Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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C~il'l'OSAN DRUG DELIVERY SYSTEM
P~I(~l~ITY
This application claims priority under 35 U.S.C. 1 l9(e) from United
States Provisional Application Serial No. 60/019,543, filed June 11, 19g6.
S E~lELD OF THE INVEN~ION
The present invention relates generally to a novel drug delivery system.
~pecifically, the invention relates to a drug delivery system comprising a
modified iron/chitosan particles and chitosan-formulated compounds which
provide enhanced drug delivery via oral a~lministration. In another aspect, the
10 present invention relates to a chitosan matrix for oral delivery of therapeutic
or prophylactic substances.
~AC~GROUND OF l~iE I~VENTION
Development of effective drug delivery systems is an integral part of
research and development in the pharmaceutical industry. To date, several
15 delivery vehicles are commercially available, including simple pills, lozenges,
intravenous solutions, ointments, nasal sprays, transdermal patches, eye drops
and others. For s~lst~in(.d release of drugs over time, delivery devices
include, among others, time-release pills, osmotic pumps and compositions
which support depot formation. Identification and synthesis of biopolymers
20 has led to development of more advanced delivery systems which improve
means for oral ~lministration of drugs ~Langer, Science 249:1527-1533
(1990)], and potentially overcome prob1ems associated with loss of bioactivity
~ due to degradation of drugs in the digestive tract which results in decreased
availability of the drug. Of particular interest are small biopolymer particles
25 which can trap drugs efficiently, protect drugs from the harsh environment ofthe digestive system, and Illtim~tely enhance absorption in the gastro-intestin~l
tract.
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Chitin, poly-~-(1~4) linked N-acetyl-D-glucosamine, is an
biopolymer abundant in nature and has several properties which make it
amenable to use as a drug delivery vehicle, including moderate solubility in
diluted acid solution and high af~mity for organic compounds. The
deacetylated form of chitin, chitosan, has structural characteristics similar toglycosaminoglycans and shows particular promise as a exogenous matrix in
reparative connective-tissue rebuilding [Muzzarelli, et al., Biomaterials 2:247-252 (1988)]. In addition, the ability of chitosan to stimulate macrophages for
tumoricidal activity and for interleukin-l production suggests it possible use
as a drug carrier for tumor patients with depressed immune systems. Id. These
properties have led investigators to test various chitosan formulations witll
hopes of providing a potential drug delivery vehicle which can itself survive
the gastric environment while protecting an associated drug, and which
permits release of the drug over extended periods of time.
Past efforts have been directed to chitosan formulations which
have been tested in limited in vitro assays to examine the formulations ability
to m~int~in integrity and provide ~ll.st~in~cl drug release. Early in vitro studies
suggested that simple dried chitosan gels may have potential use as a vehicle
for sustained drug release rMiyazaki, et al., Chem. Pharm. Bull. 29:3067-
3069 (1981)]. In later studies, chitosan beads, or gels, were prepared in
combination with, for example, glutaraldehyde cro.~.~linking tThacharodi and
Ro, Biomaterials 16:145 148 (~995); Chandy and Sharma, Biomateria7,s
14:939-944 (1993)]; ~lgin~te lAlexakis, et al., Appl. Biochem. Biotechnol.
50:93-106 (1995); Polk, et al., J. Pharm. Sci. 83: 178-185 (1994); Miyazaki
et al, Biol. Pharm. Bull. 17:745 747 (1994); Filiprovic-Grcic, et al., ~ntl. J.
Pharm. 116:39 44 ~1995); Bodmeier and Paeratakul, J. Pharm. Sci. 78:964-
967 (1989)]; ~lgin~te in the presence of counterions, for example
tripolyphosphate or calcium chloride [Bodmeier, et al., Pharm. Res. 6:413-
417 (1989)]; tripolyphosphate alone rSezer and Akbuga, Intl. J. Pharm.
121:113 116 (1995)]; and sodium hydroxide/methanol [Chandy and Sharma,
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Biomate7ials 13:949 952 (1992)~.
Chitosan formulations which incorporate iron appear to be
limited to one instance where iron was entrapped in chitosan beads and which
were then coated with either liposomes or albumin in order to modulate the
5 rate of iron release from the beads ~Chandy and Sharrna, Biom~terials 17:61
66 (1996)]. In that formulation, iron, derived from l~eC13, was the entrapped
"drug" rather than an integral component of the chitosan bead itself.
