Note: Descriptions are shown in the official language in which they were submitted.
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[SPECIFICATION]
[TITLE OF INVENTION]
ORAL DELIVERY OF PEPTIDE
[TECHNICAL FIELD]
The present invention relates to a preparation for oral administration of
peptide
and protein drugs (hereinafter, referred to as "the peptide drug") and a
method for
producing the same.
[BACKGROUND ART]
According to a rapid progress of genetic recombination technique and
solid-phase peptide synthetic technique, the kinds of peptide drugs which can
be used in
clinical field have been broadened on a sudden increasing tendency in the
1990's, which
has been accelerated due to remarkable progress of biotechnology so that about
100 or
more drugs have been developed and commercialized in the world at present.
However, most of peptide drugs as presently developed and commercialized are
in the form of injections and therefore, have some disadvantages in that 1)
injection to
the patient is accompanied by a pain, 2) it has a difficulty in storage such
as the
necessity of storage in a refrigerator, and 3) a patient must attend a
hospital when the
drug is injected. Thus, although it is urgently required to develop the oral
preparations
of the peptide drug, such a development is under the first stage to provide
only an
insignificant outcome up to the present.
If the above-mentioned disadvantages involved in the injection of the peptide
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drug can be overcome by the oral preparation, it is expected that 1) the
peptide drug can
be formulated in the most suitable form for administration to the patient, 2)
the stability
of the preparation is increased so that the conditions of production, selling
and
consumption such as distribution, establishment of the effective period, etc.
can be
conveniently and advantageously varied as compared to the prior formulation,
and 3)
the market of the oral preparation preferred by the patient will rapidly
substitute for the
market of prior drug formulations. Therefore, the technical study to develop
the oral
preparation has been actively made.
In general, the technical difficulty in developing the oral preparation of the
peptide drug has been reported in well-known papers (Raymond M. Reilly, Rommel
Domingo and Jasbir Sandhu. Drug Delivery Systems, 32, 4, 313-323, 1997;
Jeoseph A.
Fix, Pharmaceutical Research, vol. 13, No. 12, 1996; Amyn P. Sayani and Yie W.
Chien, Critical Reviews in Therapeutic Drug Carrier Systems, 13, 1&2, 85-184,
1996;
Jane P. F. Bai, Li-Ling Chang and Jian-Hwa Guo, Critical Reviews in
Therapeutic Drug
Carrier Systems, 12, 4, 339-371, 1995).
In developing the oral preparation of such peptide drugs, the major problems
to
be solved include 1) a low absorption in intestinal tract, 2) a loss of
efficacy by
proteinase present in small intestine and 3) a difficulty in maintaining the
efficacy due
to a short half life within a living body after absorption and thus, the study
places the
focus such problems.
Among these problems, first the following methods have generally been used
for solving a low absorption in intestinal tract: 1 ) Inclusion of drugs in a
nano- or
microsphere particle to increase their permeation through biomembrane (Istvan
Toth,
International Journal of Pharmaceutical, 183, 1999, 51-55; E. Bjork, U.
Isaksson, P.
Edman, J. Drug Targeting 2, 501-507, 1995; Shinji Sakuma, Norio Suzuki,
International
Journal of Pharmaceutical, 149, 1997, 93-106); 2) Method for using the drug
together
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with an absorption accelerant (Patrick J. Shinko, Yong-Hee Lee, Pharmaceutical
Research, vol. 16, No. 4, 1999, 527-533; J. C. Scott-Moncrieff, Z. Shao, J.
Pharm. Sci.
83, 1465-1469, 1994; E. A. Hosny, N. M. Khan, Drug Devel. Ind. Pharm. 2I,
1583-1589, 1995; Isao Sasaki, Hideyuki Tozaki, Biol. Pharm. Bull. 22, 6, 611-
615,
1999; Anthomy C. Chao, Joseph Vu Nguyen, International Journal of
Pharmaceutical,
191, 1999, 15-24); 3) Methods for modifying peptide structures (Deven Shah,
Wei
Chiang, Journal of Pharmaceutical Sciences, vol. 85, No. 12, 1996, 1306-1309;
Kazunori Iwanaga, Satoshi Ono, Journal of Pharmaceutical Sciences, vol. 88,
No. 2,
1999, 248-252; Istvan, John P. Malkinson, J. Med. Chem. 1999, 42, 4010-4013);
4)
Method for modifying the structures of the drugs or using the drugs together
with an
enzyme inhibitor (P. Buhlnayer, A. Caseli, W. Fuhrer, J. Med. Chem. 31, 1839,
1998; A.
