean protein with
benzenesulfonyl chloride
a. Extraction of soybean protein
3.2L of acid hydrolyzed liquid soybean protein solution
(AJI-EKI, Ajinomoto USA, Inc.) was reduced in vacuo to give 1440g
of solid powder. This solid was extracted 3 times with methanol
(2L per extraction). Methanol was removed from the pooled
extracts by evaporation. The yield of soybean protein as a dark
brown powder was 608 g. The functional groups of the soybean
protein powder was titrated using conventional procedures. See,
W094l~702 21 6 0 6 9 2 PCT~S94/04561
13
for example, "A Laboratory Manual of Analytical Methods of
Protein Chemistry," Vol. 1-3, Editors P. Alexander and R.J.
Block, Pergamon Press, 1960 and 1961. The soybean protein
contained the following functional groups: 3.7 milliequivalents-
/g of COOH; 0.44 milliequivalents/g free N-terminal NH2; and
3.48 milliequivalents/g total free NH2. The molecular weight of
the soybean protein ranged from 100 to 2000 D.
b. Modification of soybean protein
The dried soybean protein of step (a) (600 g, 2.5
equivalents of total free NH2) was dissolved in 3L of aqueous 2N
potassium hydroxide solution (2.25 mole excess) and the solution
was heated at 60~C for 30 minutes. Thereafter, benzenesulfonyl
chloride (460 g, 2.60 moles) was added dropwise to the mixture
and the reaction temperature was monitored so that it did not
exceed 65cC. The reaction continued, with stirring, for 4 hours
at 63C. The reaction mixture cooled to room temperature, then
acidified to pH 3.0 with 20~ aqueous HCl solution and modified
soybean protein precipitated out. The modified soybean protein
was then washed twice with distilled water (lL) and dissolved in
2N aqueous sodium hydroxide solution until a pH of 8.5-9
resulted. The solution was filtered to remove particulates and
the filtrate was reduced and dried in vacuo to give dry modified
product (257g, yield = 24~). The product had the following
titratable groups: 2.3 milliequivalents/g of COOH; 0.2
milliequivalents/g N-term~n~l free NH2; and 0.3 milliequivalents-
/g total free NH2.
Example 2: Modification of soybean protein with
benzoyl chloride
A commercial hydrolyzed water solution of soybean
protein (AJI-EKI, Ajinomoto USA, Inc.) was used in this Example
without further extraction. The protein in solution contained
the following functional groups: 2.6 milliequivalents/ml of COOH
and 2.0 milliequivalents/ml NH2. The molecular weight of the
W094/~702 PCT~S94/04561
21~692
.
14
soybean protein was approximately 6.5 kD.
To the soybean solution (240 mL, 0.5 equivalents f~ee
NH2) was added 107 mL of 10N aqueous potassium hydroxide solution
followed by 200 mL of distilled water. The solution was then
placed in an ice bath (5~C) and benz~oyl chloride (70 g, 0.5
moles) was added dropwise within a t~mperature range between 10
to 250C. The reaction mixture was ~en stirred for 4.5 hours at
room temperature. The pH of the reaction mixture was then
reduced from 13.2 to 2.8 with concentrated HCl. After being
allowed to settle for 1 hour, the precipitated modified soybean
protein was collected by filtration and washed with distilled
water. The soybean protein was then dissolved in 2N aqueous
sodium hydroxide solution to give a solution (pH 12.6) which was
then evaporated to afford 48 g of dried product (~ yield - 41~).
Example 3: Preparation of Empty Microspheres
With Modified SoYbean Protein
This Example illustrates a method for the preparation
and cleaning of empty modified soybean protein microspheres.
PROCEDURE
1. Reagents:
a. Modified protein powder prepared as described in
Example 1
b. Anhydrous citric acid (USP)
c. Gum acacia NF
d. Deionized water
e. Glacial acetic acid
2. Equipment
a. pH meter
b. Eppendorf pipette (0-100ul) and tips
c. Water bath, 40C
d. liquid nitrogen
e. lyophilization flasks
W094/~702 21 6 0 6 9 2 PCT~S94/04561
3. Preparation of Solutions:
a. Protein solution - Dissolve l00mg modified soybean
protein in lml deionized water (or multiples thereof).
Filter through a Whatman #l filter paper (if necessary)
and keep at 40C in a water bath. This is solution A.
- b. l.7 N citric acid with 0.5~ acacia - Dissolve 5g of
acacia and l09g of citric acid in l liter deionized
water. Incubate at 40C. This is solution B.
4. Preparation of Microspheres:
a. Add all of solution A to solution B rapidly in one step
while swirling solution B by hand, in a 40C water
bath.
Exam~le 4: Preparation of Soybean protein Microsphere
containinq Encapsulated Insulin
This Example describes a method for the preparation and
cleaning of insulin microspheres.
PROCEDURE
l. Reagents:
a. Modified protein powder prepared as described in
Example l
b. Heparin
c. Anhydrous citric acid (USP)
d. Gum acacia NF
e. Deionized water
f. Desiccant
g. Liquid nitrogen
2. Equipment:
a. Magnetic stirrer
b. Buret
c. Microscope
d. Clinical centrifuge
W094/~702 PCTtUS94tO4561
2~069~ 0
16
e. Dialysis membrane tubing (Spectrum 6, 10 mm, 50,000
M.W. Cutoff)
~. pH meter
g. Lyophilizer (Labconco #75035)
h. Lyophilizing flasks (150-300 mL)
i. Rotating shell freezer i~'
,, . ~
j. Isopropanol/dry ice bath or!liquid N2
k. Mortar and pestle
1. Storage containers (500 mL)
m. Eppendorf pipet (0-100 uL)
n. Plastic closures for dialysis tubing (Spectrum)
o. 2 mL syringe with 0.45 ~m Acrodisk
3. Preparation of Solutions:
a. Protein Solution A (80 mg/ml):
Add 160 mg of modified soybean protein and dissolve to
1 ml with deionized water. Using a 2 ml syringe ~ilter
through a 0.45 ~m Acrodisk into a 10 ml test tube.