Moreover, entrapment of iron was effected by spraying solubilized chitosan
into a sodium hydroxide/methanol solution which produced chitosan particles
10 with a size on the order of one millimeter in ~i~me~er
In vivo studies using chitosan beads are considerably more
limited. For example, Jameela, et al. [J. Biomatter. Sci. Polym. Ed.
6:621-632 (1994)] described a chitosan/~lgin~tf: composite bead which allowed
sustained release of ketoprofen for three hours after sublingual delivery in
rabbits. Illum, et al. [Pha~ es. 11:1186 1189 (1994)] demonstrated
enhanced absorption of insulin in rat and sheep mucosa following nasal spray
delivery of insulin entrapped in chitosan. To date, however, no effective
chitosan formulation useful for oral drug delivery has been identi~led.
Thus there exists a need in the art to develop effective oral drug
20 delivery vehicles which survive the harsh environment of the digestive systemand permit s~st~in~.d release of a desired drug which would otherwise be
therapeutically unavailable or have limited availability. The previously
described properties of chitosan make the abundant polysaccharide an
attractive candidate for such a vehicle.
SUl~IARY OF T~E INVE NTION
In one respect, the present invention relates to a drug delivery
composition comprising iron/chitosan particles or complex within which a
drug can be e~ ~ed. The composition is particularly useful for oral
a~1mini~tration of drugs in that the particles or complex provide protection for
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the entrapped drug against the harsh environment of the digestive tract, and
permit drug adsorption by a route that increases the circulatory half-life of the
drug. Another advantage offered by the metal/chitosan complex is a high
affinity for hydrophobic organic compounds, which permits delivery of
5 normally insoluble drugs to the circulatory system.
In a preferred embodiment, the iron/chitosan particles are less than ten
microns in size. More preferred, however, are particles less than ~lve
microns in diameter, and most preferably, all particles are less than 5 nm in
diameter, in order to facilitate endocytosis by cells of the intestin~l mucosa.
In another aspect, the present invention provides a chitosan matrix for
oral delivery of therapeutic or prophylactic substances. Substances
particularly useful in the chitosan delivery system are those which are largely
unable to survive the harsh environment of the digestive tract and are
therefore absorbed at levels generally too low to be useful in an oral delivery
15 system. As an oral delivery system, the invention may be utilized in various
forms, such as a powder, pill, caplet, capsule, gel, liquid, liquid suspension,
emulsion, elixir, syrup, and the like, as long as the manufacturing process
does not result in signi~lcant changes in bead size or in pl~pelLies of the beads
in the composition which have the plefell~d size. The form may be such that
20 the particles themselves are released over a period of time or made availableall at one time. Oral compositions are preferably desi~n~ to release the
active compound at the p ~int in the gastrointestinal tract where bioavailability
is maximized and substance degradation is ~ i"~i~ed.
While oral ~1mini~tration is the presently plerelred method of use,
25 other routes of ~imini~tration are contemplated, including for example,
subcutaneous, transdermal, intr~mllsclll~r, intravenous, intranasal,
intrapulmonary, intrarectal, intravaginal, intraperitoneal, ophth~lmic, and the
like. Optimal particle size for each mode of ~dministration will vary and is
readily determined by one of oldina-y sl~ill in the art. For example, a larger
30 particle will be more easily tolerated in an oral system than in an injectable
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system, which may be con~r~ined by the mechanical injection device and/or
constraints due to capillary size.
The presently ~. ~rel I ~d method for producing the drug delivery
particles involves dissolving chitosan powder in an acidic solution, preferably
5 cont:~ining acetic acid, and sonicating the resultant mixture with concurrent
addition of a metal salt, preferably ferric ammonium citrate. In order to
entrap a drug in the particles, an acid stable drug formulation is added initially
to the chitosan in the acidic solution prior to sonication. Preparation of the
particles, however, may be effected by numerous alternative methods.
~'or example, any of a number of commercially available grades of
chitosan may be utilized to produce the drug delivery system, as well as
chitosan at various degrees of deacetylation. Chitosan may also be produced
from chitin by deacetylation with alkali treatment well known in the art.
Presently preferred is chitosan which is deacetylated between approximately
50-80%. More preferred is chitosan deacetylated to 60-75%.
In addition, any of a number of acidic buffers may be utilized to
dissolve the chitosan powder. The concentration of the acid solution in which
chitosan is initially dissolved may vary in the range of 0.1% to 15 % . Most
preferred, however, is 2% acid solution. Likewise, pH of the acid solution
20 may vary within a pH range of 1.0 to 6.8, however to most pl~relled buffer
is in a pH range of 2 to 3.
