U.S. patent application number 11/978112 was filed with the patent office on 2008-10-02 for method and reagents for the enhancement of virus transduction in the bladder epithelium.
This patent application is currently assigned to Cell Genesys, Inc.. Invention is credited to David Frey, Bahram Memarzadeh, Nagarajan Ramesh, DeChao Yu.
Application Number | 20080241105 11/978112 |
Document ID | / |
Family ID | 32680762 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241105 |
Kind Code |
A1 |
Ramesh; Nagarajan ; et
al. |
October 2, 2008 |
Method and reagents for the enhancement of virus transduction in
the bladder epithelium
Abstract
Agents and methods for enhancing recombinant virus transduction
in the bladder epithelium are described. A first method involves
contacting the luminal surface of the bladder with a composition
comprising a transduction enhancing agent and an oncolytic virus.
Alternatively, the luminal surface of the bladder can be contacted
first with a pretreatment composition comprising a transduction
enhancing agent and, subsequently, with a composition comprising an
oncolytic virus. Bladder treatment compositions comprising a
transduction enhancing agent and an oncolytic virus are also
described.
Inventors: |
Ramesh; Nagarajan;
(Sunnyvale, CA) ; Frey; David; (Half Moon Bay,
CA) ; Memarzadeh; Bahram; (San Carlos, CA) ;
Yu; DeChao; (Foster City, CA) |
Correspondence
Address: |
ROPES & GRAY LLP
PATENT DOCKETING 39/361, 1211 AVENUE OF THE AMERICAS
NEW YORK
NY
10036-8704
US
|
Assignee: |
Cell Genesys, Inc.
|
Family ID: |
32680762 |
Appl. No.: |
11/978112 |
Filed: |
October 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11897535 |
Aug 29, 2007 |
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11978112 |
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10743813 |
Dec 24, 2003 |
7267815 |
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11897535 |
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10327869 |
Dec 26, 2002 |
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10743813 |
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Current U.S.
Class: |
424/93.6 |
Current CPC
Class: |
Y02A 50/465 20180101;
A61K 47/26 20130101; Y02A 50/393 20180101; C12N 2710/10332
20130101; A61P 35/04 20180101; A61K 31/337 20130101; A61P 35/00
20180101; A61K 31/7024 20130101; A61K 48/0083 20130101; A61K 9/0019
20130101; C12N 7/00 20130101; C12N 2710/10343 20130101; A61K 47/10
20130101; A61K 35/761 20130101; A61K 45/06 20130101; Y02A 50/30
20180101; A61P 43/00 20180101; A61K 31/337 20130101; A61K 2300/00
20130101; A61K 31/7024 20130101; A61K 2300/00 20130101; A61K 35/76
20130101; A61K 2300/00 20130101; A61K 35/761 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/93.6 |
International
Class: |
A61K 35/76 20060101
A61K035/76 |
Claims
1-93. (canceled)
94. A composition comprising: a transduction enhancing agent; and
an oncolytic virus; wherein the transduction enhancing agent has a
structure represented by the following general formula (I) or the
following general formula (II): ##STR00024## wherein x is a
positive integer; and wherein the concentration of the transduction
enhancing agent is less than 0.025 wt/% of the composition.
95. (canceled)
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/327,869, filed Dec. 26, 2002, which
application is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the treatment of
bladder cancer with viral therapy agents and, in particular, to
agents and methods for enhancing recombinant oncolytic virus
transduction of the bladder epithelium.
[0004] 2. Background of the Technology
[0005] Bladder cancer is a commonly occurring cancer and more than
50,000 new cases are diagnosed every year. Bladder cancer is a
superficial disease confined to the mucosa in the majority of
patients. Of the various therapeutic modalities available,
transurethral resectioning of the tumor is considered to be the
most effective treatment for the management of superficial bladder
cancer. However, 70% of these superficial bladder tumors will recur
after endoscopic resectioning, and 20% progress to life-threatening
invasive diseases within 2 years of cystectomy. See Raghavan, et
al., "Biology and Management of Bladder Cancer", N. Engl. J. Med.,
322, 16, 1129-1138 (1990).
[0006] Gene therapy has also been used for the treatment of bladder
cancer. See, for example, Brewster, et al., Eur. Urol. 25, 177-182
(1984); Takahashi, et al., Proc. Natl. Acad. Sci. USA 88, 5257-5261
(1991); and Rosenberg, J. Clin. Oncol., 10, 180-199 (1992).
[0007] In vitro studies using cell lines derived from human bladder
tissues have demonstrated efficient transgene expression following
infection with recombinant adenovirus. Bass, et al., Cancer Gene
Therapy 2, 2, 97-104 (1995). Experiments in vivo have also shown
adenovirus transgene expression in the urinary bladder of rodents
after intravesical administration. Bass, et al., supra; Morris. et
al., J. Urology, 152, 506-550 (1994). In vitro experiments with
wild-type adenovirus demonstrate that virus attachment and
internalization is not influenced by benzyl alcohol, but do
demonstrate an enhanced uncoating of the virion. Blixt. et al.,
Arch. Virol., 129, 265-277 (1993).
[0008] In vivo studies have demonstrated that various agents (e.g.
acetone, DMSO, protamine sulfate) can break down the protective
"mucin" layer that protects the bladder epithelium from bacteria,
viruses and other pathogens. See, for example, Monson, et al., J.
Urol., 145, 842-845 (1992) and Parsons, et al., J. Urol., 143,
139-142 (1990). Methods of modifying the bladder surface to enhance
gene transfer have also been disclosed. Siemens. et al.,
"Evaluation of Gene Transfer Efficiency by Viral Vectors to Murine
Bladder Epithelium", J. of Urology, 165, 667-671 (2001).
[0009] U.S. Pat. No. 6,165,779 discloses a gene delivery system
formulated in a buffer comprising a delivery-enhancing agent such
as ethanol or a detergent. The gene delivery system may be a
recombinant viral vector such as an adenoviral vector.
[0010] There still exists a need, however, for improved gene
therapy methods and agents which can accomplish direct, optimal, in
vivo gene delivery to the bladder epithelium.
SUMMARY OF THE INVENTION
[0011] According to a first aspect of the invention, a method for
treating cancer of the bladder is provided. According to this
aspect of the invention, the method involves: contacting the
luminal surface of the bladder with a pretreatment composition
comprising a transduction enhancing agent; and subsequently
contacting the luminal surface of the bladder with a composition
comprising an oncolytic virus; wherein the transduction enhancing
agent is a mono-, di-, or poly-saccharide having a lipophilic
substituent. The transduction enhancing agent can have the
following general formula (I) or the following general formula
(II):
##STR00001##
wherein X is a sulfur or oxygen atom, R.sup.1 is an alkyl group and
each R.sup.2 is independently hydrogen or a moiety represented
by:
##STR00002##
wherein R.sup.1 is an alkyl group. The pretreatment composition can
further include an oxidizing agent. The oncolytic virus can be an
oncolytic adenovirus such as CG8840. The oncolytic virus
composition can further include a chemotherapeutic agent such as
docetaxel.
[0012] According to a second aspect of the invention, a method for
treating cancer of the bladder is provided. According to this
aspect of the invention, the method includes contacting the luminal
surface of the bladder with a pretreatment composition comprising
about 0.01 to about 0.2% by weight sodium oxychlorosene and,
subsequently, contacting the luminal surface of the bladder with a
composition comprising an oncolytic virus.
[0013] According to a third aspect of the invention, a method of
treating cancer of the bladder is provided. According to this
aspect of the invention, the method includes: contacting the
luminal surface of the bladder with a pretreatment composition
comprising a transduction enhancing agent having a structure
represented by the chemical formula:
##STR00003##
wherein x and y are positive integers; and subsequently contacting
the luminal surface of the bladder with a composition comprising an
oncolytic virus. According to a preferred embodiment of the
invention, x is 6 and y is 8-10 and the pretreatment composition
comprises about 0.02 to about 0.05 wt. % of the transduction
enhancing agent.
[0014] According to a fourth aspect of the invention, a method of
treating cancer of the bladder is provided. According to this
aspect of the invention, the method includes: contacting the
luminal surface of the bladder with a pretreatment composition
comprising a transduction enhancing agent having a structure
represented by the following general formula (I) or the following
general formula (II):
##STR00004##
wherein x is a positive integer and subsequently contacting the
luminal surface of the bladder with a composition comprising an
oncolytic virus.
[0015] According to a fifth aspect of the invention, a composition
comprising a transduction enhancing agent and an oncolytic virus is
provided. According to this aspect of the invention, the
transduction enhancing agent is a mono-, di-, or poly-saccharide
having a lipophilic substituent. For example, the transduction
enhancing agent can be a compound having the following general
formula (I) or the following general formula (II):
##STR00005##
wherein X is a sulfur or oxygen atom, R.sup.1 is an alkyl group and
each R.sup.2 is independently hydrogen or a moiety represented
by:
##STR00006##
wherein R.sup.1 is an alkyl group. The oncolytic virus can be an
oncolytic adenovirus such as CG8840. The oncolytic virus
composition can further include a chemotherapeutic agent such as
docetaxel. A method for treating cancer of the bladder comprising
contacting the luminal surface of the bladder with a composition as
set forth above is also provided.
[0016] According to a sixth aspect of the invention, a composition
comprising sodium oxychlorosene and an oncolytic virus is provided.
The oncolytic virus can be an oncolytic adenovirus such as CG8840.
