U.S. patent application number 11/741287 was filed with the patent office on 2008-06-12 for composite for liver-specific delivery and release of therapeutic nucleic acids or drugs.
This patent application is currently assigned to MOGAM BIOTECHNOLOGY RESEARCH INSTITUTE. Invention is credited to Meehyein KIM, Soo In Kim, Mahnhoon Park, Duckhyang Shin.
Application Number | 20080138394 11/741287 |
Document ID | / |
Family ID | 39411771 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138394 |
Kind Code |
A1 |
KIM; Meehyein ; et
al. |
June 12, 2008 |
Composite For Liver-Specific Delivery and Release of Therapeutic
Nucleic Acids or Drugs
Abstract
The inventive composite having a nanoscale particle size can
specifically deliver therapeutic nucleic acids or drugs to the
liver and selectively release them into hepatic cells to manifest
potent therapeutic effects without inducing any enzymatic
abnormalities or pathological damage to the normal liver function,
when administered together with the therapeutic agents.
Inventors: |
KIM; Meehyein; (Yongin-si,
KR) ; Kim; Soo In; (Yongin-si, KR) ; Shin;
Duckhyang; (Yongin-si, KR) ; Park; Mahnhoon;
(Yongin-si, KR) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W., SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
MOGAM BIOTECHNOLOGY RESEARCH
INSTITUTE
Yongin-si
KR
|
Family ID: |
39411771 |
Appl. No.: |
11/741287 |
Filed: |
April 27, 2007 |
Current U.S.
Class: |
424/450 ;
514/44A |
Current CPC
Class: |
A61P 31/20 20180101;
A61P 31/12 20180101; A61P 31/18 20180101; A61P 35/00 20180101; A61K
31/7088 20130101; A61K 9/1272 20130101 |
Class at
Publication: |
424/450 ;
514/44 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/7088 20060101 A61K031/7088 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2006 |
KR |
10-2006-0110402 |
Claims
1. A composite comprising an apolipoprotein A-I and a
liposome-forming material.
2. The composite of claim 1, wherein the liposome-forming material
is a cationic or neutral liposome-forming material, or a mixture of
thereof.
3. The composite of claim 2, wherein the cationic liposome-forming
material is selected from the group consisting of DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane), DC-cholosterol
(3.beta.-[N-(N',N'-dimethylaminoethane)carbamyl]cholesterol), DDAB
(dimethyldioctadecylammonium bromide), and a mixture thereof.
4. The composite of claim 2, wherein the neutral liposome-forming
material is selected from the group consisting of DOPE
(L-alpha-dioleoyl phosphatidylethanolamine), cholesterol, and a
mixture thereof.
5. The composite of claim 1, wherein the weight ratio of the
apolipoprotein A-I and the liposome-forming material is in the
range of 1:0.1 to 1:1000.
6. The composite of claim 1, which further comprise a therapeutic
nucleic acid or a drug.
7. The composite of claim 6, wherein the therapeutic nucleic acid
is selected from the group consisting of a DNA, an RNA, and a
derivative thereof.
8. The composite of claim 7, wherein the RNA is an siRNA specific
for HBV or HCV genome.
9. The composite of claim 6, wherein the therapeutic drug is an
active polypeptide, an anticancer agent or an antivirus agent.
10. The composite of claim 9, wherein the active polypeptide is
selected from the group consisting of epidermal growth factor
(EGF), erythropoietin (EPO), coagulation factors VIII, IX and VIIa,
follicle stimulating hormone (FSH), granulocyte colony-stimulating
factor (GCSF), granulocyte-macrophage colony stimulating factor
(GM-CSF), insulin, insulin-like growth factor (IGF),
interferon-.alpha., -.beta. and -.gamma. (IFN-.alpha., -.beta. and
-.gamma.), interleukin-1, -2, -11, -12 and -15 (IL-1, -2, -11, -12
and -15), parathyroid hormone (PTH), platelet-derived growth factor
(PDGF), human growth hormone (hGH), tissue plasminogen activator
(tPA), vascular endothelial growth factor (VEGF), and a mixture
thereof.
11. The composite of claim 9, wherein the anticancer agent is
selected from the group consisting of carboplatin, cisplatin,
oxaliplatin, heptaplatin, etoposide, semustine, hydroxycarbamide,
citarabine, fludarabine, doxorubicin, epirubicin, idarubicin,
pirarubicin, fluorouracil (5-FU), fluoxuridine, mitomycin,
bleomycin, clofazimine, interferon, streptozocin, gemcitabine,
enocitabine, capecitabine, ursodeoxycholic acid, sorafenib,
tegafur, holmium, a holmium-chitosan complex, and a mixture
thereof.