Similarly, numerous iron salts may be used as an iron source to effect
particle formation during sonication including, for example, ferric chloride.
Other metal ions, for example, zinc, copper, or nickel, may also be useful in
25 forming chitosan particles. Any physiologically acceptable acid or metal salt is contemplated by the invention.
In another embodiment a drug may be ~lmini~tered orally in a
composition comprising chitosan, a sugar, such as, but not limited to maltose,
hexose, mannose, or glucose and the drug. The invention is also directed to
30 a rnethod for producing an oral drug delivery system, the method comprising
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the steps: (a) preparing a chitosan/sugar solution, (b~ dissolving the drug in
an organic solvent, such as, but not limited to acetone, methanol, ethanol, or
acetonitrile) (c) mixing the results of steps (a) and (b), (d) Iyophilizing the
mixture of step (c), and (e~ recon~itllting the lyophilized mixture in a suitable
5 oral excipient for ~imini.ctration to a subject. The rçsl-lting Iyophil~zed
mixture may also be ground to produce a fine powder. Further, as an oral
delively system, the invention may be utilized in various forms, such as a
powder, pill, caplet, capsule, gel, liquid, liquid suspension, emulsion, elixir,syrup, and the like. Still a further embodiment contemplated is a method for
10 oral drug ~mini~tration comprising ~1mini~tering to a patient an oral
composition comprising chitosan in combination with a sugar and a drug.
Various alternative components for the drug delivery system are also
contemplated. For example, pharmaceutically acceptable oils such as, but not
limited to canola oil, corn oil, peanut oil, olive oil, vegetable oil, mineral oil
15 and the like or lipid compositions may be included in the chitosan/drug
matrix, either prior to, during, or after the sonication step. Addition of lipids
may nrcçs~it~te addition of any of a number of physiologically acceptable
surfactants, depending on the physical characteristics of the drug to be
incorporated. Oils or lipids may provide additional protection for the
20 entrapped drug as it traverses the gastro-intestin~l tract, slow the release of the
therapeutic or prophylactic substance from the chitosan matrix, or improve
absorption in the intestines. Oils or lipids as described above may also be
included to form emulsions which can then be sprayed onto a surface, dried,
collected and compressed into a capsule or tablet. As another additional
25 component, gelatin may be included as an agent to fIX the chitosan matrix with
the entrapped substance; the fixed therapeutic or prophylactic substance-
cont~ining particles can then be collected for delivery in a capsule form.
Sonication is performed to the extent that the resulting chitosan
particles are less than ten microns in diameter, and preferably less than five
30 microns in diameter. Most preferably sonication is continued until particles
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are less than about five nanometers in diameter. The duration and power of
sonication can vary as long as particles of a preferred size are obtained. In
addition~ any type and model o~ sonicator can be utilized to produce the
chitosan particles, again, provided that the resultant particles are of a
5 preferred size. For example, either probe style or water bath types of
sonicator are adaptable to producing the drug delivery system. Depending on
the power of the sonicator, the duration of sonication may vary from one to
ten minutes. A currently preferred sonicator is a Branson Sonifier Model 250
used at a~ ately 60 % full strength for one to three minutes in duration.
1() As an alternative to use of a sonicator, the drug delivery system may
also be produced using various types of homogenizer, emulsifier, fluidizer and
the like.
For physiological delivery of therapeutic or prophylactic substance, the
preferred method for producing the chitosan matrix includes dissolving
15 chitosan in an acidic solution, to which a therapeutic or prophylactic substance
is then added. An aliquot of the mixture is withdrawn, mixed with a
pharn~celltically acceptable oil, and sonicated. The res~-lting chitosan matrix
is particularly useful for oral delivery of therapeutic or prophylactic
substances, for example, hormones (such as but not limited to insulin,
20 progesterone, estrogen, testosterone, glucocorticoids, mineralocorticoids,
growth hormone), cytokines (such as, but not limited to interleukins,
lymphokines, monnkines), chemokines, hematopoietic factors ~such as, but not
limited to erythropoietin) and other therapeutic or prophylactic substances,
polypeptides or proteins which are not ordinarily physiologically active
25 following oral delivery. Proteins or polypeptides which are useful in the
system may be native and purified from naturally occurring sources,
recombinantly produced, or chemically synth~ci7~cl
Varia~ions in the method for producing the drug delivery system are
also comprehended by the invention. For example, chitosan particles can be
30 prepared by the pl~c;ft;lled method but in the absence of a drug. Once
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prepared in this manner, the chitosan particles can then be mixed with a
solution of a desired drug, after which the mixture can be delivered as
described. Alternatively, chitosan particles can be prepared, again in the
absence of a drug, the particles Iyophilized, and subsequently rehydrated in
5 a solution cont:~ining a drug.