The oncolytic virus composition can further include a
chemotherapeutic agent such as docetaxel. A method for treating
cancer of the bladder comprising contacting the luminal surface of
the bladder with a composition as set forth above is also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention may be better understood with
reference to the accompanying drawings in which:
[0018] FIGS. 1A and 1B are photographs showing a murine bladder
after pretreatment with a 15% ethanol solution followed by
infection with Ad-LacZ wherein FIG. 1A shows the outside surface of
the bladder and FIG. 1B shows the luminal bladder surface;
[0019] FIGS. 1C and 1D are photographs showing a murine bladder
after pretreatment with a 20% ethanol solution followed by
infection with Ad-LacZ wherein FIG. 1C shows the outside surface of
the bladder and FIG. 1D shows the luminal bladder surface;
[0020] FIGS. 1E and 1F are photographs showing a murine bladder
after pretreatment with a 25% ethanol solution followed by
infection with Ad-LacZ wherein FIG. 1E shows the outside surface of
the bladder and FIG. 1F shows the luminal bladder surface;
[0021] FIGS. 1G and 1H are photographs showing a murine bladder
after pretreatment with a 30% ethanol solution followed by
infection with Ad-LacZ wherein FIG. 1G shows the outside surface of
the bladder and FIG. 1H shows the luminal bladder surface;
[0022] FIG. 2A ia a photograph showing a cross section of a murine
bladder control;
[0023] FIGS. 2B and 2C are photographs showing the cross section of
a murine bladder after pretreatment with a 30% ethanol solution
followed by infection with Ad-LacZ;
[0024] FIGS. 3A-3F are photographs showing the cross section of a
murine bladder after pretreatment with a 25% ethanol solution
followed by infection with Ad-LacZ wherein FIGS. 3A, 3C and 3E were
taken at 40.times. and FIGS. 3B, 3D and 3F were taken at 100.times.
magnification;
[0025] FIGS. 4A-4F are photographs showing the cross section of a
murine bladder after pretreatment with a 30% ethanol solution
followed by infection with Ad-LacZ wherein FIGS. 4A, 4C and 4E were
taken at 40.times. and FIGS. 4B, 4D and 4F were taken at 100.times.
magnification;
[0026] FIGS. 5A-5D are photographs showing two murine bladders
after pretreatment with a 4% poloxomer 407 solution followed by
infection with Ad-LacZ wherein FIGS. 5A and 5B show the outside and
luminal surfaces, respectively, of the first bladder and FIGS. 5C
and 5D show the outside and luminal surfaces, respectively, of the
second bladder;
[0027] FIGS. 6A-6D are photographs showing two murine bladders
after infection with a composition comprising lipofectamine and
Ad-LacZ wherein FIGS. 6A and 6B show the outside and luminal
surfaces, respectively, of the first bladder and FIGS. 6C and 6D
show the outside and luminal surfaces, respectively, of the second
bladder;
[0028] FIGS. 7A-7D are photographs showing two murine bladders
after infection with a composition comprising In vivo
geneSHUTTLE.TM. and Ad-LacZ wherein FIGS. 7A and 7B show the
outside and luminal surfaces, respectively, of the first bladder
and FIGS. 7C and 7D show the outside and luminal surfaces,
respectively, of the second bladder;
[0029] FIGS. 8A-8N are photographs showing seven murine bladders
after pretreatment with a 0.2% oxychlorosene solution for 5 minutes
followed by infection with Ad-LacZ wherein FIGS. 8A and 8B show the
outside and luminal surfaces, respectively, of the first bladder,
FIGS. 8C and 8D show the outside and luminal surfaces,
respectively, of the second bladder, FIGS. 8E and 8F show the
outside and luminal surfaces, respectively, of the third bladder,
FIGS. 8G and 8H show the outside and luminal surfaces,
respectively, of the fourth bladder, FIGS. 8I and 8J show the
outside and luminal surfaces, respectively, of the fifth bladder,
FIGS. 8K and 8L show the outside and luminal surfaces,
respectively, of the sixth bladder, and FIGS. 8M and 8N show the
outside and luminal surfaces, respectively, of the seventh
bladder;
[0030] FIGS. 9A-9N are photographs showing seven murine bladders
after pretreatment with a 0.2% oxychlorosene solution for 15
minutes followed by infection with Ad-LacZ wherein FIGS. 9A and 9B
show the outside and luminal surfaces, respectively, of the first
bladder, FIGS. 9C and 9D show the outside and luminal surfaces,
respectively, of the second bladder, FIGS. 9E and 9F show the
outside and luminal surfaces, respectively, of the third bladder,
FIGS. 9G and 9H show the outside and luminal surfaces,
respectively, of the fourth bladder, FIGS. 9I and 9J show the
outside and luminal surfaces, respectively, of the fifth bladder,
FIGS. 9K and 9L show the outside and luminal surfaces,
respectively, of the sixth bladder, and FIGS. 9M and 9N show the
outside and luminal surfaces, respectively, of the seventh
bladder;
[0031] FIGS. 10A and 10B are photographs showing the cross section
of the murine bladders of FIGS. 8C and 8I, respectively;
[0032] FIGS. 11A and 11B are photographs showing the cross section
of the murine bladders of FIGS. 9C and 9I, respectively;
[0033] FIGS. 12A-12F are photographs showing the cross section of a
murine bladder after pretreatment with a 0.2% oxychlorosene
solution for 5 minutes followed by infection with Ad-LacZ wherein
FIGS. 12A, 12C and 12E were taken at 40.times. and FIGS. 12B, 12D
and 12F were taken at 100.times. magnification;
[0034] FIGS. 13A-13F are photographs showing the cross section of a
murine bladder after pretreatment with a 0.2% oxychlorosene
solution for 15 minutes followed by infection with Ad-LacZ wherein
FIGS. 13A, 13C and 13E were taken at 40.times. and FIGS. 13B, 13D
and 13F were taken at 100.times. magnification;
[0035] FIG. 14A is a photograph showing the luminal surface of a
murine bladder after pretreatment with a 0.1% oxychlorosene
solution followed by infection with Ad-LacZ;
[0036] FIGS. 14B and 14C are photographs showing the cross section
of the murine bladder of FIG. 14A wherein FIG. 14B was taken at
40.times. and FIG. 14C was taken at 100.times. magnification;
[0037] FIG. 15A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.2% oxychlorosene
solution followed by infection with Ad-LacZ;
[0038] FIGS. 15B and 15C are photographs showing the cross section
of the murine bladder of FIG. 15A wherein FIG. 15B was taken at
40.times. and FIG. 15C was taken at 100.times. magnification;
[0039] FIG. 16A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.2% oxychlorosene
solution followed by infection with Ad-LacZ;
[0040] FIGS. 16B and 16C are photographs showing the cross section
of the murine bladder of FIG. 16A wherein FIG. 16B was taken at
40.times. and FIG. 16C was taken at 100.times. magnification;
[0041] FIG. 17A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.4% oxychlorosene
solution followed by infection with Ad-LacZ;
[0042] FIGS. 17B and 17C are photographs showing the cross section
of the murine bladder of FIG. 17A wherein FIG. 17B was taken at
40.times. and FIG. 17C was taken at 100.times. magnification;
[0043] FIG. 18A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.4% oxychlorosene
solution followed by infection with Ad-LacZ;
[0044] FIGS. 18B and 18C are photographs showing the cross section
of the murine bladder of FIG. 18A wherein FIG. 18B was taken at
40.times. and FIG. 18C was taken at 100.times. magnification;
[0045] FIG. 19A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.02% polidocanol
solution followed by infection with Ad-LacZ;
[0046] FIGS. 19B and 19C are photographs showing the cross section
of the murine bladder of FIG. 19A wherein FIG. 19B was taken at
40.times. and FIG. 19C was taken at 100.times. magnification;
[0047] FIG. 20A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.02% polidocanol
solution followed by infection with Ad-LacZ;
[0048] FIGS. 20B and 20C are photographs showing the cross section
of the murine bladder of FIG. 20A wherein FIG. 20B was taken at
40.times. and FIG. 20C was taken at 100.times. magnification;
[0049] FIG. 21A and 21B are photographs showing the outside and
luminal surfaces, respectively, of a first murine bladder after
pretreatment with a 0.05% polidocanol solution followed by
infection with Ad-LacZ;
[0050] FIGS. 21C and 21D are photographs showing the cross section
of the murine bladder of FIG. 21A wherein FIG. 21B was taken at
40.times. and FIG. 21C was taken at 100.times. magnification;
[0051] FIG. 22A and 22B are photographs showing the outside and
luminal surfaces, respectively, of a second murine bladder after
pretreatment with a 0.05% polidocanol solution followed by
infection with Ad-LacZ;
[0052] FIGS. 22C and 22D are photographs showing the cross section
of the murine bladder of FIG. 22A wherein FIG. 22B was taken at
40.times. and FIG. 22C was taken at 100.times. magnification;
[0053] FIG. 23A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.2% polidocanol
solution followed by infection with Ad-LacZ;
[0054] FIGS. 23B and 23C are photographs showing the cross section
of the murine bladder of FIG. 23A wherein FIG. 23B was taken at
40.times. and FIG. 23C was taken at 100.times. magnification;
[0055] FIG. 24A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.2% polidocanol
solution followed by infection with Ad-LacZ;
[0056] FIGS. 24B and 24C are photographs showing the cross section
of the murine bladder of FIG. 24A wherein FIG. 24B was taken at
40.times. and FIG. 24C was taken at 100.times. magnification;
[0057] FIG. 25A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.02% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ;
[0058] FIGS. 25B and 25C are photographs showing the cross section
of the murine bladder of FIG. 25A wherein FIG. 25B was taken at
40.times. and FIG. 25C was taken at 100.times. magnification;
[0059] FIG. 26A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.02% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ;
[0060] FIGS. 26B and 26C are photographs showing the cross section
of the murine bladder of FIG. 26A wherein FIG. 26B was taken at
40.times. and FIG. 26C was taken at 100.times. magnification;
[0061] FIG. 27A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.05% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ;
[0062] FIGS. 27B and 27C are photographs showing the cross section
of the murine bladder of FIG. 27A wherein FIG. 27B was taken at
40.times. and FIG. 27C was taken at 100.times. magnification;
[0063] FIG. 28A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.05% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ;
[0064] FIGS. 28B and 28C are photographs showing the cross section
of the murine bladder of FIG. 28A wherein FIG. 28B was taken at
40.times. and FIG. 28C was taken at 100.times. magnification;
[0065] FIG. 29A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.2% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ;
[0066] FIGS. 29B and 29C are photographs showing the cross section
of the murine bladder of FIG. 29A wherein FIG. 29B was taken at
40.times. and FIG. 29C was taken at 100.times. magnification;
[0067] FIG. 30A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.2% sodium salt of
dedecyl benzenesulfonic acid solution followed by infection with
Ad-LacZ;
[0068] FIGS. 30B and 30C are photographs showing the cross section
of the murine bladder of FIG. 30A wherein FIG. 30B was taken at
40.times. and FIG. 30C was taken at 100.times. magnification;
[0069] FIGS. 31A-31E are photographs showing the luminal surfaces
of murine bladders treated with alkyl maltoside and alkyl
maltopyranoside pretreating agents having various length alkyl side
chains; and
[0070] FIGS. 32A-32C are photographs showing the luminal surfaces
of murine bladders treated with sodium alkyl sulfate pretreating
agents having various length alkyl chains.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0071] The present invention is directed to the use of transduction
enhancing agents to render the bladder umbrella cell layer more
susceptible to infection with a viral gene delivery vehicle than it
would be without treatment. Exemplary transduction enhancing agents
according to the invention include: dodecyl surfactants;
dodecylmaltosides; dodecyl alcohol polyoxyethylene ethers (i.e.,
polidocanol); and sodium dodecylbenzenesulphonic acid/hypochlorous
acid complex (i.e., oxychlorosene).