12. The composite of claim 9, wherein the antivirus agent is
selected from the group consisting of atazanavir, ribavirin,
zanamivir, acyclovir, entecavir, didanosin, nevirapine,
valaciclovir, nelfinavir, efavirenz, ganciclovir, lamivudine,
famciclovir, stavudine, abacavir, indinavir, oseltamivir,
inosiplex, adefovir, and a mixture thereof.
13. A composition comprising the composite of claim 1 and a
pharmaceutically acceptable carrier.
14. The composition of claim 13, which further comprise a
therapeutic nucleic acid or a drug.
15. The composition of claim 14, wherein the therapeutic nucleic
acid is selected from the group consisting of a DNA, an RNA, and a
derivative thereof.
16. The composition of claim 15, wherein the RNA is an siRNA
specific for HBV or HCV genome.
17. The composition of claim 14, wherein the therapeutic drug is an
active polypeptide, an anticancer agent or an antivirus agent.
18. The composition of claim 17, wherein the active polypeptide is
selected from the group consisting of epidermal growth factor
(EGF), erythropoietin (EPO), coagulation factors VIII, IX and VIIa,
follicle stimulating hormone (FSH), granulocyte colony-stimulating
factor (GCSF), granulocyte-macrophage colony stimulating factor
(GM-CSF), insulin, insulin-like growth factor (IGF),
interferon-.alpha., -.beta. and -.gamma. (IFN-.alpha., -.beta. and
-.gamma.), interleukin-1, -2, -11, -12 and -15 (IL-1, -2, -11, -12
and -15), parathyroid hormone (PTH), platelet-derived growth factor
(PDGF), human growth hormone (hGH), tissue plasminogen activator
(tPA), vascular endothelial growth factor (VEGF), and a mixture
thereof.
19. The composition of claim 17, wherein the anticancer agent is
selected from the group consisting of carboplatin, cisplatin,
oxaliplatin, heptaplatin, etoposide, semustine, hydroxycarbamide,
citarabine, fludarabine, doxorubicin, epirubicin, idarubicin,
pirarubicin, fluorouracil (5-FU), fluoxuridine, mitomycin,
bleomycin, clofazimine, interferon, streptozocin, gemcitabine,
enocitabine, capecitabine, ursodeoxycholic acid, sorafenib,
tegafur, holmium, a holmium-chitosan complex, and a mixture
thereof.
20. The composition of claim 17, wherein the antivirus agent is
selected from the group consisting of atazanavir, ribavirin,
zanamivir, acyclovir, entecavir, didanosin, nevirapine,
valaciclovir, nelfinavir, efavirenz, ganciclovir, lamivudine,
famciclovir, stavudine, abacavir, indinavir, oseltamivir,
inosiplex, adefovir, and a mixture thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a composite for
liver-specific delivery of a therapeutic nucleic acid or a drug, a
process for preparing the same and a composition comprising the
same with a pharmaceutically acceptable carrier.
BACKGROUND OF THE INVENTION
[0002] A tissue-specific gene and drug delivery system has long
been considered important for drug discovery and pharmaceutical
advancement because most drugs are systemically delivered and
circulated in the body when administered to a patient, which might
adversely affect healthy organs or cells. The tissue-specific
delivery system allows the accumulation of a high drug
concentration at the target tissue which eliminating adverse side
effects, leading to efficient treatment of tissue-specific
diseases.
[0003] Some liver diseases arise from infection by pathogenic
viruses, e.g., HBV (hepatitis B virus) and HCV (hepatitis C virus),
while non-infectious liver diseases result from exposure to
liver-toxic materials, or genetic or environmental disorders. The
progression of early-stage liver diseases caused by biological
stimuli ultimately leads to chronic hepatitis, liver cirrhosis or
hepatocellular carcinoma (HCC). Among several drug or gene delivery
systems currently studied in the treatment of such liver diseases,
a lipoprotein system, mainly that of HDL (high density
lipoprotein), has advantages over other delivery systems which use
viral vectors (Wang X., et al., Gene Ther. (2006), 13: 1097-1103),
non-viral complexes (Landen C. N., et al., Cancer Res. (2005), 65:
6910-6918; Morrissey D. V., et al., Nat. Biotechnol. (2005), 23:
1002-1007; Sorensen D. R., et al., J. Mol. Biol. (2003), 327:
761-766; and Urban-Klein B., et al., Gene Ther. (2005), 12:
461-466) and antibodies (Song E., et al., Nat. Biotechnol. (2005),
23: 709-717). For example, the lipoprotein can be preferably
recognized and taken up via cell surface receptors specific for
liver cells (Firestone R. A., Bioconjug. Chem. (1994), 5: 105-113;
de Smidt P. C., et al., Crit. Rev. Ther. Drug Carrier Syst. (1990),
7: 99-120; and Filipowska D., et al., Cancer Chemother Pharmacol.