DESCRIPTION OF THE DRAWIN~:S
Figure l . sets forth the in vilro release rates of bromthymol blue (BTB)
from iron (Fe), chitosan (Ch), or iron/chitosan particle complexes.
Figure 2. shows the bioavailability of progesterone after oral
10 ~iminiAtration of progesterone/iron/chitosan particles.
Figure 3. sets forth the bioavailability of proge~lervlle after oral
~lminictration of chitosan-formulated progesLelol1e as compared to micronized
progesterone.
Figure 4. sets forth data illustrating blood glucose levels in
15 ~ll~Lo~otocin-treated ~nim~1c after oral ~(iminictration of either a
insulin/chitosan composition or a insulinlPBS composition.
DETAILED DESCRIPTION OF IHE D~VENTIQN
The present invention is illustrated by the following examples relating
to the preparation and use of an iron/chitosan drug delivery composition and
20 chitosan/protein drug delivery systems. Example 1 describes preparation of
iron/chitosan particles. Example 2 illustrates in vitro retention and release ofa compound in and from iron/chitosan particles. Example 3 shows in vivo
delivery of a compound by iron/chitosan particles after a set period of time.
Example 4 describes in vivo release of a compound by iron/chitosan particles
25 or release of a chitosan-formulated compound as a function of time. Example
5 describes use of a chitosan matrix to orally ~dminictPr insulin.
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E~ample 1
Preparation of Iron/Chito~n Par~icles
A 1% Sç~nMer N2000 grade chitosan (CTC Organics, Atlanta, GA)
solution was initially prepared by dissolving powdered chitosan in 2 % acetic
5 acid and the solution was autoclaved. To prepare iron/chitosan particles, a 1
to 5 ml chitosan solution was sonicated for one to three minutes while 0.2-0.3
ml of a 4 % ferric ammonium citrate stock solution/ml of chitosan solution was
added dropwise. The stock ferric ammonium citrate solution was initially
prepared in water. The end result of these steps was in a very fine suspension
10 of chitosan particles. Typical}y, the density of the chitosan particles was
approximately 0.3 g wet weight per ml chitosan solution, or approximately 18
mg lyophilized solid per recon~titnting ml buffer. Particles in the suspension
were measured on a periodic basis using a micrometer and found to range
from about 2 to about 10 microns in diameter. In order to incorporate small
15 molecules in the iron/chitosan particles, the above procedure was modi~led as described below.
Example 2
In vitro Dru~ Release
In order to initially determine release rate from a small molecule
20 entrapped in chitosan/iron particles produced as described above, an i7~ vitro
dialysis assay was performed as described below.
A. Preparation of Iron/Chitosan Palticles Co,~ in~ Bromthymol Blue
A 5 mg/ml solution of bromthymol blue (}3TB) (Sigma, St. Louis,
- MO~ was prepared in water and an equivalent of 200 ,ug BTB was mixed
25 thoroughly with 500 ~ul iron/chitosan solution pl~aled as described in
Example 1. Alternativeiy, an equivalent of 400 ,ug BTB per milliliter of 1%
chitosan solution was mixed. The resulting mixture was sonicated three
minutes while 0.2 to 0.4 ml of 4% ferric ammonium citrate in water solution
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was added dropwise during sonication, which produced orange colored
particles comprising bromthymol blue entrapped in the chitosan beads.
B. In Vitro Release of Bromthymol Blue From Iron chitosan Particles
In order to determine the rate at which the BTB was released to the
5 dialysis buffer, the iron/chitosan particles cont~inin~ bromthymol blue
prepared as described above were placed individually in dialysis bags having
a molecular weight cutoff of 12 kD and each bag immersed in 45 ml
phosphate buffered saline (PBS) in a 50 ml conical tube. The tube was placed
on an end to end shaker and release rate of the bromthymol blue into the
buffer was measured over time by absorption at 595 nm with a Spectronic-20.
Several controls were run in parallel: one cont~ining an equal amount of
bromthymol blue alone in a dialysis bag; another cont~ining bromthymol blue
mixed (but not incorporated as described in section A above, but still res--lting
in a uniform orange solution) with chitosan; and another bag co~ g
15 bromthymol blue mixed with ferric ammonium citrate solution.