[0072] According to the invention, the luminal surface of the
bladder can be treated with a composition comprising a transduction
enhancing agent prior to infection with a viral gene delivery
vehicle. The viral gene delivery vehicle can be an oncolytic virus
used to treat bladder cancer. Oncolytic viruses for use in
practicing the invention include, but are not limited to,
adenovirus, herpes simplex virus (HSV), reovirus, vesicular
stomatitis virus (VSV), newcastle disease virus, vacinia virus,
influenza virus, West Nile virus, coxsackie virus, poliovirus and
measles virus. Of particular interest in practicing the invention
are oncolytic viruses that exhibit preferential expression in
particular tissue types (i.e., in the bladder urothelium). An
oncolytic adenovirus of this type is disclosed, for example, in
Zhang. et al., "Identification of Human Uroplakin II Promoter and
Its Use in the Construction of CG8840, a Urothelium-specific
Adenovirus Variant that Eliminates Established Bladder Tumors in
Combination with Docetaxel", Cancer Research, 62, 3743-3750 (2002)
and in co-owned U.S. patent application Ser. No. 09/814,292, which
is expressly incorporated by reference herein. Chemotherapeutic
agents for use in combination therapy with oncolytic viruses are
described, for example, in co-owned U.S. patent application Ser.
No. 09/814,357, which is expressly incorporated by reference
herein.
[0073] Alternatively, the viral gene delivery vehicle can be any
gene therapy delivery vehicle known in the art for use in gene
therapy, including, but not limited to, an adenovirus, an
adeno-associated virus (AAV), a lentivirus, a retrovirus, a herpes
virus, etc. Exemplary gene therapy adenoviral agents are disclosed
in U.S. Pat. No. 6,165,779. The present inventors have found that
pre-treating mouse bladders with aqueous solutions of various
compounds consistently increased transduction to greater than 60%
of the bladder surface, versus an untreated percent transduction of
no more than 10%.
[0074] In addition to pre-treatment of the bladder surface with the
transduction enhancing agent, the present invention includes
co-administration of the viral gene delivery vehicle and the
transduction enhancing agent to the bladder and to co-formulations
of any one of the transduction enhancing agents with a recombinant
viral gene delivery vehicle.
Composition and Chemistry of Reagents Used to Enhance Adenovirus
Transduction in the Bladder Epithelium
[0075] Several classes of compounds, surfactants, and pre-made
reagents were tested in order to find those which increased gene
transfer or transduction by a viral gene delivery vehicle in the
bladder. An oncolytic adenovirus, CG884, was used as an exemplary
viral gene therapy vehicle. The reagents evaluated can be
classified by their physical or chemical properties and
structure.
[0076] First, the reagents can be grouped as a single compound or
as a mixed reagent (i.e., a mixture of compounds). Single compounds
evaluated include non-ionic surfactants, alcohols, polymers and
ionic surfactants. The ionic surfactants evaluated included: 4%
Poloxamer 407 (Pluronic.RTM. 127); 4% poloxamer 188 (Pluronic.RTM.
F68); 0.02%-0.5% Polidocanol; 0.1% n-dodecyl-b-D-glucopyranoside
(which can also be classified as a sugar-based surfactant);
0.02-0.5% n-dodecyl-b-D-maltoside (which can also be classified as
a sugar-based surfactant); 0.1% Tween.RTM. 20; 0.1% Triton.RTM.
X-100; 0.1% Forlan.RTM. C-24 (PEG Cholesterol); 0.1%
decyl-b-D-maltoside (which can also be classified as a sugar-based
surfactant); 0.1% 6-cyclohexylhexyl-.beta.-D-maltoside (which can
also be classified as a sugar-based surfactant); and 0.1%
Tromboject.RTM. (sodium tetradecyl sulfate).
[0077] Alcohols evaluated include 0.1%-3% benzyl alcohol and
10%-30% ethanol. Polymers evaluated include 0.4% HPMC 2910; 0.4%
PVA; 0.4% PVP; and 100 mg/ml Poly-Lysine. Ionic surfactants
evaluated include: 0.1% DC-Chol [Cholesteryl
3b-N-(dimethylaminoethyl) carbamate]; 0.2% sodium salt of Dodecyl
benzenesulfonic acid; and 0.1% sodium dodecyl sulfate. Mixed
reagents evaluated include: In vivo GeneSHUTTLE.TM. (a reagent
comprising DOTAP+Cholesterol available from Qbiogene of Carlsbad,
Calif.) and 0.1%-0.4% Oxychlorosene (sodium dodecylbenzenesulphonic
acid/hypochlorous acid complex).
Effect of Ethanol Pretreatment on Adenovirus-Mediated Gene Transfer
and Expression in the Bladder Epithelium of Rodents
[0078] A study was conducted to evaluate the effect of ethanol
pretreatment on adenovirus-mediated gene transfer and expression in
the bladder epithelium of rodents.
Test Materials
[0079] Ad-.beta.gal virus was made as a frozen formulation using
standard conditions known in the art for freezing and formulation
of adenovirus. The vehicle for the virus arm was PBS plus 10%
glycerol. Pretreatment agents were 5%, 10%, 15%, 20% and 30% GLP
grade ethanol, respectively, in PBS-10% glycerol solution.
Animals
[0080] 80 female BALB/c mice were used for this study. Female
animals are chosen because of the ease of urethral cannulation and
vesicle instillation. The mice were approximately 10 to 12 weeks on
the day of the start of the experiment.
Treatment Regimen
[0081] Animals were assigned to each group as shown in the
following table.
TABLE-US-00001 TABLE 1 Effect of Ethanol pretreatment Animals Virus
dose Group per Test Ethanol (particles/ No. group Article Route
pretreatment animal) Dose Regimen 1 8-10 Vehicle Intravesical -- --
100 ml PBS- 10% glycerol on Day 1 2 8-10 Ad-.beta.gal Intravesical
-- 1.3 .times. 10.sup.11 Ad-.beta.gal on Day 1 3 8-10 Ad-.beta.gal
Intravesical 5% ethanol 1.3 .times. 10.sup.11 5% ethanol
pretreatment followed by virus administration on Day 1 4 8-10
Ad-.beta.gal Intravesical 10% ethanol 1.3 .times. 10.sup.11 10%
ethanol pretreatment followed by virus administration on Day 1 5
8-10 Ad-.beta.gal Intravesical 15% ethanol 1.3 .times. 10.sup.11
15% ethanol pretreatment followed by virus administration on Day 1
6 8-10 Ad-.beta.gal Intravesical 20% ethanol 1.3 .times. 10.sup.11
20% ethanol pretreatment followed by virus administration on Day 1
7 8-10 Ad-.beta.gal Intravesical 25% ethanol 1.3 .times. 10.sup.11
25% ethanol pretreatment followed by virus administration on Day 1
8 8-10 Ad-.beta.gal Intravesical 30% ethanol 1.3 .times. 10.sup.11
30% ethanol pretreatment followed by virus administration on Day
1
For the data in Table 1, the concentration of Ad-.beta.gal virus
was 1.3.times.10.sup.12 vp/mi as determined by optical density
measurements.
Treatment Procedure
[0082] 1. Animals were anesthetized with isoflurane and a 24 g
catheter introduced through the urethra into the bladder.
[0083] 2. Residual urine was emptied and the bladder was flushed 3
times with 100-150 .mu.l each of PBS.
[0084] 3. In test animals, bladders were pretreated for 20 minutes
with 0.1 ml of 5, 10, 15, 20, 25 or 30% ethanol solution,
respectively, and then rinsed 3 times with 100-150 .mu.l of
PBS.
[0085] 4. Ad-.beta.gal viruses diluted in 0.1 ml of PBS-10%
glycerol were administered intravesically into the bladder and
retained in the bladder for 45 minutes. A knot was placed around
the urethral orifice to prevent leakage of the virus and to prevent
the catheter from dislodging.
[0086] 5. Treatment was stopped by withdrawing the virus and
flushing the bladders 3 times with 100-150 .mu.l of PBS. If the
catheter became clogged, the washing step was avoided so that the
virus was flushed out in the urine. However, the use of this
procedure may prevent determination of the viral resident time in
the bladder.
Measurement/Determinations
[0087] The clinical condition of the animals was observed before
dosing on the day of treatment and the animals were observed daily
during the experimental period.
Assessment of .beta.-Galactosidase Activity
[0088] Animals were killed 48 hours after treatment. Bladders were
filled with 0.1 ml whole organ fixative: 2% Neutral buffered
formalin, 2% glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4.
Bladders were then removed and immersed in whole organ fixative for
1 hr. Thereafter, the bladders were cut open longitudinally, rinsed
(2 mM MgCl.sub.2, 0.1% deoxycholate, 0.2% Triton) for 24 hours at
4.degree. C., and submerged into X-gal staining solution. Transgene
expression in the luminal epithelium of the longitudinally opened
bladders was empirically determined.
Histopathology
[0089] Bladders fixed in whole organ fixative were sectioned and
stained with hematoxylin-eosin for histologic examination.
Results
[0090] Pretreatment of the luminal bladder surface with various
concentrations of ethanol (i.e., 15%, 20%, 25%, and 30 wt. %) for
20 minutes resulted in 10-20% transduction. FIGS. 1-4 show
transduction of murine bladders after pretreatment with ethanol.
FIGS. 1A and 1B are photographs showing a murine bladder after
pretreatment with a 15% ethanol solution followed by infection with
Ad-LacZ. FIG. 1A shows the outside surface of the bladder and FIG.
1B shows the luminal bladder surface. FIGS. 1C and 1D are
photographs showing a murine bladder after pretreatment with a 20%
ethanol solution followed by infection with Ad-LacZ. FIG. 1C shows
the outside surface of the bladder and FIG. 1D shows the luminal
bladder surface. FIGS. 1E and 1F are photographs showing a murine
bladder after pretreatment with a 25% ethanol solution followed by
infection with Ad-LacZ. FIG. 1E shows the outside surface of the
bladder and FIG. 1F shows the luminal bladder surface. FIGS. 1G and
1H are photographs showing a murine bladder after pretreatment with
a 30% ethanol solution followed by infection with Ad-LacZ. FIG. 1G
shows the outside surface of the bladder and FIG. 1H shows the
luminal bladder surface. As can be seen from FIG. 1, higher
concentrations of ethanol resulted in greater levels of
transduction as measured by staining.