(1992), 29: 396-400), and it is an endogenous product which is not
detrimental to human and does not trigger immunological responses
in clinical applications (Pussinen P. J., et al., Biochem. Biophys.
Acta. (2000), 1485: 129-144).
[0004] Recently, there has been a report that a recombinant high
density lipoprotein (HDL) can be used as a carrier for delivering a
lipophilic antitumor drug into human hepatocellular carcinoma cells
by taking advantage of the hydrophobic cholesterol ester-loading
properties of HDL (Lou B., et al., World J. Gastroenterol. (2005),
11: 954-959). However, it has merely been demonstrated in vitro,
but not in vivo, that the cellular uptake of the HDL carrier by a
hepatoma cell line, SMMC-7721, is higher in compared with a normal
liver cell line, L02, and the limitation in tissue-specific
targeting remains to be solved.
[0005] The present inventors have therefore endeavored to develop
an effective system for liver-specific delivery of a therapeutic
drug, and have found that a composite comprising an apolipoprotein
A-I and a liposome-forming material can specifically deliver and
release therapeutic drugs to the liver.
SUMMARY OF THE INVENTION
[0006] Accordingly, it is an object of the present invention to
provide a composite capable of specifically delivering and
releasing a therapeutic nucleic acid or a drug to the liver when
administered via a systemic route.
[0007] It is another object of the present invention to provide a
process for the preparation of said system.
[0008] It is further object of the present invention to provide a
composition for liver-specific delivery of a therapeutic nucleic
acid or drug, comprising said composite.
[0009] In accordance with one aspect of the present invention,
there is provided a composite comprising an apolipoprotein A-I (Apo
A-I) and a liposome-forming material.
[0010] In accordance with another aspect of the present invention,
there is provided a process for the preparation of the
composite.
[0011] In accordance with further another aspect of the present
invention, there is provided a composition comprising the composite
and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The above and other objects and features of the present
invention will become apparent from the following description of
the invention, when taken in conjunction with the accompanying
drawings, which respectively show:
[0013] FIG. 1A: Purified human Apo A-I from adult blood separated
by 4-20% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis;
[0014] FIG. 1B: In vivo images of a mouse intravenously injected
with Apo A-I labeled with an infrared fluorescent dye at several
times after the injection;
[0015] FIG. 1C: Percent uptake rate of Apo A-I in the livers of
mice injected with Apo A-I at different time points (n=4) after the
injection;
[0016] FIG. 2A: Whole body images for radioiodine signals captured
using a gamma camera in a mouse intravenously injected with the
inventive composite (DTC-Apo*/RLuc) which contains a Renilla
luciferase expression plasmid, phRL-CMV and .sup.131I label;
[0017] FIG. 2B: Whole body images captured several times after mice
were intravenously injected with the inventive composite
(DTC*-Apo/RLuc) and a comparative composite (DTC*/RLuc), which are
labeled with rhodamine, respectively;
[0018] FIG. 2C: Luciferase levels measured in tissue homogenates
from heart, lung, kidney and liver of mice (n=3) which were
intravenously administered with a mock control (5% dextrose), naked
DNA, or the inventive or a comparative composite containing a
Renilla luciferase expression plasmid, phRL-CMV (DTC/RLuc or
DTC-Apo/RLuc);
[0019] FIG. 3A: Relative levels of secreted HBsAg determined by
ELISA in mice which were intravenously injected with a mock control
(5% dextrose); naked siHBV; comparative composites containing
HBV-specific siRNA (DTC/siHBV) and irrelevant control siRNA
(DTC/siCont); and the inventive composites containing HBV-specific
siRNA (DTC-Apo/siHBV) and irrelevant control siRNA
(DTC-Apo/siCont), respectively, at days 2, 4, 6 and 8 after the
injection;
[0020] FIG. 3B: Serum HBsAg levels measured by ELISA in in vivo