The release rates are shown in Figure 1. It can be seen from the graph
that the dialysis bag cont~inin~ bromthymol blue entrapped in iron/chitosan
particles release the bromthymol blue at a much slower rate than the bags
cont~ining bromthymol blue alone, bromthymol blue mixed with ferric
20 ammonium citrate, or blu~ ylllol blue mixed with chitosan. The relatively
slow release from the iron/chitosan particles is observed in the initial stages
of incubation and the slow release rate becomes considerably more
pronounced after longer periods of incubation. These data inflic~te an ability
of the iron/chitosan particles to significantly retain the initially trapped
25 molecules and to initially release the molecules at a slow rate.
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Example 3
In vivo Drn~ Release
In view of the in vitro results presented in Example 2 above,
experiments were ~signecl in order to determine if the iron/chitosan particles
5 were also capable of providing slow release of a substance in vivo. A water
soluble fungicide, nystatin, was chosen for the initial studies.
A. Preparation of Iron/Chitosan Particles Cont~inin~ Nystatin
A 2 mg/ml suspension of nystatin (Sigma) was prepared in distilled
water and 100 fil was mixed thoroughly with 200 ,ul of the chitosan solution
10 prepared as described in Example 1. The rçsl-lting mixture was sonicated for
two to three minutes while 4% ferric ammonium citrate in water was added
(100 ,uVml chitosan:nystatin mixture) producing yellowish particles. The
suspension was centrifuged, the supernatant removed by aspiration, and the
particles resuspended in 200 ~bl phosphate buffered saline (PBS). Following
15 centrifugation the supel.lal~l~l was clear, sug~e~Ling a high degree of drug
incorporation, as nystatin is generally insoluble in water and gives a fine
particulate appearance in aqueous solution. Drug incorporation in the chitosan
particles was determined by HPLC (Waters) on a C-18 column and by
extracting entrapped nystatin from the particles with cold methanol.
20 B. Serum Levels of Nystatin Following Oral
~<lministration of Nystatin Iron/Chitosan Particles
A test group of adult female mice were orally ~Amini~tered
iron/chitosan particles cont~inin~ ny~tin with a feeding needle fitted on 1 ml
syringe. A first control group of mice was fed ny~Latill suspended in PBS and
25 a second control group was fed a chitosan suspension mixed with nystatin to
which S ,ul glutaraldehyde was added to obtain a gel. Each mouse received
an equivalent of a~ro~ ately 4 mg nystatin in a volume of 200-400 ,ul.
The mice were bled at 0, 1 and 5 hr interval through retro-orbital
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puncture. The blood was allowed to clot and serum was separated. After
extracting the serum with cold methanol, the serum levels of nystatin were
measured using HPLC as described above
Blood nystatin concentration values at five hours are shown in Table
S I below; nystatin was not ~letect~hle at zero and one hour after 7l~lministration.
The mice that received nystatin entrapped in iron/chitosan particles produced
higher serum nystatin levels after rlve hours as compared to mice that received
nystatin alone or merely mixed with chitosan. In addition, comparison of
serum nystatin levels between groups in which mice received either OMI or
10 intMperitoneal ~dminictration of nystatin in iron/chitosan particles, it was
observed that mice receiving oMI ?~clminictration had greater than 150% more
serum nystatin than the intraperitoneal group. This result suggests that
association with chitosan provided a higher level of available drug.
Table 1 - Blood Nystatin ConcentMtion Following
Oral ~1mini~stration IJsing Various Delivery Systems
FORMULATION CONCENTRATION
(~/ml)
Nystatin in PBS 0.938
Nystatin mixed with 0.338
20Chitosan and Glutaraldehyde
Nystatin in 2.964
Iron/Chitosan Particles
Nystatin in 1.787
Iron/Chitosan Particles
25 This fonnulation is identical to the formu1ation immediately above, except
that a~mini~tration was intraperitoneal.
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Example 4
I~l v~vo D~-~ Release
In order to further evaluate the effectiveness of iron/chitosan particles
for in vivo delivery and release of a drug, various groups of rats were orally
S ?~lmini.cte,red progesterone prepared as described below (A-B). This
experiment differed from that in Example 3 in that the effect of water
solubility of the entrapped drug was also ~ess~d Further, in order to
evaluate the effectiveness of a chitosan-forrnulated compound to be delivered
and released in vivo, two groups of rats were orally ~-lmini.ctered pro~e~el~,ne10 prepared as described below (C-D).