Yamamato, T. Taniguchi, K. Rikyu, T. Tsuji, Pharm. Res. I 1, 1496-1500, 1994);
and 5)
Inclusion of the drugs in a microsphere or modification of the drugs in order
to increase
the stability of drugs in a living body (David F. Ranney, Biochemical
Pharmacology,
vol. 59, 105-114, 2000; Ian M. Chapman, Ora H. Pescoviz, Gail Murphy, Journal
of
Clinical Endocrinology & Metabolism, 1997, vol. 82, No. 10, 3455-3463).
Further, the
study to develop the preparation of the peptide drug has been actively
conducted
through numerous ways (Z. Aydin, J. Akbuga, International Journal of
Pharmaceutics
131, 101-103, 1996; K. Aledeh, E. Gianasi, I. Orienti and V. Zecchi, J.
Microcapsulation, 1977, vol. 14, No. 5, 567-576; A. Pork, B. Amsden, K. de
yao,
Journal of Pharmaceutical Science vol. 83, No. 2, 1994, 178-185).
Further, as one method for developing the oral preparation of such peptide
drugs a liposome has been utilized. This method has some advantages that 1)
the drugs
can be relatively readily included in a nano- or microsphere particle and 2) a
liposome
can be easily absorbed in intestinal mucous membrane, and therefore, has been
widely
studied in recent years. Prior arts relating to such method include a
technique
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disclosed in a patent by Lipotec, S.A. (EP-0855179 A2). However, this method
has
several disadvantages in using the liposome as the medicinal product that in
preparing
the liposome 1 ) the use of water may cause the problem relating to a
stability of the
drugs, 2) the procedures for lyophilization or drying must be used in order to
prepare
the liposome in the form of a powder, 3) its stability in aqueous solution is
low because
of the properties of liposome itself including aggregation, sedimentation,
fusion,
oxidation, phospholipid hydrolysis, etc., and 4) it is difficult to provide
the
reproductivility and sterilization during the production procedures.
[DETAILED DESCRIPTION OF INVENTION)
In order to overcome the problems involved in the prior art as mentioned above
the present inventors have studied and examined the preparation. of peptide
drugs with
laying emphasis on the physical modification rather than chemical modification
of
peptide drugs for a long time, and thus completed the present invention.
Thus, the present invention provides a method for improving the problems
involved in the prior art as mentioned above, in which 1 ) water-soluble
chitosan is used
to prepare proliposome as the precursor of liposome thereby increasing the
absorption
into intestinal mucous membrane, 2) an agent for controlling pH is used to
increase a
stability of peptide drugs in aqueous intestinal juice, 3) an additive such as
absorption
accelerator, etc. is added in order that peptide drugs can be smoothly
absorbed into
intestinal mucous membrane, and then 4) the product is formulated into a
preparation
suitable to oral administration and at the same time, S) the preparation is
covered with
an enteric coating so that the drug can be readily migrated and absorbed into
the
intestine without destruction.
That is, the present invention can prepare a proliposome, as the precursor of
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liposome, in a high yield within a short time without conducting the procedure
for
lyophilization or evaporation in order to prepare liposome in the form of a
powder,
which may cause the problems occurring in the prior method for preparing the
oral
preparation using liposome as disclosed in EP-0855179, and therefore, has some
advantages that the procedure of the process is simple, a stability of peptide
drugs to
moisture and temperature, which constitutes the disadvantage of peptide drugs,
is
increased, and chitosan is used as a carrier for preparing proliposome to
increase a
bioavailabilty.
Specifically, according to the present invention
1 ) using the water solubility of water-soluble chitosan, the organic solvent
is used to
prepare the peptide drag coated onto water-soluble chitosan in the form of a
powder;
2) an enteric coating is applied to the drug in order to protect the peptide
drug from
gastric acid;
3) when the drug reaches the intestine, the powdery proliposome in the form of
a
powder forms a liposome within the intestine owing to the physical properly of
water-soluble chitosan to be dissolved in water, so that the drug can be
smoothly
absorbed into intestinal mucous membrane and at the same time, chitosan having
a
free cation provides a property of attaching to intestinal mucous membrane;
4) in order to prevent an easy decomposition of the drug in the intestine
after oral
administration due to an instability of most peptide drugs in an aqueous
solution, an
agent for controlling pH level is added to increase a stability of the drug in
the
intestine by taking an advantage that the most of peptide drugs are stable at
pH level
of 3 to 4; and
5) in order that peptide drugs can be readily absorbed into intestinal mucous
membrane
after a paratrans pattern between cells, an additive such as absorption
accelerator is
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combined so that the peptide drugs can be easily absorbed into intestinal
mucous
membrane.