mouse models of HBV replication which were intravenously injected
with the inventive composites containing HBV-specific siRNA
(DTC-Apo/siHBV) and irrelevant control siRNA (DTC-Apo/siCont),
respectively, at doses of 0.5, 1 or 2 mg/kg, at day 4 after the
injection;
[0021] FIG. 4A: In vivo images of luciferase gene expression in
mice which were administered with a luciferase expression plasmid,
pEGFPLuc, and one day after administration, intravenously injected
with the inventive composites containing luciferase-specific siRNA
(DTC-Apo/siLuc) or irrelevant control siRNA (DTC-Apo/siCont),
respectively; and
[0022] FIG. 4B: Relative luciferase expression levels measured by
counting bioluminescent signals emitted from the liver of the mice
shown in FIG. 4A.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The composite of the present invention may be in the form of
nanoparticles having an average particle size ranging from 50 to
400 nm, preferably 100 to 250 nm, and the apolipoprotein A-I (Apo
A-I) used in the present invention may be obtained from human blood
by cold ethanol precipitation in accordance with a conventional
method (e.g., Lerch, P. G., et al., Vox. Sang. (1996), 71:
155-164).
[0024] The liposome-forming material employed in the inventive
composite may be a cationic or neutral liposome-forming material,
or a mixture thereof, which play a role of avoiding undesirable
interactions between the inventive composite and unknown serum
components. Representative examples of the cationic
liposome-forming material include DOTAP
(1,2-dioleoyl-3-trimethylammonium-propane), DC-cholosterol
(3.beta.-[N-(N',N'-dimethylaminoethane)-carbamyl]cholesterol), DDAB
(dimethyldioctadecylammonium bromide), and a mixture thereof, and
the neutral liposome-forming material may be DOPE (L-alpha-dioleoyl
phosphatidylethanolamine), cholesterol, or a mixture thereof.
[0025] The inventive composite may comprise Apo A-I and the
liposome-forming material at a weight ratio ranging from 1:0.1 to
1:1000, preferably 1:1 to 1:100.
[0026] The composite of the present invention may further comprise
a therapeutic nucleic acid and/or drug.
[0027] The therapeutic nucleic acid may be a DNA such as plasmid
and PCR product, an RNA such as siRNA and ribozyme, or a derivative
thereof obtained by chemical modification, preferably siRNA
specific for HBV or HCV genome.
[0028] The therapeutic drug may be an active polypeptide,
anticancer agent, or antivirus agent, which does not limit the
scope of the present invention.
[0029] The active polypeptide used in the inventive composition may
be selected from the group consisting of epidermal growth factor
(EGF), erythropoietin (EPO), coagulation factors VIII, IX and VIIa,
follicle stimulating hormone (FSH), granulocyte colony-stimulating
factor (GCSF), granulocyte-macrophage colony stimulating factor
(GM-CSF), insulin, insulin-like growth factor (IGF),
interferon-.alpha., -.beta. and -.gamma. (IFN-.alpha., -.beta. and
-.gamma.), interleukin-1, -2, -11, -12 and -15 (IL-1, -2, -11, -12
and -15), parathyroid hormone (PTH), platelet-derived growth factor
(PDGF), human growth hormone (hGH), tissue plasminogen activator
(tPA), vascular endothelial growth factor (VEGF), and a mixture
thereof, which does not limit the scope of the present
invention.
[0030] Further, the anticancer agent may be selected from the group
consisting of carboplatin, cisplatin, oxaliplatin, heptaplatin,
etoposide, semustine, hydroxycarbamide, citarabine, fludarabine,
doxorubicin, epirubicin, idarubicin, pirarubicin, fluorouracil
(5-FU), fluoxuridine, mitomycin, bleomycin, clofazimine,
interferon, streptozocin, gemcitabine, enocitabine, capecitabine,
ursodeoxycholic acid, sorafenib, tegafur, holmium and a
holmium-chitosan complex, and the antivirus agent may be selected
from the group consisting of atazanavir, ribavirin, zanamivir,
acyclovir, entecavir, didanosin, nevirapine, valaciclovir,
nelfinavir, efavirenz, ganciclovir, lamivudine, famciclovir,
stavudine, abacavir, indinavir, oseltamivir, inosiplex, and
adefovir, which does not limit the scope of the present
invention.