A. Preparation of Iron/Chitosan Particles Con~ining Progesterone
Progesterone (Sigma Chemical Co., St. Louis, MO) was dissolved in
dimethyl sulfoxide (DMSO; Sigma) to a ~mal concentration of 10 mg/ml and
1 ml of the solution was mixed with an equal volume of 1% iron/chitosan
cont~ining 0.1% Tween 20 and 1 ml mineral oil. The resulting emulsion was
sonicated one to three minutes while 50 ,ul ferric ammonium citrate stoc~
solution was added dropwise. Particles in the rçslllting solution were
measured under the microscope and were determined to range in size from
two to ten microns. Rats receiving aclmini.~tered this ~ )ald~ion were
20 ~lesi~n~tecl Group 1.
Control preparations included: progesterone suspended in PBS and the
resulting was suspension sonicated (~lmini~tered to Group 2); pro~e~efolle
suspended in PBS to which ferric ammonium citrate was added during
sonication (~limini~tered to Group 3); and a suspension of progesterone
25 without ferric ammonium citrate (~lmini~tered to Group 4).
In addition to the above controls, two other ~r~aldtions comprising
water soluble progesterone were l~tili7e,-1 In a water soluble form, the steroidis trapped in 2-hydroxy propyl-~-cyclodextrin and the water soluble form is
commercially available from Sigma (St. Louis MO). In a first pl~dlalion of
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water soluble progesterone, a 10 mg/ml steroid solution was mixed with 1%
chitosan suspension (at a ratio of 10 mg progesterone equivalent/ml chitosan
solution), and the resulting mixture was sonicated. This preparation was
~rlmini~tered to rats in Group 5. The second preparation comprising water
5 soluble progesterone was prepared similar to the first except that 50 ~bl 4%
fernc ammonium citrate was added during sonication to obtain iron/chitosan
particles with entrapped steroid. This preparation was orally ~lmini~tered to
rats in Group 6.
B. Bioavailability of progesterone after oral 7~(1mini~tration
of pro~esterone iron chitosan micro particles
Adult female rats (ovaries removed) weighing between 300 and 350
grams were separately ~dministered each of the various progesterone
forrnulations described in section (A) with each dosage cont;~ining the
e~uivalent of 1 mg progesterone/rat. Dosages were ~mini~tered orally with
a feeding needle attached to a I ml syringe. The rats were bled by retro-
orbital puncture at 0, 2, 4, and 20 hr after ~1mini~tration and serum
progesterone levels were measured by radioi~ oassay (RIA) using a Coat-
A-Count Progesterone solid phase radioimmunoassay kit (Diagnostic Product
Corporation, Los Angeles, CA) .
The results are shown in Figure 2. Serum progesterone levels were
found to be highest in the Group 1 rats which were ~(lmini~tered insoluble
progesterone in iron/chitosan particles, with the highest levels detected four
hours after ~imini~tration. Similarly high levels (particularly four hours after7~riministration) were also detected in ~nim~l~ from Group 6 which were
a-~mini~1~red water soluble progesterone en~ld~ ed in iron/chitosan. While
rats which received progesterone in several of the other form~ tinns showed
highest serum progesterone levels 2 hours after ~-iministration~ the level of
detectable progesterone reduced to near control levels at four hours. Based
on a generally recognized short half-life for progesterone, this observation
suggests either a difrel~;nlial release rate for progesterone in the various
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formulations, or a different route of absorption for the steroid (either native
or water soluble) entrapped in the iron/chitosan particles.
C. Preparation of Chitosan-Formulated Progesterone
200 mg of progesterone (Sigma Chemical Co., St. Louis MO) were
S dissolved in 5 ml of acetone (reagent grade; Fisher Scientific, Pittsburgh, PA)
while 0.3 g of maltose (Fisher Scientific) were dissolved in 10 ml of a 2%
chitosan solution (prepared by dissolving powdered SeaSanMer N2000 Grade
Chitosan (Cl[C Organics, Atlanta, GA) in 0.25 M citric acid, (Fisher
Scienti~lc)). 5 ml of the progesterone/acetone solution were mixed with 5 ml
of the chitosan/maltose solution. The reslllting ~ Lule was rapidly frozen in
liquid nitrogen and lyophilized. After Iyophilization, the weight equivalent of
progesterone in the ground mixture was calculated (usually 3.03 to 3.05 mg
of the fine powder = 1 mg of progesterone alone). The resulting sponge-like
dried chitosan-formulated progesterone may also be ground into a fine
powder. The resulting lyophilized mixture was recon~tihlte~l in deionized
water for oral ~lmini~tration to test ~nim~lc As discussed previously, the
invention may also be utilized in various forms, such as a powder, pill, caplet,capsule, gel, liquid, liquid suspension, emulsion, elixir, syrup, and the like.