[0091] FIG. 2A is a photograph showing a cross section of a murine
bladder control (i.e., no pretreatment). FIGS. 2B and 2C are
photographs showing the cross section of a murine bladder after
pretreatment with a 30% ethanol solution followed by infection with
Ad-LacZ.
[0092] FIGS. 3A-3F are photographs showing the cross section of
three murine bladders after pretreatment with a 25% ethanol
solution followed by infection with Ad-LacZ. FIGS. 3A and 3B are
photographs showing the cross-section of the first murine bladder,
FIGS. 3C and 3D are photographs showing the cross-section of the
second murine bladder, and FIGS. 3E and 3F are photographs showing
the cross-section of the third murine bladder. FIGS. 3A, 3C and 3E
were taken at 40.times. and FIGS. 3B, 3D and 3F were taken at
100.times. magnification.
[0093] FIGS. 4A-4F are photographs showing the cross section of
three murine bladders after pretreatment with a 30% ethanol
solution followed by infection with Ad-LacZ. FIGS. 4A and 4B are
photographs showing the cross-section of the first murine bladder,
FIGS. 4C and 4D are photographs showing the cross-section of the
second murine bladder, and FIGS. 4E and 4F are photographs showing
the cross-section of the third murine bladder. FIGS. 4A, 4C and 4E
were taken at 40.times. and FIGS. 4B, 4D and 4F were taken at
100.times. magnification.
Effect of Chemical Agent Pretreatment on Adenovirus-Mediated Gene
Transfer and Expression in the Bladder Epithelium of Rodent
[0094] A study was conducted to evaluate the effect of chemical
agent pretreatment on adenovirus-mediated gene transfer and
expression in the bladder epithelium of rodents.
Test Materials
[0095] Ad-.beta.gal virus was made at CGI, as a frozen formulation
using standard conditions known in the art for freezing and
formulation of adenovirus. The vehicle for the virus arm was PBS
plus 10% glycerol.
Animals
[0096] 152 female BALB/c mice were used this study. Female animals
were chosen because of the ease of urethral cannulation and vesicle
instillation. The mice were approximately 10 to 12 weeks on the day
of the start of the experiment.
Treatment Regimen
[0097] Animals were assigned to each group shown in the following
table. The route of administration of the chemical agent and virus
was intravesical.
TABLE-US-00002 TABLE 2 Effect of Chemical Agent Pretreatment
Animals Virus dose Group per Test (particles/ No. group Article
Chemical agent animal) Dose Regimen 1 2 Vehicle 4% Poloxamer 407 --
100 ml of 4% (Pluronic 127) Poloxamer 407 (Pluronic 127) in PBS-
10% glycerol on Day 1 2 6-8 Ad-.beta.gal 4% Poloxamer 407 1.3
.times. 10.sup.11 4% Poloxomer 407 (Pluronic 127) (Pluronic 127)
pretreatment followed by virus administration on Day 1 3 2 Vehicle
4% Poloxamer 188 100 ml of 4% (Pluronic F68) Poloxamer 188
(Plluronic F68) in PBS-10% glycerol on Day 1 4 6-8 Ad-.beta.gal 4%
Poloxamer 188 1.3 .times. 10.sup.11 4% Poloxomer 188 (Pluronic F68)
(Pluronic F68) pretreatment following by virus administration on
Day 1 5 2 Vehicle Lipofectamine 100 ml of 2000 Lipofectamine 2000
(20 mg/ml) in PBS 10% glycerol. 6 6-8 Ad-.beta.gal Lipofectamine
0.65 .times. 10.sup.11 1.25 mg of 2000 Lipofectamine 2000 mixed
with virus administration on Day 1 7 2 Vehicle 3% Benzyl 100 .mu.l
of 3% Benzyl Alcohol Alcohol in PBS-10% glycerol on Day 1 8 6-8
Ad-.beta.gal 3% Benzyl 1 .times. 10.sup.11 3% Benzyl Alcohol
Alcohol pretreatment followed by virus administration on Day 1 9 2
Vehicle 0.2% 0.2% Oxychlorosene Oxychlorosene in PBS-10% glycerol
on Day 1 10 6-8 Ad-.beta.gal 0.2% 1.3 .times. 10.sup.11 0.2%
Oxychlorosene Oxychlorosene pretreatment (only wash) followed by
virus administration on Day 1 11 6-8 Ad-.beta.gal 0.2% 1.3 .times.
10.sup.11 0.2% Oxychlorosene Oxychlorosene pretreatment (5 min)
followed by virus administration on Day 1 12 6-8 Ad-.beta.gal 0.2%
1.3 .times. 10.sup.11 0.2% Oxychlorosene Oxychlorosene pretreatment
(15 min) followed by virus administration on Day 1 13 2 Vehicle
0.05% Polidocanol 0.05% Polidocanol in PBS-10% glycerol on Day 1 14
6-8 Ad-.beta.gal 0.05% Polidocanol 1.3 .times. 10.sup.11 0.05%
Polidocanol pretreatment followed by virus administration on Day 1
15 2 Vehicle 0.1% DC-Chol 0.1% DC-Chol in PBS-10% glycerol on Day 1
16 6-8 Ad-.beta.gal 0.1% DC-Chol 1.3 .times. 10.sup.11 0.1% DC-Chol
pretreatment followed by virus administration on Day 1 17 2 Vehicle
In vivo Gene 4 mM solution in PBS Shuttle (DOTAP + Cholesterol) 18
6-8 Ad-.beta.gal In vivo Gene 0.65 .times. 10.sup.11 4 mM of In
vivo Gene Shuttle (DOTAP + Shuttle mixed with Cholesterol) virus.
Administration on Day 1. (Dilute 60 ml of Lipid with 90 ml of
water. Then add 150 ul of Ad-bgal) 19 2 Vehicle 0.5% Polidocanol
0.5% Polidocanol in PBS-10% glycerol on Day 1 20 6-8 Ad-.beta.gal
0.5% Polidocanol 1.3 .times. 10.sup.11 0.5% Polidocanol
pretreatment followed by virus administration on Day 1 21 2 Vehicle
0.4% HPMC 2910 0.4% HPMC 2910 in PBS-10% glycerol on Day 1 22 6-8
Ad-.beta.gal 0.4% HPMC 2910 0.5 .times. 10.sup.11 0.8% HPMC 2910
mixed with an equal volume of the virus and then administered on
Day 1 23 2 Vehicle 100 mg/ml Poly- 100 ug/ml Poly-Lysine Lysine in
PBS-10% glycerol on Day 1 24 6-8 Ad-.beta.gal 100 mg/ml Poly- 0.5
.times. 10.sup.11 200 ug/ml Poly-Lysine Lysine mixed with an equal
volume of the virus and then administered on Day 1 25 2 Vehicle
0.1% n-dodecyl-b- 0.1% n-dodecyl-b-D D glucopyranoside
glucopyranoside in PBS-10% glycerol on Day 1 26 6-8 Ad-.beta.gal
0.1% n-dodecyl-b- 1 .times. 10.sup.11 0.1% n-dodecyl-b-D D
glucopyranoside glucopyranoside pretreatment followed by virus
administration on Day 1 27 2 Vehicle 0.4% PVA 0.4% PVA in PBS- 10%
glycerol on Day 1 28 6-8 Ad-.beta.gal 0.4% PVA 0.5 .times.
10.sup.11 0.8% PVA mixed with an equal volume of the virus and then
administered on Day 1 29 2 Vehicle 0.4% PVP 0.4% PVP in PBS- 10%
glycerol on Day 1 30 6-8 Ad-.beta.gal 0.4% PVP 0.5 .times.
10.sup.11 0.8% PVP mixed with an equal volume of the virus and then
administered on Day 1 31 2 Vehicle 0.1% Cholesterol- 0.1%
Cholesterol- Cyclodextrin Cyclodextrin reagent reagent in PBS-10%
glycerol on Day 1 32 6-8 Ad-.beta.gal 0.1% Cholesterol- 0.5 .times.
10.sup.11 0.2% Cholesterol- Cyclodextrin Cyclodextrin reagent
reagent mixed with an equal volume of the virus and then
administered on Day 1 33 2 Vehicle 0.05% n-Dodecyl 0.05% n-Dodecyl
b-D- b-D-Maltoside Maltoside in PBS-10% glycerol on Day 1 34 6-8
Ad-.beta.gal 0.05% n-Dodecyl 1 .times. 10.sup.11 0.05% n-Dodecyl
b-D- b-D-Maltoside Maltoside pretreatment followed by virus
administration on Day 1 35 2 Vehicle 0.3% Benzyl 100 .mu.l of 0.3%
Benzyl Alcohol Alcohol in PBS-10% glycerol on Day 1 36 6-8
Ad-.beta.gal 0.3% Benzyl 1 .times. 10.sup.11 0.3% Benzyl Alcohol
Alcohol pretreatment followed by virus administration on Day 1 37 2
Vehicle 0.1% Benzyl 100 .mu.l of 0.1% Benzyl Alcohol Alcohol in
PBS-10% glycerol on Day 1 38 6-8 Ad-.beta.gal 0.1% Benzyl 1 .times.
10.sup.11 0.1% Benzyl Alcohol Alcohol pretreatment followed by
virus administration on Day 1 39 2 Vehicle 0.1% 0.1% Oxychlorosene
Oxychlorosene in PBS-10% glycerol on Day 1 40 6-8 Ad-.beta.gal 0.1%
1 .times. 10.sup.11 0.1% Oxychlorosene Oxychlorosene pretreatment
(5 min) followed by virus administration on Day1 41 2 Vehicle 0.4%
0.4% Oxychlorosene Oxychlorosene in PBS-10% glycerol on Day 1 42
6-8 Ad-.beta.gal 0.4% 1 .times. 10.sup.11 0.4% Oxychlorosene
Oxychlorosene pretreatment (5 min) followed by virus administration
on Day1 43 2 Vehicle 0.02% Polidocanol 0.02% Polidocanol in PBS-10%
glycerol on Day 1 44 6-8 Ad-.beta.gal 0.02% Polidocanol 1 .times.
10.sup.11 0.02% Polidocanol pretreatment followed by virus
administration on Day 1 45 2 Vehicle 0.2% Polidocanol 0.2%
Polidocanol in PBS-10% glycerol on Day 1 46 6-8 Ad-.beta.gal 0.2%
Polidocanol 1 .times. 10.sup.11 0.2% Polidocanol pretreatment
followed by virus administration on Day 1 47 2 Vehicle 0.02%
n-Dodecyl 0.02% n-Dodecyl b-D- b-D-Maltoside Maltoside in PBS-10%
glycerol on Day 1 48 6-8 Ad-.beta.gal 0.02% n-Dodecyl 1 .times.