Hereinafter, the present invention will be more specifically explained.
Chitosan is a natural high molecular material and is a substance made from
polysaccharide chitin which is widely distributed in shells of crustacean such
as crab,
shrimp, etc. and insect outer skins, mushrooms, cell walls of fungi and plays
a role of
supporting and preventing the living body containing them.
Specifically, chitin is a high molecular material in a very long chain
structure
without side chains, which is similar to celluloses, wherein carbon atom at 2-
position is
substituted with acetylamino radical (-NHCOCH3) in place of hydroxyl radical (-
OH).
Chitosan is a material produced by deacetylation of acetyl radical attached to
amino
group present on carbon atom at 2-position in chitin and can have a lot of
free canons so
that it possesses various physiological activities.
For this reason, chitosan in a high quality has been developed and widely used
in various industrial field such as food, cosmetics, pharmaceuticals and
absorbents,
activating agent for plant cells, aggregating agent for waste water disposal,
etc.
Particularly, in recent years it has been disclosed that an ability of
chitosan to combine
fat is far better than that of vegetable fibrin, and chitosan improves the
immunity and
resistance to diseases and further, has no toxicity in a living body and
therefore, is
harmless.
However, since chitosan which has been widely used in various field in these
days cannot be dissolved in water or an alcohol but can be dissolved only in
an aqueous
solution of an organic acid such as formic acid, lactic acid, acetic acid,
etc., or an
inorganic acid such as dilute hydrochloric acid, it has the limited
applicability in the
field of pharmaceutical preparations.
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Thus, the present inventors have studied to prepare the oral preparation of
peptide drugs using water-soluble chitosan (JK FM-O1: Ja Kwang Co., Ltd.), and
then
completed the present invention. Water-soluble chitosan used in the present
invention
is characterized in that 1) the degree of deacetylation is 85%-99%, 2) the
solubility in
water is 99.99% or more, 3) the molecular weight is 100,000 to 500,000 to be
possibly
used as the excipient in pharmaceutical products, and 4) it meets the standard
requirements for a food additive.
That is, the major characteristic feature of chitosan used in the present
invention is the water solubility of 99.99% or more. The reasons why the water
solubility of chitosan used in the present invention is higher than that of
the prior
chitosan are that 1) the method for preparing chitosan by treating chitin with
an aqueous
alkaline solution, and conducting deacetylation procedure by hydrolyzing
acetylamino
group to amino group followed by a mufti-step separation membrane procedure
allows
an increase in the degree of acetylation to 85%-99% while maintaining the
water-solubility of chitosan, 2) chitosan can be prepared in a purity of 90%
or more
through a mufti-step separation membrane procedure, and 3) since amino group
is
activated during the deacetylation procedure for carbon atom at position 2 of
chitosan,
chitosan can be readily dissolved in water while maintaining the
characteristic
properties of chitosan when chitosan is contacted with water molecules.
Furthermore, the characteristic feature of the present invention is to prepare
the
oral preparation of peptide drugs by removing the problems of liposome which
has been
used in the field of pharmaceuticals and cosmetics and simultaneously taking
advantages of water-soluble chitosan which can be dissolved in an aqueous
solution
while reserving the advantages of chitosan.
That is, since in 1965 liposome was reported as a mono-layer or mufti-layer
phospholipid double-membrane closing vesicle by Bamgham et al., (Diffusion of
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Univalent Ions across the Lamellae of Swollen Phospholipids, J. Mol. Biol.,
13,
238-252, 1965), during the past scores of years liposome has been prepared
according
to numerous methods for preparation thereof by modifying 1 ) a surface charge,
2) a size
and 3) a lipid content so as to meet with the use purpose and end use of
liposome, and
used in food, cosmetics and pharmaceutical products.