[0031] The composite of the present invention may be prepared by a
method comprising (i) dispersing liposome-forming materials in an
organic solvent to form a liposome, (ii) dispersing the liposome in
a dextrose solution, and sonicating the mixture to obtain a
liposome suspension, and (iii) adding a solution containing Apo A-I
thereto to allow forming the inventive composite. The method of the
present invention may further comprise (iv) adding a therapeutic
nucleic acid or a drug to the suspension of the inventive composite
obtained in step (iii).
[0032] In accordance with further aspect of the present invention,
there is provided a composition for liver-specific delivery of a
therapeutic nucleic acid or drug, comprising the inventive
composite and a pharmaceutically acceptable carrier. The inventive
composition may further comprise the therapeutic nucleic acid or
drug as described above.
[0033] The composition of the present invention may be formulated
for oral or parenteral administration according to any one of the
procedures well known in the art, so as to take the form of
sterilized aqueous solution, hydrophobic solvent, suspension,
emulsion, lyophilized formulation or suppository. In the
formulation of the inventive composition, the hydrophobic solvent
or suspension may further comprise a vegetable oil such as
propylene glycol, polyethylene glycol and olive oil; an ester such
as ethyloleate; or a mixture thereof, and the suppository may
further comprise witepsol, macrogol, Tween 61, cacao butter, laurel
oil, glycerol, gelatine, or a mixture thereof.
[0034] Further, a proposed daily dose of the composition of the
present invention for administration to a human (of approximately
70 kg body weight) is about from 0.1 mg to 1000 mg, more preferably
about from 1 mg to 500 mg. It should be understood that the daily
dose should be determined in light of various relevant factors
including the condition to be treated, the severity of the
patient's symptoms, the route of administration, or the
physiological form of the anticancer agent; and, therefore, the
dosage suggested above does not limit the scope of the invention in
anyway.
[0035] The following Examples are intended to further illustrate
the present invention without limiting its scope.
TEST EXAMPLE 1
Liver-specificity of Purifed Apo A-I
[0036] High purity human apolipoprotein A-I (Apo A-I, 28 kDa) was
obtained from serum fractions of normal healthy adults not infected
with viral pathogens such as HBV, HCV or HIV by cold ethanol
precipitation according to the established protocol (Lerch, P. G.,
et al., Vox. Sang. (1996), 71: 155-164).
[0037] After sodium dodecyl sulfate-polycrylamide gel
electrophoresis (SDS-PAGE), and the purified Apo A-I was
characterized by Coomassie blue staining. The result is shown in
FIG. 1A. The identity of the purified Apo A-I was confirmed by
western blot analysis using a goat anti-human Apo A-I antibody
(Academy Biomedical Company, USA) which has cross-reactivity to
mouse Apo A-I, and a secondary antibody, rabbit anti-goat IgG-HRP
(KPL, USA).
[0038] For in vivo imaging, the purified protein (0.6 mg) was
labeled with an infrared dye using IRDye 800 CW in vivo imaging
agent (LI-COR Biosciences, USA), and purified using a dextran
desalting column (Pierce Biotechnology, Inc., USA) to remove
unincorporated dye. The labeled Apo A-I (200 .mu.g) was
administered to 6- to 8-week-old female nude mice (Charles River
Laboratories, Inc., USA) via tail vein injection. After 7, 40, 90,
150, 240 or 360 min, the test mice anesthetized with 2% isoflurane
were placed in a supine position in a light tight chamber, and
their whole body images were obtained using IVIS 200 imaging system
(Xenogen, USA) and Living Image Software (Xenogen, USA). The
resulting images are shown in FIG. 1B.
[0039] As shown in FIG. 1B, Apo A-I can be specifically delivered
to and stably maintained in the liver for at least 6 hours when
systemically administered.
[0040] Further, the photon intensities in the liver of the test
mice were measured using Living Image Software (Xenogen, USA) at
each time point after the administration, and the result is shown
in FIG. 1C.
[0041] FIG. 1C reveals that the uptake yield of the administered
Apo A-I by liver are maximal at approximately 45% within 150 min
after administration.
[0042] Taken together, these data demonstrate that the purified Apo
A-I maintains its native conformation required for cell-surface
receptor recognition and catabolic circulation in vivo, suggesting
that it might be applicable as a potent candidate carrier for
targeting the liver in feasibility studies for therapeutic drug
delivery.