10.sup.11 0.02% n-Dodecyl b-D- b-D-Maltoside Maltoside pretreatment
followed by virus administration on Day1 49 2 Vehicle 0.2%
n-Dodecyl b- 0.2% n-Dodecyl b-D- D-Maltoside Maltoside in PBS-10%
glycerol on Day 1 50 6-8 Ad-.beta.gal 0.2% n-Dodecyl b- 1 .times.
10.sup.11 0.2% n-Dodecyl b-D- D-Maltoside Maltoside pretreatment
followed by virus administration on Day1 51 2 Vehicle 0.2% sodium
salt 0.2% sodium salt of of Dodecyl Dodecyl benzenesulfonic
benzenesulfonic acid acid in PBS-10% glycerol on Day 1 52 6-8
Ad-.beta.gal 0.2% sodium salt 1 .times. 10.sup.11 0.2% sodium salt
of of Dodecyl Dodecyl benzenesulfonic benzenesulfonic acid acid
pretreatment followed by virus administration on Day 1 53 2 Vehicle
0.1% sodium 0.1% sodium dodecyl dodecyl sulphate sulphate in
PBS-10% glycerol on Day 1 54 6-8 Ad-.beta.gal 0.1% sodium 1 .times.
10.sup.11 0.1% sodium dodecyl dodecyl sulphate sulphate
pretreatment followed by virus administration on Day1 55 2 Vehicle
0.1% Tween 20 0.1% Tween 20 in PBS-10% glycerol on Day 1 56 6-8
Ad-.beta.gal 0.1% Tween 20 1 .times. 10.sup.11 0.1% Tween 20
pretreatment followed by virus administration on Day 1 57 2 Vehicle
0.1% Triton X-100 0.1% Triton X-100 in PBS-10% glycerol on Day 1 58
6-8 Ad-.beta.gal 0.1% Triton X-100 1 .times. 10.sup.11 0.1%
Triton
X-100 pretreatment followed by virus administration on Day 1 59 2
Vehicle 0.1% Forlan C-24 0.1% Forlan C-24 in (PEG Cholesterol)
PBS-10% glycerol on Day 1 60 6-8 Ad-.beta.gal 0.1% Forlan C-24 1
.times. 10.sup.11 0.1% Forlan C-24 (PEG Cholesterol) pretreatment
followed by virus administration on Day 1 61 2 Vehicle 0.1%
Decyl-b-D- 0.1% Decyl-b-D- Maltoside Maltoside in PBS-10% glycerol
on Day 1 62 6-8 Ad-.beta.gal 0.1% Decyl-b-D- 1 .times. 10.sup.11
0.1% Decyl-b-D- Maltoside Maltoside pretreatment followed by virus
administration on Day1 63 2 Vehicle 0.1% 6- 0.1% 6-
Cyclohexylhexyl- Cyclohexylhexyl-b-D- b-D-Maltoside Maltoside in
PBS-10% glycerol on Day 1 64 6-8 Ad-.beta.gal 0.1% 6- 1 .times.
10.sup.11 0.1% 6- Cyclohexylhexyl- Cyclohexylhexyl-b-D-
b-D-Maltoside Maltoside pretreatment followed by virus
administration on Day1 65 2 Vehicle 0.1% Tromboject 0.1% Tromboject
in (Sodium PBS-10% glycerol on Tetradecyl Sulfate) Day 1 66 6-8
Ad-.beta.gal 0.1% Tromboject 1 .times. 10.sup.11 0.1% Tromboject
(Sodium pretreatment followed Tetradecyl Sulfate) by virus
administration on Day 1 67 2 Vehicle 0.1% Phenyl B-D- 0.1% Phenyl
B-D- Glucopyranoside Glucopyranoside in PBS-10% glycerol on Day 1
68 6-8 Ad-.beta.gal 0.1% Phenyl B-D- 1 .times. 10.sup.11 0.1%
Phenyl B-D- Glucopyranoside Glucopyranoside pretreatment followed
by virus administration on Day 1 69 2 Vehicle 0.1% Sucrose 0.1%
Sucrose Monolaurate Monolaurate in PBS- 10% glycerol on Day 1 70
6-8 Ad-.beta.gal 0.1% Sucrose 1 .times. 10.sup.11 0.1% Sucrose
Monolaurate Monolaurate pretreatment followed by virus
administration on Day 1 71 2 Vehicle 0.1% 1-O-dodecyl- 0.1%
1-O-dodecyl- rac-glycerol rac-glycerol in PBS- 10% glycerol on Day
1 72 6-8 Ad-.beta.gal 0.1% 1-O-dodecyl- 1 .times. 10.sup.11 0.1%
1-O-dodecyl- rac-glycerol rac-glycerol pretreatment followed by
virus administration on Day 1
The concentration of Ad-.beta.gal virus for the data in Table 2 was
1.3.times.10.sup.12 vp/ml (1st preparation, particle: PFU: 30) and
1.times.10.sup.12 vp/ml (2.sup.nd preparation, particle: PFU: 30)
as determined by optical density measurements.
Treatment Procedure
[0098] 1. The animals were anesthetized with isoflurane and a 24 g
catheter was introduced through the urethra into the bladder.
[0099] 2. Residual urine was emptied and the bladder was flushed 3
times with 100 .mu.l each of PBS.
[0100] 3. Based on the reagent being tested, bladder pretreatment
was performed as follows:
[0101] Poloxomer 407 procedure: Washed 2 times with 100 .mu.l each.
Retained the 3.sup.rd wash for 5 min and gave one additional wash.
Performed 3 times PBS wash prior to virus instillation.
[0102] Poloxomer 188 procedure: Washed 2 times with 100 .mu.l each.
Retained the 3.sup.rd wash for 5 minutes and gave one additional
wash. Performed 3 times PBS wash prior to virus instillation.
[0103] Lipofectamine 2000 procedure: Added 5 .mu.l of stock
Lipofectamine (1 mg/ml) to 195 .mu.l of PBS-10% glycerol. Mixed
with an equal volume of Ad-.beta.gal virus and incubated for 15
minutes. Administered 100 .mu.l of the mixture intravesically and
retained in the bladder for 30 minutes.
[0104] Benzyl Alcohol procedure: Washed 2 times with 100 .mu.l
each. Retained the 3.sup.rd wash for 15 minutes and then gave one
additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0105] Oxychlorosene procedure: Washing performed as mentioned in
the dose regimen (i.e., 3 washes of 100 .mu.l each, one wash but
retained for 5 min., one wash but retained for 15 min). Performed 3
times PBS wash prior to virus instillation.
[0106] Polidocanol procedure: Washed 2 times with 100 .mu.l each.
Retained the 3rd wash for 5 min and then gave one additional wash.
Performed 3 times PBS wash prior to virus instillation.
[0107] DC-Cho procedure: Washed 2 times with 100 .mu.l each.
Retained the 3.sup.rd wash for 5 min and then gave one additional
wash. Performed 3 times PBS wash prior to virus instillation.
[0108] 0.4% HPMC 2910 procedure: No pretreatment. An equal volume
of the virus was mixed with 0.8% solution of HPMC2910 and the
mixture was instilled into the bladder for 30 minutes.
[0109] 100 mg/ml Poly-Lysine procedure: No pretreatment. An equal
volume of the virus was mixed with 100 mg/ml solution of
Poly-Lysine and the mixture was instilled into the bladder for 30
minutes.
[0110] 0.4% polyvinyl alcohol (PVA) procedure: No pretreatment. An
equal volume of the virus was mixed with 0.8% solution of PVA and
the mixture was instilled into the bladder for 30 minutes.
[0111] n-dodecyl-.beta.-D glucopyranoside procedure: Washed 2 times
with 100 .mu.l each. Retained the 3.sup.rd wash for 5 min and then
gave one additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0112] 0.4% PVP procedure: No pretreatment. An equal volume of the
virus was mixed with 0.8% solution of PVP and the mixture was
instilled into the bladder for 30 min.
[0113] 0.1% cholesterol-cyclodextrin reagent procedure: No
pretreatment. An equal volume of the virus was mixed with 0.2%
solution of Cholesterol-Cyclodextrin and the mixture was instilled
into the bladder for 30 minutes.
[0114] n-dodecyl-.beta.-D-maltoside procedure: Washed 2 times with
100 .mu.l each. Retained the 3.sup.rd wash for 5 min and then gave
one additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0115] Sodium salt of dodecyl benzenesulfonic acid procedure:
Washed 2 times with 100 .mu.l each. Retained the 3.sup.rd wash for
5 min and then gave one additional wash. Performed 3 times PBS wash
prior to virus instillation.
[0116] 0.1% sodium dodecyl sulphate procedure: Wash 2 times with
100 .mu.l each. Retained the 3.sup.rd wash for 5 min and then gave
one additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0117] 0.1% Tween 20 procedure: Washed 2 times with 100 .mu.l each.
Retained the 3.sup.rd wash for 5 min and then gave one additional
wash. Performed 3 times PBS wash prior to virus instillation.
[0118] 0.1% Triton.RTM. X-100 procedure: Washed 2 times with 100
.mu.l each. Retained the 3.sup.rd wash for 5 min and then gave one
additional wash. Perform 3 times PBS wash prior to virus
instillation.
[0119] 0.1% Forlan C-24 procedure: Washed 2 times with 100 .mu.l
each. Retained the 3.sup.rd wash for 5 min and then gave one
additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0120] 0.1% decyl-b-D-maltoside procedure: Washed 2 times with 100
.mu.l each. Retained the 3.sup.rd wash for 5 min and then gave one
additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0121] 0.1% 6-cyclohexylhexyl-b-D-maltoside procedure: Washed 2
times with 100 .mu.l each. Retained the 3.sup.rd wash for 5 min and
then gave one additional wash. Performed 3 times PBS wash prior to
virus instillation.
[0122] 0.1% sodium tetradecyl sulfate (Tromboject.RTM., Omega
Laboratories Ltd.) procedure: Washed 2 times with 100 .mu.l each.
Retained the 3.sup.rd wash for 5 min and then gave one additional
wash. Performed 3 times PBS wash prior to virus instillation.