Particularly, in recent years chitosan has been used in the pharmaceutical
field
to develop various pharmaceutical formulation for the purpose of 1 ) the
sustained
activity and targeting of the drug, 2) the alleviation of acute toxicity and
the alleviation
or enhancement of immunoreaction, 3) the stabilization of the drug and 4) the
change of
dosage formulation. However, Iiposomes which have been utilized for
development of
pharmaceutical formulations have disadvantages insufficient to be effectively
used in
the pharmaceutical products that their stability in the aqueous solution is
low due to the
properties of Iiposome itself including I) aggregation, 2) sedimentation, 3)
fusion, 4)
oxidation and 5) phospholipid hydrolysis, and 6) the efficiency of
sterilization and
reproductibility during the process for mass production is low.
For the reasons above, the present inventors have extensively studied to
remove
the problems involved in such prior liposome preparations for a long time, and
as a
result, surprisingly found that all the problems of the prior liposome
preparations can be
removed by preparing a proliposome in the form of a solid powder as the
precursor of
liposome using water-soluble chitosan as a carrier. Thus, we have completed
the
present invention.
Therefore, the purpose of the present invention is to provide a proliposome of
the peptide drug prepared by using a water-soluble chitosan.
Another purpose of the present invention is to provide an oral preparation
formulated from a proliposome of the peptide drug produced using a water-
soluble
chitosan according to a conventional pharmaceutical method.
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Further purpose of the present invention is to provide a process for preparing
an
oral preparation formulated from a proliposome of the peptide drug produced
using a
water-soluble chitosan according to a conventional pharmaceutical method.
In general, the carrier for preparing proliposome must have the following
properties: I) it must be water-soluble since it is dissolved in water to be
converted
into liposome from proliposome, and 2) its solubility in the organic solvent
used in the
course of the process must be low. Water-soluble chitosan used -in the present
invention not only has the carrier properties of proliposome as mentioned
above but
also can increase the absorption of peptide drug in the living body in case of
the oral
administration of the drug due to an effect of increasing the adhesive
property caused by
an ability of free cations contained in chitosan itself to combine with lipid
when it is
dissolved by the action of water within the intestine.
Typical examples of peptide drugs which can be used in the present invention
include aprotinin, buserelin, calcitonin, desmopressin, elcatonin, glucagon,
gonadotropin, gonadorelin, goserelin, hirudin, leuprolein, lypressin,
nafarelin, octreotide,
oxytocin, protirelin, salcatonin, sermorelin, somatostatin, somatropin,
terlipressin,
tetracosacrin, thymopentin, triptorelin, vasopressin, albumin, insulin,
interferon,
immunoglobulin, GM-CSF, G-CSF, glycoprotein, etc. In addition, of course,
other
peptide and protein drugs can also be included within the scope of the present
invention.
Phospholipids used in preparing the proliposome containing peptide drug
according to the present invention include a pharmaceutically acceptable
phospholipid
conventionally used for preparation of liposome, for example,
phosphatidyl-DL-glycerol-dimyristoyl, L-aegg phosphatidyl choline, soybean
phosphatidyl choline (lecithin), dipalmitoyl phosphatidyl choline (DPPC),
dimyristoyl
phosphatidyl choline (DMPC), cholesterol, stearylamine, diacetylphosphate,
phosphatidyl serine, methoxypolyethylene glycol distearoyl phosphatidyl-
ethanolamine,
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etc.
The peptide drugs composed of amino acids are generally contain asparagines
or glutamine residue in their structure and these residues occur the reaction
such as
deamidation, beta-elimination, disulfide exchange, racemization, oxidation,
etc., which
may readily induce the structural degeneration to ultimately cause the lose of
an activity
as peptide drugs. As the method for preventing such problem, the present
invention
uses an agent for controlling pH level to pH 3-4 at which asparagines and
glutamine
residues do not occur chemical reaction when the oral preparation according to
the
present invention is readily dissolved in the intestine, to stably maintain
the peptide
drugs in the intestine.
As the agent for controlling pH level in the present invention, any agent
which
can be generally used for oral administration in the pharmaceutical products
can be used
and the typical example thereof includes citric acid, sodium citrate, adipic
acid, sodium
monohydrogen phosphate, etc. The agent for controlling pH level is preferably
used in
an amount of 1.0% to 50% on the basis of the total amount.
Another characteristic feature of the present invention is to stimulate the
absorption of peptide drugs into the small intestine when the drug is
administered via
oral route, by using the absorption accelerator originated from natural
products or food
products which do not cause any damage to the epithelial cells of intestinal
tract. The
absorption accelerators which can be used in the present invention include
fatty acids or
salts thereof such as capric acid, oleic acid, etc., cholate as the salt of
bile acid,
deoxycholic acid, salicylate-based absorption accelerator, or monoglyceride-
based
absorption accelerator. The absorption accelerator generally used in Example
part of
the present invention is preferably deoxycholate as the cholic acid-based salt
which is
preferably used in an amount of 0.1% to 10%.