D. Bioavailability of progesterone after oral ~-lmini~tration
of pro~esterone chitosan micro particles
Adult female rats (with ovaries removed) weighing between 300 and
350 grams were divided into two treatment groups (7 ~nim~l~ per group).
Animals receiving the recon~tih-te~l chitosan-formnl~t~d progesterone, as
described in ~C), were ~le~ign~te~i Group A (test group), while ~nim~l~
receiving micronized (powdered form) progesterone were ~ecign~terl Group
B (control group). Dosages, which were equivalent to 5mg proge~Lelone/rat,
were ~lmini~tered orally with a feeding needle attached to a 1 ml syringe.
The rats were bled via tail at (), 1, 5~ 24, and 48 hrs after ~1mini~tration andserum progesterone levels were measured by radioimmunoassay (RIA) using
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a Coat-A-Count Progesterone solid phase radioimmuno~c~y kit (Diagnostic
Product Corporation, Los Angeles, CA).
Results, which are shown in Figure 3, indicate a significantly higher
serum level of progesterone in ~nim~lc receiving the chitosan-formulated
5 progesterone mixture as compared to the control ~n;m~l~ (micronized
progesterone) at 1 hour and 48 hours post-~lmini~stration (p-0.013) and 24
post-~q(lmini~tration (p=O.OOl).
Example 5
In Vivo Protein Dr~-~ Release
In view of the results which demonstrated the usefulness of a chitosan
matrix for physiological delivery of drugs, the ability of chitosan to de~iver
proteins via the same oral a(lmini~tration route was investig;ltt-cl
A. Preparation of Chitosan Particles Cont~inin~ Insulin
A 1% chitosan solution was prepared in 2% acetic acid (pH 3.9~ as
15 described in Example 1, above, except that no ferric ammonium citrate was
added to the mixture. A 1 ml aliquot of the chitosan solution was combined
with 12 mg bovine pancreatic insulin (Sigma, approximately 300 units) and
the resulting solution was mixed well. A second 1 ml aliquot of the
chitosan/acetic acid solution cont:~ining 0.1 % polyoxyethylenesorbitan
20 monolaurate (Tween 20) (Sigma) was added and the ~ Ul~ vortexed. A 1
ml aliquot of this final solution was removed and added to î.5 ml Canola oil
(Hunt-Wesson, Fullerton, CA) and the mixture sonicated for one minute. The
final insulin concentration in the resulting cloudy mixture was 60 units/ml.
As a control, 6 mg insulin (approximately 154 U.S.P. units) was added
25 to l ml PBS and the solution mixed well. Canola oil (1.5 ml) was added to
the solution and the resulting mixture sonicated for 1 minute. The res-llting
control solution, like the test solution above, appeared as a white emulsion-
like mixture, however, the control mixture tended to separate into layers while
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the test solution appeared as a stable emulsion.
B. Insulin Bioavailability From Oral
Delivery in Normal Animals
Two groups of three rats were used to dete~nine the availability of
5 insulin following oral delivery in the chitosan matrix prepared as described
above. In the assay protocol, each rat was fed 0.3 ml of a 0.625 g/ml glucose
solution. Five minute~ later each rat was fed 0. 3 ml of either the
insulin/chitosan mixture or the insulin control mixture. Blood was drawn
from the tail vein of each rat at 0, 30, 60, and 120 minutes after
10 7~mini.~tration of insulin, and blood glucose levels were determined using an ExacTech blood glucose testing system (MediSense, Waltham, MA).
The results are presented in Table 2 which indicate that insulin
delivered orally in the chitosan matrix was able to cause a transient decrease
in blood glucose levels that was not detected when insulin was orally delivered
15 alone. These results, however, did not provide any information as to whether
the observed drop in blood glucose was cause by absorbed chitosan alone.