[0123] 0.1% phenyl-.beta.-D-glucopyranoside procedure: Washed 2
times with 100 .mu.l each. Retained the 3.sup.rd wash for 5 min and
then gave one additional wash. Performed 3 times PBS wash prior to
virus instillation.
[0124] 0.1% sucrose monolaurate procedure: Washed 2 times with 100
.mu.l each. Retained the 3.sup.rd wash for 5 min and then gave one
additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0125] 0.1% 1-O-dodecyl-rac-glycerol procedure: Washed 2 times with
100 .mu.l each. Retained the 3.sup.rd wash for 5 min and then gave
one additional wash. Performed 3 times PBS wash prior to virus
instillation.
[0126] In vivo geneSHUTTLE.TM. procedure. Mixed 4 mM of In vivo
geneSHUTTLE.TM. with virus. Administration on Day 1. Diluted 60 ml
of Lipid with 90 ml of water. Then added 150 .mu.l of
Ad-.beta.gal.
[0127] 4. Virus treatment (45 min) stopped by withdrawing the virus
and flushing the bladders 3 times with 100 .mu.l of PBS.
Measurement/Determinations
[0128] The clinical condition of the animals were observed before
dosing on the day of treatment, and animals were observed daily
during the experimental period.
Assessment of .beta.-Galactosidase Activity
[0129] Animals were killed 48 hours after treatment. The bladders
were filled with 0.1-ml whole organ fixative: 2% Neutral buffered
formalin, 2% glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4.
The bladders were then removed and immersed in whole organ fixative
for 1 hour. Thereafter, each bladder was cut open longitudinally,
rinsed (in 2 mM MgCl.sub.2, 0.1% deoxycholate, 0.2% Triton) for 24
hours at 4.degree. C., and submerged into X-gal staining solution.
Transgene expression in the luminal epithelium of the
longitudinally opened bladders was empirically determined.
Histopathology
[0130] Bladders fixed in whole organ fixative were sectioned and
stained with hematoxylin-eosin for histologic examination.
Results
[0131] The results of the above experiments can be summarized as
follows:
[0132] Pre-treatment of the bladder with 4% Poloxamer 407 (Pluronic
127) for 5 minutes resulted in <5% transduction.
[0133] Treatment of the bladder with a lipofectamine and virus
mixture (no pretreatment) resulted in <5% transduction.
[0134] Treatment of the bladder with an In vivo geneSHUTTLE.TM. and
virus mix (no bladder pretreatment) resulted in <5%
transduction.
[0135] A pre-treatment of the bladder with 0.1% oxychlorosene for 5
minutes resulted in >90% transduction of the urothelium. The
pathologists report indicated mild submucosal edema with intact
epithelial layer.
[0136] A pre-treatment of the bladder with 0.2% oxychlorosene for 5
minutes resulted in >90% transduction of the urothelium. The
pathologists report indicated minimal submucosal edema and
perivascular lymphocytes.
[0137] A pre-treatment of the bladder with 0.2% oxychlorosene for
15 minutes resulted in >90% transduction of Urothelium. The
pathologists report indicated focal severe ulceration with
suppurtative exudate, hemorrhage and edema in the submucosa.
[0138] A pre-treatment of the bladder with 0.4% oxychlorosene for 5
minutes resulted in >90% transduction of Urothelium. The
pathologists report indicated moderate submucosal edema with focal
large ulcer.
[0139] A pre-treatment of the bladder with 0.02% polidocanol for 5
minutes resulted in 10-20% transduction of the urothelium. The
pathologists report indicated an intact mucosa.
[0140] A pre-treatment of the bladder with 0.05% polidocanol for 5
minutes resulted in 30-40% transduction of the urothelium. The
pathologists report indicated minimal submucosal edema.
[0141] A pre-treatment of the bladder with 0.2% polidocanol for 5
minutes resulted in 50-80% transduction of Urothelium. The
pathologists report indicated erosions and focal ulcer as well as
mucosal compromise.
[0142] A pre-treatment of the bladder with 0.02% n-dodecyl
.beta.-D-maltoside for 5 minutes resulted in 50-80% transduction of
the urothelium. The pathologists report indicated no significant
lesions.
[0143] A pre-treatment of the bladder with 0.05% n-dodecyl
.beta.-D-maltoside for 5 minutes resulted in >90% transduction
of the urothelium. The pathologists report indicated no significant
lesions.
[0144] A pre-treatment of the bladder with 0.2% n-dodecyl
.beta.-D-maltoside for 5 minutes resulted in >90% transduction
of the urothelium. The pathologists report indicated erosions,
focal ulcer, moderate submucosal edema with mucosal compromise.
[0145] A pre-treatment of the bladder with 0.2% dodecyl
benzenesulfonic acid for 5 minutes resulted in 20-40% transduction
of the urothelium.
[0146] As can be seen from the above results, several single
compounds and one mixed reagent showed significantly increased
transduction as measured by the levels of final blue stain (LacZ).
Several other single compounds resulted in enhanced but smaller
levels of transduction. An ethanol pre-treatment was used as a
reference to validate each chemical tested. Even with an ethanol
percentage as high as 30%, only 10-20% transduction was observed.
The "strong responders" were those transduction enhancing agents
which exhibited significantly better (i.e., 70-90% staining) than
the ethanol pre-treatment controls, which exhibited 10-20%
staining. The weak responders had significantly less stained area
compared to the ethanol control group.
[0147] The strongest response (i.e., highest level of transduction)
was observed following pretreatment of the bladder surface with:
0.02%-0.5% polidocanol; 0.02-0.5% n-dodecyl-b-D-maltoside; 0.1%
6-cyclohexylhexyl-b-D-maltoside; 0.1%-0.4% oxychlorosene; 0.2%
sodium salt of dodecyl benzenesulfonic acid; and 0.1% sodium
dodecyl sulphate.
[0148] The "weak responders" included 0.1% decyl-b-D-maltoside and
0.1% Triton.RTM. X-100.
[0149] Although not wishing to be bound by theory, the mechanism of
action can be hypothesized by analyzing the physical and chemical
properties of successful transduction enhancing reagents. The
transduction enhancing reagent in general is a surfactant. The
surfactant can be ionic or non-ionic. The surfactant preferably has
both hydrophilic and lipophilic sections. The hydrophilic portion
of the molecule contributes to water solubility while the
lipophilic (i.e., hydrophobic) portion helps molecular interactions
with lipids. The hydrophilic/lipophilic balance or HLB ratio is an
indication of the relative size of each part of the molecule.
Sugar Based Surfactants (Saccharides)
[0150] The transduction enhancing agent according to the invention
can be a sugar (e.g., a mono-, di-, or poly-saccharide) having a
lipophilic substituent. The transduction enhancing agent can be any
mono-, di-, or poly-saccharide having a lipophilic substituent.
According to a preferred embodiment of the invention, the
transduction enhancing agent is a di-saccharide having a lipophilic
substituent. Exemplary di-saccharides include maltose or sucrose.
Other di-saccharides having lipophilic substituents, however, can
also be used including lactose, isomaltose, trehalose or
cellobiose.
[0151] The lipophilic substituent can be linear (e.g., a straight
chain n-alkane or alkene) or non-linear (e.g., cyclic or branched
chain alkanes or alkenes). The lipophilic substituent can also be
an alkanoic acid residue. The length of the lipophilic substituent
can be varied to achieve the desired hydrophilic-lipophilic
balance. Tests on various maltoside substituted compounds indicated
that a sufficient lipophilic length resulted in improved
transduction efficacy. For example, both
n-dodecyl-.beta.-D-maltoside and
6-cyclohexylhexyl-.beta.-D-maltoside increased transduction
significantly. In contrast, n-decyl-.beta.-D-maltoside had only a
slight effect on transduction.
[0152] Results for bladder pretreatment with
n-dodecyl-.beta.-D-maltoside are shown in FIGS. 25-29. FIG. 25A is
a photograph showing the luminal surface of a first murine bladder
after pretreatment with a 0.02% n-dodecyl .beta.-D-maltoside
solution followed by infection with Ad-LacZ. FIGS. 25B and 25C are
photographs showing the cross section of the murine bladder of FIG.
25A. FIG. 25B was taken at 40X and FIG. 25C was taken at 100.times.
magnification. FIG. 26A is a photograph showing the luminal surface
of a second murine bladder after pretreatment with a 0.02%
n-dodecyl .beta.-D-maltoside solution followed by infection with
Ad-LacZ. FIGS. 26B and 26C are photographs showing the cross
section of the murine bladder of FIG. 26A. FIG. 26B was taken at
40.times. and FIG. 26C was taken at 100.times. magnification. FIG.
27A is a photograph showing the luminal surface of a first murine
bladder after pretreatment with a 0.05% n-dodecyl
.beta.-D-maltoside solution followed by infection with Ad-LacZ.
FIGS. 27B and 27C are photographs showing the cross section of the
murine bladder of FIG. 27A. FIG. 27B was taken at 40.times. and
FIG. 27C was taken at 100.times. magnification. FIG. 28A is a
photograph showing the luminal surface of a first murine bladder
after pretreatment with a 0.05% n-dodecyl .beta.-D-maltoside
solution followed by infection with Ad-LacZ. FIGS. 28B and 28C are
photographs showing the cross section of the murine bladder of FIG.
28A. FIG. 28B was taken at 40.times. and FIG. 28C was taken at
100.times. magnification. FIG. 29A is a photograph showing the
luminal surface of a first murine bladder after pretreatment with a
0.2% n-dodecyl .beta.-D-maltoside solution followed by infection
with Ad-LacZ. FIGS. 29B and 29C are photographs showing the cross
section of the murine bladder of FIG. 29A. FIG. 29B was taken at
40.times. and FIG. 29C was taken at 100.times. magnification.
[0153] The chemical formula for n-dodecyl-.beta.-D-maltoside and
n-decyl-.beta.-D-maltoside is given below:
##STR00007##
where n is 11 and 9, respectively. The chemical formula for
6-cyclohexylhexyl-.beta.-D-maltoside is:
##STR00008##
where n is 6.
[0154] The transduction experiments demonstrated that a small
reduction in the size of the lipophilic side chain (i.e.,
CH.sub.2--CH.sub.2) can limit the efficacy of the molecule for
transduction enhancement to a great degree. It is important to note
that all of the above compounds had good solubility in both water
and PBS buffer.