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[BRIEF DESCRIPTION OF DRAWINGS]
Figure 1 is a photograph showing a shape of the proIiposome formed from
salmon calcitonin and water-soluble chitosan and the inclusion of the drug;
Figure 2 is a photograph showing the process of dissolution in water as
obtained from observation of the hydration process of the proliposome using
water-soluble chitosan by mean of a microscope, wherein Figure 2a is a
photograph
showing proliposome powder and Figure 2b is a photograph showing the
production of
liposome from proliposome;
Figure 3 is a graph showing the particle size and distribution of liposome
formed from hydration of the proliposome;
Figure 4 is a graph showing a change in the content of salmon calcitonin in
the
proliposome of salmon calcitonin, as one embodiment according to the present
invention, in course of time; and
Figure 5 is a graph showing the drug permeability of various formulations
containing the proliposome of salmon calcitonin, as one embodiment according
to the
presentinvention.
[EMBODIMENT FOR CARRYING OUT THE INVENTION]
The present invention will be more specifically illustrated by the following
examples and experiments. However, the scope of the present invention is not
limited
by these examples and experiments.
Example 1
Preparation of proliposome (Example of preparing salmon calcitonin)
4 g of water-soluble chitosan powder (106 Nm - 300 Nm) passed through a
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sieve was introduced into a 100 ml round-bottomed flask, mounted on a rotary
evaporator and then dried under reduced pressure for 30 minutes at room
temperature.
267 mg of egg lecithin as phospholipid was dissolved in 30 ml of chloroform
and then
mixed with a solution of 27 mg of salmon calcitonin, which was previously
dissolved in
ml of methanol.
While the temperature in a 100 ml round-bottomed flask containing
water-soluble chitosan is maintained at 20-30°C, a given amount (about
1 ml) of a
mixed solution of liposome constituents dissolved in the organic solvent was
added and
then, the rotation of rotary evaporator was maintained at 120-150 rpm. When
water-soluble chitosan is completely dried to be a free-flow state, the mixed
solution of
organic solvent was added again to repeatedly apply the coating. The final
solution
was added and dried and then, the product was re-dried in a freezing drier for
24 hours
to remove the organic solvent (Yield: 95% or more).
Example 2
Preparation of proliposome (Example of preparing desmopressin)
3g of water-soluble chitosan powder (106 Nm - 300 Nm) passed through a
sieve was introduced into a 100 ml round-bottomed flask, mounted on a rotary
evaporator and then dried under reduced pressure for 30 minutes at room
temperature.
160 mg of phosphatidyl-DL-glycerol-dimyristoyl as phospholipid was dissolved
in 24
ml of chloroform and then mixed with a solution of 20 mg of desmopressin ,
which was
previously dissolved in 12 ml of ethanol.
While the temperature in a 100 ml round-bottomed flask containing
water-soluble chitosan is maintained at 20-30°C, a given amount (about
1 ml) of a
mixed solution of liposome constituents dissolved in the organic solvent was
added and
then, the rotation of rotary evaporator was maintained at 120-150 rpm. When
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water-soluble chitosan is completely dried to be a free-flow state, the mixed
solution of
organic solvent was added again to repeatedly apply the coating.
The final solution was added and dried and then, the product was re-dried in a
freezing drier for 24 hours to remove the organic solvent (Yield: 96% or
more).
Example 3-33
The respective drug selected from the group consisting of aprotinin,
buserelin,
elcatonin, glucagon, gonadotropin, gonadorelin, goserelin, hirudin,
leuprolein, lypressin,
nafarelin, octreotide, oxytocin, protirelin, salcatonin, sermorelin,
somatostatin,
somatropin, terlipressin, tetracosacrin, thymopentin, triptorelin,
vasopressin, albumin,
insulin, interferon, immunoglobulin, GM-CSF, G-CSF and glycoprotein was used
according to the similar method to Example 1 or 2 to prepare the corresponding
proliposome containing respective peptide drug.