Keep at 40 C.
b. Solution B (1.7 N citric acid with 1~ gum):
Dissolve 10 g of acacia and 109 g of citric acid in 1
liter deionized water.
c. Solution C (Heparin solution):
Dissolve heparin in Solution B at 150 mg/mL and keep at
40 C.
4. Preparation of Microspheres:
a. Add all of solution A to solution C quickly while
swirling solution C slowly, by hand, in a 40C water
bath.
5. Cleaning of Microspheres:
~ or multiples thereof.
W094/~702 21 6 0 ~ 9 2 PCT~594104561
a. Transfer the suspension with a syringe (no needle) to
dialysis tubing and seal with plastic closures. Tubing
should be no more than 70~ .ull.
b. Discard any amorphous material sedimented and/or
aggregated on the surface.
~ c. Dialyze the microsphere suspension against acetic acid
(using 20 mL of acetic acid solution per ml of micro-
sphere suspension) while stirring the acetic acid
solution with a magnetic stirrer.
d. Replace the acetic acid solution every hour. Continue
dialyzing for a total of 3 hours.
6. Lyophilization:
a. Add one part of 50~ Trehalose (Sigma) into nine parts
of dialyzed microsphere solution. Flash freeze
microspheres in a freeze-drying flask using the shell
freezer adjusted to rotate at ca. l90 rpm and immersed
in a liquid nitrogen bath.
b. Freeze dry for 24 hours or until dry as evidenced by
lack of self-cooling.
c. Record weight of dry microspheres.
d. Grind to a fine powder with mortar and pestle.
e. Transfer to amber container, seal with desiccant, and
store at room temperature.
7. Resuspension:
a. Weigh the lyophilized powder and calculate the amount
of protein in the powder.
b. Add 0.85 N citric acid into the lyophilized powder at
40C. The final concentration of protein is 80 mg/ml.
Example 5: Evaluation of Insulin Microspheres in Rats
In this Example, the insulin microspheres prepared in
accordance with Example 3 were evaluated in rats. Twelve rats
w094/~702~ ~16 a 6 9 2 PCT~S9~/04561
18
were divided into two groups as follows:
l. oral insulin microspheres: 3 mg insulin/kg body
weight by oral gavage (six rats);
2, Raw insulin (no encaps,ulation): 3 mg insulin/kg
body weight by oral ga~age (six rats).
Oral gavage dosing of ~ts was performed. Insulin
microspheres were prepared immedi~ately prior to dosing and Group
l rats each receive an appropriate dosage of the microsphere
suspension. Group 2 rats received the unencapsulated insulin.
Approximately 0.5 ml of blood was withdrawn from each rat just
prior to dosing ("0" time) and l to 6 h post-dosing. Serum from
the blood samples were stored at -20C.
The glucose levels of thawed serum taken from the rats
were analyzed by conventional methods. As shown in Figure l,
sharp decreases in serum glucose levels were observed in groups
l rats receiving the encapsulated insulin. In contrast, the
serum glucose levels in group 2 rats slightly increased from t ~
0. The results show that encapsulated insulin had a greater
biological effect, when administered orally, in contrast to
unencapsulated insulin.
Example 6: Preparation of Microsphere
Encapsulated Calcitonin
Encapsulation of salmon calcitonin in soybean protein
microspheres were performed in the same manner described in
Example 3. Calcitonin was obtained from Sandoz (Basil, Switzer-
land) and a 150 mg/mL calcitonin solution in l.7 N citric acid
solution with l~ gum was prepared as described in Example 3.
Example 7: Evaluation of Calcitonin Micros~heres in Rats
In this Example, the calcitonin microspheres prepared
in accordance with Example 5 were evaluated in rats. Twelve rats
were divided into two groups as follows:
l. oral calcitonin microspheres: 60 ~g calcitonin/kg
body weight by oral gavage (six rats).
W094/~702 21 6 0 69 ~ PCT~S94/04561
19
2. oral unencapsulated microspheres: 60 ~g calci-
tonin/kg body weight by oral gavage (3 rats)
(Control).
Oral gavage dosing of rats was performed. Calci~onin
microspheres were prepared immediately prior to dosing and Group
1 rats received an appropriate dosage of the microsphere
suspension. Group 2 rats received the unencapsulated calcitonin.
Approximately 0.5 ml of blood was withdrawn from each rat just
prior to dosing ("0" time) and 1 h, 2 h and 3 h post-dosing.
Serum from the blood samples were stored at -20C.
The calcium levels of thawed serum taken from group 1
and 2 rats were analyzed by conventional methods. As shown in
Figure 2, sharp decreases in serum calcium levels were observed
in group 1 rats receiving the encapsulated calcitonin. In
contrast, the calcium levels in group 2 rats slightly decreased
from t = 0. The results show that encapsulated calcitonin had a
greater biological effect, when administered orally, in contrast
to unencapsulated calcitonin.