[0155] Compounds in this class of surfactants having a shorter
hydrophilic moiety were also evaluated. The results for
n-dodecyl-.beta.-D-glucopyranoside showed little or no enhancement
of transduction. The chemical formula for
n-dodecyl-.beta.-D-glucopyranoside is:
##STR00009##
where n is 11. While not wishing to be bound by theory, the
relative sizes of the hydrophilic and lipophilic portions of the
molecule appear to influence transduction enhancement. Therefore,
shorter chain n-alkyl-.beta.-D-glucopyranosides (e.g.,
n-hexyl-.beta.-D-glucopyranoside) may exhibit improved
transduction.
[0156] Any mono-, di-, or poly-saccharide having a lipophilic
substituent can be used as a transduction enhancing agent according
to the invention. Exemplary di-saccharide compounds include
sucrose, lactose, maltose, isomaltose, trehalose, and cellobiose.
The lipophilic substituent preferably comprises an alkyl or alkenyl
group. According to a preferred embodiment of the invention, the
lipophilic substituent is an alkanoic acid residue.
[0157] Although the .beta.-forms of the mono- and di-saccharides
are described above, the .alpha.-forms of these and other mono-,
di-, or poly-saccharide compounds can also be used according to the
invention. Exemplary .alpha.-saccharide transduction enhancing
agents according to the invention include
n-dodecyl-.alpha.-D-maltoside, n-hexyl-.alpha.-D-glucopyranoside
and 6-cyclohexylhexyl-.alpha.-D-maltoside. Additionally, either the
D- or L-forms of the mono-, di-, or poly-saccharides may be used as
transduction enhancing agents according to the invention.
[0158] FIGS. 31A-31E are photographs showing the luminal surfaces
of murine bladders treated with alkyl maltoside and alkyl
maltopyranoside pretreating agents having various length alkyl side
chains. FIG. 31A is a photograph of the luminal surface of a murine
bladder treated with n-dodecyl-.beta.-D-maltoside (C12 alkyl side
chain) prior to infection with Ad-LacZ (10.sup.9 vp). FIG. 31B
shows the luminal surface of a murine bladder treated with
tridecyl-.beta.-D-maltopyranoside (C13 alkyl side chain) prior to
infection. FIG. 31C shows the luminal surface of a murine bladder
treated with n-tetradecyl-.beta.-D-maltoside (C14 alkyl side chain)
prior to infection. FIG. 31D shows the luminal surface of a murine
bladder treated with n-decyl-.beta.-D-maltoside (C10 alkyl side
chain) prior to infection. FIG. 3 1E shows the luminal surface of a
murine bladder treated with n-octyl-.beta.-D-maltopyranoside (C8
alkyl side chain) prior to infection.
Ionic Alkyl Surfactants
[0159] Ionic alkyl surfactants can also be used as a transduction
enhancing compounds according to the invention. Exemplary ionic
alkyl surfactants include sodium dodecyl sulfate which has a
formula represented by:
##STR00010##
[0160] Another exemplary ionic surfactant is the sodium salt of
dodecyl-benzenesulfonic acid which has a chemical formula
represented by:
##STR00011##
[0161] Surfactants of the above type were evaluated and were found
to exhibit enhanced transduction comparable to the non-ionic
reagents set forth above. These results are shown in FIG. 30 for
dodecyl benzenesulfonic acid sodium salt. As can be seen by FIGS.
30A-30C, dodecyl benzenesulfonic acid sodium salt, enhanced the
transduction of Ad-LacZ in murine bladders. FIG. 30A is a
photograph showing the luminal surface of a first murine bladder
after pretreatment with a 0.2% sodium salt of dedecyl
benzenesulfonic acid solution followed by infection with Ad-LacZ.
FIGS. 30B and 30C are photographs showing the cross section of the
murine bladder of FIG. 30A. FIG. 30B was taken at 40.times. and
FIG. 30C was taken at 100.times. magnification.
[0162] FIGS. 32A-32C are photographs showing the luminal surfaces
of murine bladders treated with sodium alkyl sulfate pretreating
agents having various length alkyl chains. FIG. 32A shows the
luminal surface of a bladder treated with sodium dodecyl sulfate
(SDS) (C12 alkyl side chain) prior to infection with Ad-LacZ
(10.sup.9 vp). FIG. 32B shows the luminal surface of a bladder
treated with sodium decyl sulfate (C10 alkyl side chain) prior to
infection. FIG. 32C shows the luminal surface of a bladder treated
with sodium octyl sulfate (C8 alkyl side chain) prior to
infection.
[0163] The ionic alkyl surfactants consist of two portions, a
hydrophilic portion and a lipophilic portion. The arrangement of
these portions of the molecule is similar to the sugar-based
enhancing agents described above. According to the invention,
compounds similar to those set forth above and having variations in
alkyl substitution can also be used.
Alkyl(Ether) Alcohols
[0164] Also according to the invention, an alkyl ether compound can
be used as a transduction enhancing compound. Polidocanol, an alkyl
ether having the following chemical formula:
.about.C.sub.12H.sub.62O--(CH.sub.2--CH.sub.2--O).sub.-9
and a total formula of .about.C.sub.30H.sub.62O.sub.10, was
evaluated. The polidocanol used in the evaluation was sold under
the name Thesit.RTM., which is a registered trademark of
Desitin-Werk, Carl Klinke GmbH, Hamburg, Germany). There are
several other chemical names for polidocanol such as
polyethyleneglycoldodecyl ether [9002-92-0], lauryl alcohol, and
macrogol lauryl ether.
[0165] Results for pretreatment of the bladder surface with various
concentrations of polidocanol are shown in FIGS. 19-24. Results for
pretreatment of the bladder surface with 0.02% polidocanol are
shown in FIGS. 19 and 20. FIG. 19A is a photograph showing the
luminal surface of a first murine bladder after pretreatment with a
0.02% polidocanol solution followed by infection with Ad-LacZ.
FIGS. 19B and 19C are photographs showing the cross section of the
murine bladder of FIG. 19A. FIG. 19B was taken at 40.times. and
FIG. 19C was taken at 100.times. magnification. FIG. 20A is a
photograph showing the luminal surface of a second murine bladder
after pretreatment with a 0.02% polidocanol solution followed by
infection with Ad-LacZ. FIGS. 20B and 20C are photographs showing
the cross section of the murine bladder of FIG. 20A. FIG. 20B was
taken at 40.times. and FIG. 20C was taken at 100.times.
magnification.
[0166] Results for pretreatment of the bladder surface with 0.05%
polidocanol are shown in FIGS. 21 and 22. FIG. 21A and 21B are
photographs showing the outside and luminal surfaces, respectively,
of a first murine bladder after pretreatment with a 0.05%
polidocanol solution followed by infection with Ad-LacZ. FIGS. 21C
and 21D are photographs showing the cross section of the murine
bladder of FIG. 21A. FIG. 21B was taken at 40.times. and FIG. 21C
was taken at 100.times. magnification. FIG. 22A and 22B are
photographs showing the outside and luminal surfaces, respectively,
of a second murine bladder after pretreatment with a 0.05%
polidocanol solution followed by infection with Ad-LacZ. FIGS. 22C
and 22D are photographs showing the cross section of the murine
bladder of FIG. 22A. FIG. 22B was taken at 40.times. and FIG. 22C
was taken at100.times. magnification.
[0167] Results for pretreatment of the bladder surface with 0.2%
polidocanol are shown in FIGS. 23 and 24. FIG. 23A is a photograph
showing the luminal surface of a first murine bladder after
pretreatment with a 0.2% polidocanol solution followed by infection
with Ad-LacZ. FIGS. 23B and 23C are photographs showing the cross
section of the murine bladder of FIG. 23A. FIG. 23B was taken at
40.times. and FIG. 23C was taken at 100.times. magnification. FIG.
24A is a photograph showing the luminal surface of a second murine
bladder after pretreatment with a 0.2% polidocanol solution
followed by infection with Ad-LacZ. FIGS. 24B and 24C are
photographs showing the cross section of the murine bladder of FIG.
24A. FIG. 24B was taken at 40.times. and FIG. 24C was taken at
100.times. magnification.
[0168] Triton.RTM. X-100, having a general formula of:
##STR00012##
wherein x=10 was also evaluated and was also found to enhance
transduction. A similar compound having a cyclohexane ring rather
than a benzene ring can also be used as a transduction enhancing
agent according to the invention. This compound has the following
chemical structure:
##STR00013##
wherein x=10. Compounds of the above type wherein x is any positive
integer can also be used according to the invention.
[0169] Similar alkyl(ether) compounds having the general structure
of:
##STR00014##
are also commercially available. The trade name for these compounds
is "Brij". The compound shown above is designated "Brij 56". Brij
56 has the chemical formula C.sub.20H.sub.42O.sub.5. Another
commercially available compound, "Brij 58", has the chemical
formula C.sub.56H.sub.114O.sub.21.
[0170] Any of the above mentioned alkyl(ether) compounds can be
used as transduction enhancing agents according to the
invention.
Sodium Oxychlorosene
[0171] A composition comprising a sodium salt of
dodecylbenzenesulfonic acid and hypochlorous acid (i.e., sodium
oxychlorosene) at a pH of about 6.5 to 6.9 was evaluated. The
sodium oxychlorosene used in these evaluations was sold under the
name Clorpactin WCS-90 (manufactured by Guardian Labs and sold by
Cardinal Health). Sodium oxychlorosene has been used to treat
urinary tract infections and in abdominal and plastic surgery.
[0172] Results for pretreatment of the bladder surface with sodium
oxychlorosene are shown in FIGS. 8-18. FIGS. 8A-8N are photographs
showing seven murine bladders after pretreatment with a 0.2%
oxychlorosene solution for 5 minutes followed by infection with
Ad-LacZ. FIGS. 8A and 8B show the outside and luminal surfaces,
respectively, of the first bladder, FIGS. 8C and 8D show the
outside and luminal surfaces, respectively, of the second bladder,
FIGS. 8E and 8F show the outside and luminal surfaces,
respectively, of the third bladder, FIGS. 8G and 8H show the
outside and luminal surfaces, respectively, of the fourth bladder,
FIGS. 8I and 8J show the outside and luminal surfaces,
respectively, of the fifth bladder, FIGS. 8K and 8L show the
outside and luminal surfaces, respectively, of the sixth bladder,
and FIGS. 8M and 8N show the outside and luminal surfaces,
respectively, of the seventh bladder.