EXAMPLE 1
Preparation of the Inventive Composite
[0043] An equimolar mixture of
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti Polar
Lipids, USA) and cholesterol (Sigma, USA) was dispersed in
chloroform and mixed to form a cationic liposome of
DOTAP/cholesterol (DTC). After the liposome assembly was formed,
the organic solvent was removed by evaporation under a stream of
N.sub.2 gas and the residue was kept in a vacuum desiccator for 2
hours to ensure the removal of the residual organic solvent. The
resulting dried film was hydrated in a 5% dextrose solution and the
suspension thus obtained was sonicated using a bath sonicator. A
solution containing 10% the Apo A-I purified in Test Example 1 was
added thereto at a DTC: Apo A-I mix ratio of 10:1 (w/w), and the
mixture was kept overnight at 4.degree. C. to obtain the inventive
composite (DTC-Apo).
EXAMPLE 2
Preparation of the Inventive Composite Containing Nucleic Acid
[0044] 40 .mu.g of HBV X gene-specific siRNA (SEQ ID NOs: 1 (sense)
and 2 (antisense); Shin, D., Virus Res. (2006), 119: 146-153) was
mixed with 400 .mu.g of the inventive composite, DTC-Apo, in 200
.mu.l of 5% dextrose solution and the mixture was incubated at room
temperature for 30 min, to obtain the inventive composite
containing HBV X gene-specific siRNA, named DTC-Apo/siHBV.
EXAMPLE 3
Preparation of the Inventive Composite Containing Nucleic Acid
[0045] The procedure of Example 2 was repeated except for using
firefly luciferase-specific siRNA (SEQ ID NOs: 3 (sense) and 4
(antisense); Elbashir, S. M., Nature (2001), 411: 494-498) instead
of HBV-specific siRNA, to obtain the inventive composite containing
firefly luciferase-specific siRNA, named DTC-Apo/siLuc.
EXAMPLE 4
Preparation of the Inventive Composite Containing Nucleic Acid
[0046] The procedure of Example 2 was repeated except for using 0.3
mg of a plasmid phRL-CMV encoding Renilla luciferase (Promega, WI)
and 3 mg of DTC-Apo instead of 40 .mu.g of HBV-specific siRNA and
400 .mu.g of DTC-Apo, to obtain the inventive composite containing
a plasmid phRL-CMV, named DTC-Apo/RLuc.
EXAMPLE 5
Preparation of the Inventive Composite Containing Nucleic Acid
[0047] The procedure of Example 2 was repeated except for using
control double stranded RNA (SEQ ID NOs: 5 (sense) and 6
(antisense)) instead of HBV-specific siRNA, to obtain the inventive
composite containing control double stranded RNA, named
DTC-Apo/siCont.
COMPARATIVE EXAMPLES 1 TO 4
Preparation of the Comparative Composite Containing Nucleic
Acid
[0048] The procedures of Examples 2 to 5 were repeated except for
using DTC instead of DTC-Apo, to obtain comparative composites
named DTC/siHBV, DTC/siLuc, DTC/RLuc and DTC/siCont,
respectively.
TEST EXAMPLE 2
Characterization of the Inventive and Comparative Composite
[0049] The inventive and comparative composites obtained in
Examples 2 to 5 and Comparative Examples 1 to 4 were characterized
by measuring their size and charge using a Zetasizer 3000 apparatus
(Malvern Instruments, Malvern, Worcestershire, United Kingdom),
respectively, to determine the average diameters and zeta potential
values thereof. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Formulation Size (nm) .zeta. pot (mV) DTC
176.5 .+-. 1.4 53.3 .+-. 4.0 DTC with DNA 205.5 .+-. 4.2 42.7 .+-.
1.8 DTC with siRNA 196.0 .+-. 1.8 44.6 .+-. 2.2 DTC-Apo 147.9 .+-.
2.8 49.5 .+-. 6.3 DTC-Apo with DNA 179.5 .+-. 3.4 38.6 .+-. 4.0
DTC-Apo with siRNA 177.1 .+-. 1.4 39.1 .+-. 2.8
[0050] As shown in Table 1, the inventive composite has an average
particle size in the nanoscale range, suitable for systemic
administration, and it is positive charged no matter whether it
contained a nucleic acid or not, which showed that the inventive
composite would not occur undesirable interaction with unknown
serum components.