Experiment 1
Identification of inclusion of the drug within proliposome (Cryo-SEM
identification)
In order to comfirm the inclusion of the drug within proliposome containing
salmon calcitonin, the proliposome not including the drug and the proliposome
including the drug were comparatively observed by means of an ultra-low
temperature
electron microscope. For pretreatment of the sample, first, one drop (about 3
N1) of the
liquid sample (liposome hydrated from proliposome) was dropped on a discus
sample
table having a diameter of 1 cm, and liquid nitrogen was filled in a nitrogen
slushing
chamber of Cryo-transfer system (CT 1 S00 Cryotrans, Oxford Instrument Ltd.,
UK).
Then, the sample was rapidly introduced in the chamber and fixed for one
minute in
vacuum.
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Next, the sample was transferred to a cryo-chamber controlled to -
170°C and
the vacuum state was confirmed. Then, the cooled sample was voluntarily broken
and
cleaved, and the resulting broken sample was transferred to a sample table of
a scanning
electron microscope (JSM-5410LV, JEOL LTD., Japan) connected to the cryo-
transfer
system and the temperature was controlled to -70°C at which the sample
was maintained
for 5 minutes to sublimate the moisture form the sample surface.
After completion of sublimation, the sample was transferred again to the
cryo-chamber and then subjected to gold-coating. Under the accelerating
voltage of 20
kV, the broken and cleaved section of the sample was observed to confirm the
shape of
liposome as formed and the inclusion of the drug of which the photograph is
shown in
Figure 1.
Experiment 2
Observation of dissolution of proliposome
In order to observe the hydration process of proliposome produced using
water-soluble chitosan by means of an optical microscope, the proliposome
granules
were mounted on a slide of the microscope and the focus was adjusted. At 400X
magnifications, one drop of water was dropped on the granule and after one
minute the
hydration process could be observed to confirm the dissolution process of the
proliposome in water of which the photograph is shown in Figure 2.
Experiment 3
Analysis of the particle size of liposome as produced
ml of distilled water was added to 5 mg of water-soluble chitosan
proliposome as prepared in Example 1 and shaken with vortex to dissolve the
proliposome, which was then hydrated for 30 minutes. The particle size and
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distribution of liposome thus prepared was measured by means of a particle
size
analyzer. The result thereof is shown as the graph in Figure 3.
Experiment 4
Qantitative analysis and stability of salmon calcitonin within the proliposome
In order to identify the stability and conduct the quantitative analysis, the
prepared proliposome corresponding to about 5 Ng of salmon calcitonin was
accurately
weighed, dissolved in 300 p1 of methanol and then completely dissolved with
vortex.
After filtration through a 0.45 Nm filter, an aliquot of 40 ~l was applied to
HPLC to
quantify the content of salmon calcitonin in the proliposome. The result from
determination of the stability at room temperature and under refrigerator is
shown in
Figure 4. The conditions for HPLC utilized for analyzing the content of the
drug are as
follows:
HPLC conditions: Column - C 18 (Sucelpo Inc.)
Flow rate - 1 ml/min.
Solvent A - 0.1% trifluoro acetic acid /acetonitrile
Solvent B - 0.1 % trifluoro acetic acid /water
Experiment 5
Preparation and enteric coating of oral formulation (tablet)
The tablet was prepared by a direct compression in the manner of dry process
rather than wet process according to the general method for preparing a tablet
as defined
in the part of General Provisions For Preparation in the Korea Pharmacopeia.
The
compositions of respective samples prepared in order to examine the effect of
the
present invention are shown in the following Table 1 and the respective
samples
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prepared by a direct compression were coated with Cellulose Acetate
phthalate(CAP) to
provide an enteric coating. The result of coating was confirmed by practicing
the
disintegrating test in artificial gastric juice(solution I) and artificial
intestinal
juice(solution 2) using the general test method as defined in the Korea
Pharmacopeia
and is described in the following Table 2.
Table 1
ClassificationFormulationFormulationFormulationFormulationFormulation
I 2 3 4 5
Proliposome24% 24% 24% ~ 24% 24%
containing
salcatonin
Deoxycholic 5% 5% 16%
acid
Citric 19% 48% 60%
acid
Lactose 76% 71% 57% 23%
Total 100% 100% 100% 100% 100%
Table 2
ClassificationFormulationFormulationFormulationFormulationFormulation
I 2 3 4 5
Solution Not dissolveNot dissolveNot dissolveNot dissolveNot dissolve
1
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Solution 2 Dissolve Dissolve Dissolve Dissolve Dissolve
In order to verify the effect of the present invention, the samples were used
to
practice both in vivo and in vitro tests.