Table 2
Blood Glucose Levels Followin~ Insulin ~dmini~tration
GLUCOSE CONCENTRATION
TIME (mg/dl ~t SD)
(minutes) Control Test
0 84 ~ 7 90 + 9
119 ~t 18 50 ~t 5
114+18 63~t3
120 109 ~t 11 106 + 34
25 C. Insulin Bioavailability From Oral Delivery
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In Normal and Streptozotocin-Treated Animals
In order to determine if the previously observed decrease in blood
glucose resulted from an increase in bioavailability of insulin or from
increased absorption of chitosan itself, the following experiments were
5 performed using both normal rats and rats indllced into a diabetic state by
~lmini~tration of streptozotocin (Sigma, St. Louis, MO) [Rakieten, et al.,
Cancer Chemotherapy Reports 29:91-98 (1963)]. Briefly, diabetes was
induced in rats by intravenous ~(lminic1ration of streptozotocin dissolved in
norrnal saline buffered with anticoagulant acid citrate dextrose (ACD) solution
(1:50 dilution of ACD with 0.9~ NaCl). The final streptozotocin
concentration of the solution was 20 mg/ml, pH 5Ø The diluted solution was
sterilized by filtration through a 0.22 micron filter and used within 10 to 15
minutes of pl~al~llion. Rats were anesthetized with halothane and
a(lminictered a single intravenous injection via the tail vein at a dosage of
approximately 50 mg/kg body weight. Typically, prior to ~rlministration~
blood glucose levels were in the range of 80 to 110 mg/dl blood. One day
after ~rlmini~tration, blood glucose levels were typically elevated to more than600 mg/dl, and six days after ~lministration, blood glucose levels were
typically 350-450 mg/dl.
~ats were a~lmini~t~ red bovine insulin in the oral drug delivery system
described in (A) and resultant changes in blood glucose was G~sermined. As
controls in various experiments, rats from each group were also orally
?Irlmini~te.red: (i) insulin in PBS, ~ii) insulin prepared as in (A) above without
addition of chitosan; and (iii) the ~lcpa.dtion described in ~A) above except
that no insulin was added. As an additional positive control, bovine insulin
in PBS was ~mini~tered intramuscularly.
In the initial experiment, 18.4 units of insulin prepared as described in
(A) suppressed blood glucose levels for almost 2 hours, while intr~mll~c~ r
injection of 0.26 units of insulin depressed blood glucose for almost one hour.
When chitosan was omitted from the oral delivery system, or when insulin
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was orally a(lmini~tered in PBS alone, no change was observed in blood
glucose levels.
In subsequent experiments (see Figure 3), 18.4 units of insulin
prepared as described above in (A) suppressed blood glucose levels for over
5 2.5 hours. Animals treated with insulin in PBS saw no change in blood
glucose levels.
D. Oral Delivery of Insulin in Treating Diabetes
The previous results indicated that insulin delivered in chitosan via an
oral route was capable of suppressing blood glucose levels. In order to assess
10 the ability of orally delivered insulin to treat diabetes, an animal model was
utilized wherein rats were induced into a diabetic state by intravenous
?i~lmini~tration of streptozotocin as ~ cu~ed above. The onset of diabetes was
monitored by m~ ring blood glucose levels. When sustained blood glucose
was measured above 300 mg/dl, the animals were orally :~(1mini~te.red insulin
15 in the following experiment.
Insulin prepared as described in ~A) above was ~lministered to a group
of rats and blood glucose levels were measured and compared to the levels in
a group ~lmini~te.red insulin in PBS. In each group, 18.4 units of insulin
were provided. In rats fed only insulin in PBS, no change in blood glucose
20 levels were detected. In rats fed insulin in chitosan, however, blood glucoselevels were suppressed over the course of the first three hours which the rats
were monitored. These data in-lic~te that the oral delivery of insulin in a
chitosan matrix is useful for treating diabetes in an animal model, suggesting
usefulness of the same drug delivery system for treating diabetes in hllm~n~.
Finally, the above described methods and formulations may be used
with other drugs and therapeutic or prophylactic substances. Such drugs or
- substances may be (but are not limited to) those that are difficult to place in
solution such as many psychoactive drugs (e.g. clozapine) or hormones (e.g.
insulin, progesterone, estrogen, testosterone, glucocorticoids,
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mineralocorticoids, growth hormone) , cytokines (e. g., interleukins,
lymphokines, monokines), chemokines, hematopoietic factors ( e.g.,
erythropoietin) or other therapeutic or prophylactic substances.
Numerous modifications and variations in the invention as set forth in
5 the above illustrative examples are expected to occur to those skilled in the
art. Consequently only such limit~tions as appear in the appended claims
should be placed on the invention.