TEST EXAMPLE 3
Liver-specific Gene Delivery In Vivo
[0051] In order to facilitate the systemic and sensitive detection
of the in vivo migration route, Apo A-I of DTC-Apo/RLuc obtained in
Example 4 was labeled with 0.6 mCi .sup.131I (The Korea Atomic
Energy Research Institute, Daejeon, South Korea) by the
chloramines-T method (named DTC-Apo*/RLuc). 200 .mu.Ci of the
purified DTC-Apo*/RLuc was intravenously injected into nude mice
(Charles River Laboratories), and the radioactivity from the whole
body of each mouse was monitored using a gamma-camera (Medical
imaging Electronics, USA) at 40, 120 and 240 min postinjection,
respectively. The results are shown in FIG. 2A.
[0052] FIG. 2A clearly shows that the inventive composite is
accumulated in the hepatic tissue at 40 min after
administration.
[0053] Further, the cationic liposomes of the comparative and
inventive composites, DTC/RLuc and DTC-Apo/RLuc obtained in
Comparative Example 3 and Example 4, respectively, were labeled
with a fluorescent dye, rhodamine using lissamine rhodamine
B-diacyl phosphatidylethanolamine (Avanti Polar Lipids), to obtain
labeled composities, DTC*/RLuc and DTC*-Apo/RLuc, respectively. The
labeled composites were each injected into nude mice (Charles River
Laboratories), and the whole body was monitored using IVIS 200
imaging system (Xenogen, USA) at 20, 60 and 100 min
postinjection.
[0054] As shown in FIG. 2B, the accumulation level of the inventive
composite is enhanced in the liver more prominently than that of
the comparative composite. The fluorescent signal noise detected at
the ends of the limb may be due to the overlapped emission
wavelengths between rhodamine and red blood cells.
[0055] Further, in order to examine nucleic acid release by the
inventive composite after systemic injection, mice (Charles River
Laboratories) were intravenously treated with 200 .mu.Ci of the
unlabeled DTC/RLuc or DTC-Apo/RLuc, or naked phRL-CMV or a 5%
dextrose solution, as controls, and sacrificed the following day.
Heart, lung, kidney and liver were each harvested from each mouse
and homogenized. The bioluminescent intensity of each tissue
homogenate was measured using a renilla luciferase assay system
(Promega) to determine the luciferase expression level per total
protein. The results are shown in FIG. 2C.
[0056] As shown in FIG. 2C, consistently with liver-specific
accumulation of isotope or rhodamine-labeled DTC-Apo composites
(FIGS. 2A and 2B), luminescence signals are particularly prominent
in the liver of mice injected with DTC-Apo/RLuc in an amount
ranging from 6,700 to 50,300 RLU/mg. In contrast, in mice treated
with DTC/RLuc, luciferase signals were strong in the lung and
kidney but only modest in the liver.
[0057] The results suggest that the inventive composite can
liver-specifically deliver a therapeutic gene or drug to hepatic
cells the therapeutic gene being expressed therein.
TEST EXAMPLE 4
Therapeutic Effect of the Inventive Composite with Nucleic Acid
[0058] To examine the therapeutic activity of the inventive
composite, in vivo antiviral effect of DTC-Apo containing
HBV-specific siRNA (DTC-Apo/siHBV) was examined using a mouse model
for acute HBV infection as follows.
[0059] First, in order to establish an acute HBV-infected mouse
model, HBV replication competent plasmid, pCpGHBV-MBRI, was created
by excision of the viral genome from the mother clone pHBV-MBRI
(Shin, D., et al., Virus Res. (2006), 119: 146-153) and religated
into SpeI and XbaI digested pCpG-mcs (InvivoGen, USA), which is
known to be not inducible nonspecific inflammatory responses in
mammalian hosts. Then, 10 .mu.g of pCpGHBV-MBRI was
hydrodynamically injected into the tail veins of female C57BL/6
mice (Charles River Laboratories) of 8-9 weeks of age weighing
approximately 20 g to induce the acute HBV infectious. After 8
hours, the HBV-infected model mice were intravenously administered
with 2 mg/kg (i. e., 40 .mu.g of nucleic acid per mouse) of
DTC-Apo/siHBV, DTC-Apo/siCont, DTC/siHBV and DTC/siCont obtained in
Examples 2 and 5, and Comparative Examples 1 and 4, respectively. 2
mg/kg of naked HBV-specific siRNA or 5% dextrose solution was also
administrated to control mice groups. Serum samples were collected
from each treated mouse on days 2, 4, 6 and 8 after injection, and
the serum HBV surface antigen (HBsAg) level, one of the major viral
structural proteins, was determined by ELISA (DiaSorin, USA) to
monitor the viral protein level secreted into the blood. The
results are shown in FIG. 3A.