Experiment 6
In vitro test using cells
(1) Preparation of a complete media
1 L x 2 of DMEM (Dulbecco's modified eagle medium), 7.4 g of sodium
bicarbonate and 2.6 g of HEPES were added to 2 L of distilled water which was
previously sterilized and cooled, and then stirred for about 1-1.5 hours in a
plate stirrer.
After stirring, the pH level was adjusted with 1 N hydrochloric acid to 7.4 by
means of a pH- meter. The solution was filtered through a filter (Corning
Filter
430015) and then dispensed into 500 ml bottles 450 ml in each bottle by means
of a 50
ml pipette.
To a 500 ml bottle containing 450 ml of the solution were added 50 ml of fetal
bovine serum(FBS), 5 ml of streptomycin and 5 ml of mem nonessential amino
acid
solution(NEAA). In order to confirm whether the prepared complete medium was
contaminated or not, about 6 ml of the complete medium was introduced into a
25 ml
T-flask, and after observation by a microscope, was incubated for one day or
more in an
incubator to ascertain the contamination.
(2) Defrosting of CaC02 cells
The complete medium, which was prepared and then preserved in a refrigerator,
was warmed in a water bath at 37°C for about 20 minutes and then mixed
for 4 to 5
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times using a 5 ml pipette. 5 ml of the medium was then introduced into a 1 S
ml
centrifuge tube. Cell line (ATCC HTB-37, LOT 944495) was taken out of a nitro
tank
and thawed in a water bath at 37°C with slightly opening a cap of the
bottle. After
identifying that the cell line was completely thawed, the cap of the bottle
reserving cell
line was opened and mixed 5 to 6 times with a 5 ml pipette. The whole content
of the
bottle was introduced into the above 15 ml centrifuge tube.
I Sm1 centrifuge tube was centrifuged by means of a centrifuge at 1000 rpm for
7 minutes to separate the supernatant, which was taken off then removed. I ml
of the
medium was added thereto and then mixed 4 to S times with a pipette to lose
the cells.
The whole content of the solution contained in I S ml conical tube was
transferred to a
25 ml T-flask which was then preserved in an incubator.
(3) Change of feeding cell media
A 25 ml T-flask which was preserved in the incubator was taken out of the
incubator and the media on the bottom of the flask was removed and replaced by
warming the compete medium, which was previously prepared and preserved in a
refrigerator, in a water bath at 37°C for about 20 minutes and then
introducing 5 ml
aliquot of the medium into 25 ml T-flask with taking care of not touching the
pipette
with the bottom of the flask.
(4) Subculture
Trypsin EDTA container and free media, complete medium, which were
preserved in a refrigerator, were warmed in a water bath at 37°C for
about 20 minutes,
and then the medium contained in 25 ml T-flask preserved in the incubator was
removed and 2 ml of trypsin EDTA was introduced into 25 ml T-flask.
18
CA 02420032 2003-02-10
Trypsin EDTA was allowed to uniformly distribute and the flask was then
preserved in a water bath at 37°C for 4 minutes. 10 ml of the complete
medium was
added to 25 ml T-flask and the flask was shaken to separate the cells. The
separated
solution was transferred to 15 ml conical tube by means of a pipette,
centrifuged at 1000
rpm for 5 minutes to remove the supernatant. 2 ml of the complete medium was
added
to T-flask and the cells were allowed to be well loosen and then re-incubated
in the
incubator (In this experiment, the cells over 40 passages were used.).
(5) Coating of transwell membrane with collagen
ml of sterilized 0.1% acetic acid was added to 25 mg of rat tail collagen,
which was then dissolved by stirring for 3 to 5 hours with addition of a
magnetic bar.
The resulting solution was diluted with 60% ethanol solution in the ratio of
I:1.5 to
make the final concentration of 0.3 mg/ml. Then, 50 ~I of collagen solution
was
uniformly distributed on the upper membrane of a transwell (Costar 3401) and
dried by
standing under UV light form 4 to 5 hours in a laminar flow with opening the
cap.
(6) Seeding cells onto the transwell plate
0.5 ml and 1.5 ml of the complete medium were introduced into the upper
compartment and the lower compartment, respectively, of the coated transwell
and
incubated for 15 minutes in the incubator. Then, the medium was removed and
1.5 ml
of the complete medium was re-introduced into the lower compartment of the re-
coated
transwell. Cells over 40 passages under subculture were diluted to the
concentration of
2.5-3 x 105 cells/ml and then introduced into the upper compartment of the
coated
transwell in an amount of 1.5 ml and incubated for 2 to 3 weeks so as to be
used in the
experiment.
19
CA 02420032 2003-02-10
(7) Identification of the properties of CaC02 cell monolayer
After 2 to 3 weeks from the inoculation of cells onto the transwell,
transepithelial electrical resistance (TEE) was measured by means of a
Millicell-ERS
Resistance System to confirm the resistance of 250 f2cm2 or more which means
that the
monolayer of cells was well established. When the permeability experiment was
conducted using C14-mannitol, the value of (<receiver dpm> / <donor dpm>) / hr
/ cm2
was below 0.4%, which means that the cell monolayer was well established. The
specific content of the experiment are as follows.
Drug concentration as used: 2-10 ~M of C~4-mannitol having a specific activity
of 50 mCi/mmol
Quantification of C'4-mannitol: 100 ~tl of the sample was taken and added to a
LSC vial to which 2 ml of LSC cocktail was added and, after vortex for 10
seconds, the
content of C'4-mannitol was measured in dpm by means of a liquid scintillation
counter
(LSC).
(8) Test for drug permeation (in case of saIcatonin)
The examined medium present in the upper compartment of the transwell was
removed using a pipette. The proliposome prepared in Example 1 above was
dissolved
in a transbuffer to the concentration of the drug (4.0 Ng/ml) and then
introduced into
the upper compartment of the transwell, which was transferred to the new well
containing a flashbuffer in intervals of 15 minutes after addition of the drug
while
slowly shaking at 85 rpm in a shaking water bath of 37°C.
The drug was sampled from the well in the donor part of transwell and then
quantitatively analyzed with respect to salcatonin by means of ELISA and RIA
kit (Pen.
CA 02420032 2003-02-10
Lab.). The result is shown in Figure 5.
Experiment 7
In vivo test using rats
SD male rats (5 rats per group) weighing 250 g-300 g were received only
drinking water from one day before the start of the experiment and adapted to
the
experimental environment. In order to insert cannula into femoral vein and
artery 400
Nl of the mixture of ketamine and lumpun in the ratio of 1:4 as an anesthetic
agent was
intramuscularly injected and then rats were fixed on the operating bed and
femoral vein
and artery were found in a leg using scissor and pincette and cannulated.
After cannulation, blood was sucked using a syringe from vein and artery to
identify the blood collection. Then, the samples of formulation l, 2, 3, 4 or
5 was
orally administered to rats using an oral capsule sonde and blood was
collected from
femoral artery in an amount of 500 NI in each case in interval of a given
time. The
blood sample was collected while the corresponding amount of saline was
injected into
femoral vein.
The collected blood was centrifuged in a centrifuge at 10,000 rpm for 10
minutes to separate the supernatant from which the content of the drug was
quantified
using ELISA and RIA Kit (Pen. Lab.).
Separately, in order to compare an increase in bioavailability with each
other,
the same amount of the drug as orally administered was injected to femoral
vein and the
blood was pre-treated according to the similar manner to the method for
collecting
blood in case of each formulation. Then, the drug was quantified to calculate
the
bioavailability, which is described in the following Table 3.
21
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Table 3
FormulationFormulationFormulationFormulationFormulation
1 2 3 4 5
Bioavailability0.1% 0.6% 2.5% 7.5% 5.8%
Y
[INDUSTRIAL APPLICABIITY)
Thus, the present invention provides a method wherein 1) water-soluble
chitosan is used to prepare proliposome as the precursor of liposome thereby
increasing
the absorption into intestinal mucous membrane, 2) an agent for controlling pH
is used
to increase a stability of peptide drugs in aqueous intestinal juice, 3) an
additive such as
absorption accelerator, etc. is added in order that peptide drugs can be
smoothly
absorbed into intestinal mucous membrane, and then 4) the product is
formulated into a
preparation suitable to oral administration and at the same time, 5) the
preparation is
covered with an enteric coating so that the drug can be readily migrated and
absorbed
into the intestine without destruction.
That is, the present invention can prepare a proliposome, as the precursor of
liposome, in a high yield within a short time without conducting the procedure
for
lyophilization or evaporation in order to prepare liposome in the form of a
powder,
which may cause the problems occurring in the prior method for preparing the
oral
preparation using liposome as disclosed in EP-0855179, and therefore, has some
advantages that the procedure of the process is simple, a stability of peptide
drugs to
moisture and temperature, which constitutes the disadvantage of peptide drugs,
is
increased, and chitosan is used as a carrier for preparing proliposome to
increase a
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