[0060] As shown in FIG. 3A, there is a significant reduction of
serum HBsAg in mice administered with a single dose of
DTC-Apo/siHBV particles, as shown by the average inhibitions degree
of 65.1% (P=0.014), 63.4% (P=0.047), 74.9% (P=0.015) and 72.8%
(P=0.034) on days 2, 4, 6 and 8 post-injection, respectively,
relative to the matched DTC/siHBV or DTC-Apo/siCont treated
mice.
[0061] Further, in order to examine the dose-dependent activity of
DTC-Apo/siHBV, the mice with acute HBV replication were treated
with 0.5, 1, or 2 mg/kg doses of DTC-Apo/siHBV, while 2 mg/kg of
DTC-Apo/siCont or a 5% dextrose solution was also administrated to
the model mice as control groups. The serum viral antigen levels in
each mouse was monitored at day 4 post-injection. The results are
shown in FIG. 3B.
[0062] As shown in FIG. 3B, the treatment of the inventive
composite containing HBV-specific siRNA reduces the viral antigen
expression in mice with acute HBV replication in a does-dependent
manner, unlike the control groups.
[0063] These in vivo data indicate that the inventive composite can
promote the hepatic tissue-specific delivery of a therapeutic
nucleic acid or drug and lead to potent therapeutic effects in
vivo, only through a intravenous treatment of the inventive
composite containing a therapeutic nucleic acid or drug.
TEST EXAMPLE 5
Therapeutic Effect of the Inventive Composite with Nucleic Acid
[0064] In order to confirm that the target-specific effect of the
inventive composite comprising a therapeutic nucleic acid or drug
occurs selectively and mainly in the hepatic tissue, 6-7-week-old
female Balb/c mice (Charles River Laboratories) were
hydrodynamically injected with 10 .mu.g of pEGFPLuc plasmid
(Clontech), which is known to express the firefly luciferase and
also to facilitate in vivo image analysis, respectively.
[0065] After one day, DTC-Apo/siLuc or DTC-Apo/siCont obtained in
Example 3 or 5, or a 5% dextrose solution control was injected at a
dose of 1 mg/kg via the tail veins of the mice under ambient
pressure, and the following day, the treated mice were
anaesthetized with 2% isoflurane, and intraperitoneally injected
with 200 .mu.l of 15 mg/ml D-luciferin (Molecular Imaging Products
Company, USA). Ten minutes later, photon signals from the whole
body of each mouse was analyzed using an IVIS imaging system
(Xenogen). The results are shown in FIGS. 4A and 4B.
[0066] As shown in FIGS. 4A and 4B, there is no signal change
suggesting luciferase expression inhibition in mice injected with
DTC-Apo/siCont, while a dramatic reduction in luciferase activity
(about 70%) was observed for mice treated with DTC-Apo/siLuc as
early as day 1 after treatment.
[0067] Taken together, these results show that the selective target
of the inventive composite administered systemically is the liver
and that the therapeutic nucleic acid or drug delivered by the
inventive composite is specifically released into hepatic cells to
manifest a potent therapeutic effect, without inducing any
enzymatic abnormalities or pathological damage of the normal liver
function.
[0068] While the embodiments of the subject invention have been
described and illustrated, it is obvious that various changes and
modifications can be made therein without departing from the spirit
of the present invention which should be limited only by the scope
of the appended claims.
Sequence CWU 1
1
6119RNAArtificial Sequencesense strand of siRNA specific for HBV
1gaggacucuu ggacucuca 19 219RNAArtificial Sequenceantisense strand
of siRNA specific for HBV 2ugagagucca agaguccuc 19319RNAArtificial
Sequencesense strand of siRNA specific for luciferase 3cuuacgcuga
guacuucga 19419RNAArtificial Sequenceantisense strand of siRNA
specific for luciferase 4ucgaaguacu cagcguaag 19519RNAArtificial
SequenceControl siRNA 5gcaccuauaa caacgguag 19619RNAArtificial
SequenceControl siRNA 6cuaccguugu uauaggugc 19
* * * * *