[0173] FIGS. 9A-9N are photographs showing seven murine bladders
after pretreatment with a 0.2% oxychlorosene solution for 15
minutes followed by infection with Ad-LacZ. FIGS. 9A and 9B show
the outside and luminal surfaces, respectively, of the first
bladder, FIGS. 9C and 9D show the outside and luminal surfaces,
respectively, of the second bladder, FIGS. 9E and 9F show the
outside and luminal surfaces, respectively, of the third bladder,
FIGS. 9G and 9H show the outside and luminal surfaces,
respectively, of the fourth bladder, FIGS. 9I and 9J show the
outside and luminal surfaces, respectively, of the fifth bladder,
FIGS. 9K and 9L show the outside and luminal surfaces,
respectively, of the sixth bladder, and FIGS. 9M and 9N show the
outside and luminal surfaces, respectively, of the seventh
bladder.
[0174] FIGS. 10A and 10B are photographs showing the cross section
of the murine bladders of FIGS. 8C and 8I, respectively. FIGS. 11A
and 11B are photographs showing the cross section of the murine
bladders of FIGS. 9C and 9I, respectively.
[0175] FIGS. 12A-12F are photographs showing the cross section of
three murine bladders after pretreatment with a 0.2% oxychlorosene
solution for 5 minutes followed by infection with Ad-LacZ. FIGS.
12A and 12B are photographs showing the cross-section of the first
murine bladder, FIGS. 12C and 12D are photographs showing the
cross-section of the second murine bladder, and FIGS. 12E and 12F
are photographs showing the cross-section of the third murine
bladder. FIGS. 12A, 12C and 12E were taken at 40.times. and FIGS.
12B, 12D and 12F were taken at 100.times. magnification.
[0176] FIGS. 13A-13F are photographs showing the cross section of
three murine bladders after pretreatment with a 0.2% oxychlorosene
solution for 15 minutes followed by infection with Ad-LacZ. FIGS.
13A and 13B are photographs showing the cross-section of the first
murine bladder, FIGS. 13C and 13D are photographs showing the
cross-section of the second murine bladder, and FIGS. 13E and 13F
are photographs showing the cross-section of the third murine
bladder. FIGS. 13A, 13C and 13E were taken at 40.times. and FIGS.
13B, 13D and 13F were taken at 100.times. magnification.
[0177] FIG. 14A is a photograph showing the luminal surface of a
murine bladder after pretreatment with a 0.1% oxychlorosene
solution followed by infection with Ad-LacZ. FIGS. 14B and 14C are
photographs showing the cross section of the murine bladder of FIG.
14A. FIG. 14B was taken at 40.times. and FIG. 14C was taken at
100.times. magnification;
[0178] FIG. 15A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.2% oxychlorosene
solution followed by infection with Ad-LacZ. FIGS. 15B and 15C are
photographs showing the cross section of the murine bladder of FIG.
15A. FIG. 15B was taken at 40.times. and FIG. 15C was taken at
100.times. magnification.
[0179] FIG. 16A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.2% oxychlorosene
solution followed by infection with Ad-LacZ. FIGS. 16B and 16C are
photographs showing the cross section of the murine bladder of FIG.
16A. FIG. 16B was taken at 40.times. and FIG. 16C was taken at
100.times. magnification.
[0180] FIG. 17A is a photograph showing the luminal surface of a
first murine bladder after pretreatment with a 0.4% oxychlorosene
solution followed by infection with Ad-LacZ. FIGS. 17B and 17C are
photographs showing the cross section of the murine bladder of FIG.
17A. FIG. 17B was taken at 40.times. and FIG. 17C was taken at
100.times. magnification.
[0181] FIG. 18A is a photograph showing the luminal surface of a
second murine bladder after pretreatment with a 0.4% oxychlorosene
solution followed by infection with Ad-LacZ. FIGS. 18B and 18C are
photographs showing the cross section of the murine bladder of FIG.
18A. FIG. 18B was taken at 40.times. and FIG. 18C was taken at
100.times. magnification.
Polymers with Alternating Hydrophilic and Lipophilic Units
[0182] Polymeric compounds comprising repeating sequences of
alternating or identical monomers were also tested. One such
compound tested was Poloxamer 407 (Pluronic 127) having a structure
represented by the following formula:
##STR00015##
[0183] Poloxamers polymers come in a wide range of HLB values. Both
of the compounds tested, however, had only a minimal effect on the
transduction of adenovirus. While not wishing to be bound by
theory, it is believed that compounds having separated, longer
hydrophilic and lipophilic chains are more effective at enhancing
transduction of the bladder epithelium.
Additional Transduction Enhancing Compounds
[0184] Additional compounds can also be used as transduction
enhancing agents according to the invention.
[0185] These compounds include
.omega.-undecylenyl-.beta.-D-maltopyranoside, which has a structure
represented by:
##STR00016##
Sugar based thiolic compounds such as
alkyl-.beta.-D-thioglucopyranosides having a general structure
represented by:
##STR00017##
may also be employed.
[0186] Additionally, alkyl-.beta.-D-thiomaltopyranosides having a
general structure represented by:
##STR00018##
may also be used as transduction enhancing compounds according to
the invention.
[0187] Further, compounds having a positive charge such as
##STR00019##
can also be used.
[0188] Additionally, compounds wherein the lipophilic and
hydrophilic parts are connected via a carboxylic bond can also be
employed. An exemplary compound of this type is
6-O-methyl-n-heptylcarboxyl-.alpha.-D-glucopyranoside:
##STR00020##
[0189] Sugar based compounds having alkyl groups with side groups
or other modifications may also be used. Exemplary compounds of
this type include 2-propyl-1-pentyl-.beta.-D-maltopyranoside having
a structure represented by:
##STR00021##
[0190] Sarcosine compounds may also be used as transduction
enhancing agents according to the invention. Exemplary sarcosine
compounds include sodium alkyl sarcosine having a structure
represented by:
##STR00022##
[0191] Various substituted sugars can also be used as transduction
enhancing compounds. An exemplary substituted sugar which can be
used as a transduction enhancing compound is a sucrose mono alkyl
ester having a chemical structure represented by:
##STR00023##
[0192] Exemplary compounds of this type include compounds wherein
n-10 (i.e., sucrose monolaurate).
[0193] Also according to the present invention, methods of treating
the luminal surface of the bladder are provided. According to a
preferred embodiment of the invention, the bladder is treated by
instillation using bladder catheterization. According to this
embodiment, any urine in the bladder is first removed and the
bladder is optionally washed with a buffer (e.g., PBS). A
composition comprising the transduction enhancing agent is then
applied to the luminal surface of the bladder (e.g., by
instillation). The transduction enhancing solution may be incubated
for some specified time or drained immediately. Multiple treatments
with the composition comprising the transduction enhancing agent
can be performed. After treatment with the transduction enhancing
agent, the luminal surface of the bladder may be washed with a
buffer (e.g., PBS). A solution comprising the adenovirus can then
be introduced into the bladder (e.g., by instillation). The
solution comprising the adenovirus can be removed immediately or,
alternatively, the solution can be allowed to incubate for a
certain amount of time. After treatment with the adenovirus, the
bladder surface can again be washed with a buffer solution (e.g.,
PBS). According to a preferred embodiment of the invention, about
50 to about 500 ml of the transduction enhancing composition is
delivered to the bladder by instillation for each treatment.
[0194] Alternatively, a composition comprising the transduction
enhancing agent and the adenovirus can be used to treat the luminal
bladder surface. According to this embodiment of the invention, any
urine in the bladder is first removed and the bladder is then
optionally washed with a buffer (e.g., PBS). A composition
comprising the transduction enhancing agent and the adenovirus is
then applied to the luminal surface of the bladder. The solution
may be incubated for some specified time or drained immediately.
After treatment, the luminal surface of the bladder may again be
washed with a buffer (e.g., PBS).
[0195] Although phosphate buffered saline (PBS) is the preferred
buffer, any other pharmaceutical buffer can be used according to
the invention. Exemplary buffers include sodium phosphate/sodium
sulfate, Tris buffer, glycine buffer, sterile water and other
buffers known in the art, including those described by Good, et
al., Biochemistry 5, 467 (1966). The pH of the buffer can be in the
range of 6.4 to 8.4, preferably 7 to 7.5, and most preferably 7.2
to 7.4.
[0196] The composition comprising the transduction enhancing agent
according to the invention preferably also comprises an oxidizing
agent. Exemplary oxidizing agents include, but are not limited to,
chlorite compounds, hypochlorous acid, hydrogen peroxide, and
peroxyacetic acid. According to a preferred embodiment of the
invention, any of the single compound transduction enhancing agents
can be combined with an oxidizing agent and used as a transduction
enhancing agent.
[0197] As set forth above, the viral gene therapy vehicle can be an
oncolytic virus, for example an oncolytic adenovirus exemplified
herein by CG8840. The adenovirus composition can further comprise a
chemotherapeutic agent such as Docetaxel. The adenovirus
composition preferably comprises from about 1.times.10.sup.11 to
about 1.times.10.sup.14 viral particles.
[0198] Various additional studies were conducted and are described
below. In these studies, all percent values that are presented are
weight percent values unless otherwise indicated.
Effects of Adenovirus Formulation with Different Concentrations of
Dodecyl-.beta.-D-Maltoside on the Infectivity of Mice Bladder
Urothelium [0199] Species: Female Balb/c Mouse (Taconic
Laboratory)-2/group [0200] Study Design: To test the effect of
formulating Ad-.beta.gal virus with different concentrations of
Dodecyl-.beta.-D-Maltoside and the resultant infection of mice
bladder urothelium
Dose/Route (Viral Particles #/Dose):
[0201] 1.times.10.sup.10 vp/dose of Ad.CMV.LacZ (Lot#1351.122)
[0202] 0.4%, 0.2%, 0.1%, and 0.05% of n-Dodecyl-.beta.-D-Maltoside
(Lot#100K5308).
[0203] An equal volume of 2.times. DDM and 2.times. Ad.CMV.LacZ was
mixed together immediately before instillation into the bladder.
100 .mu. of the mixture was instilled into the bladder for 10 min,
20 min, and 45 min. [0204] Endpoints: Bladders were harvested 48
hrs post virus infection with 0.1 ml whole organ fixative (2%
Neutral buffered formalin, 2% glutaraldehyde, 2 mM MgCl.sub.2, 10
mM PBS, pH 7.4). [0205] Results: Formulating Ad-.beta.gal virus
with different concentrations of Dodecyl-.beta.-D-Maltoside
resulted in a linear Adenovirus transduction rate (0.1% >0.05%
>0.025%). For the 20 min. instillation, 0.1% DDM formulated with
Ad-.beta.gal virus resulted in about 80% gene expression in
bladder, while 0.05% DDM resulted in about 40% gene expression. But
with 45 min. instillation, all animals with 0.05%-0.2% DDM with
Ad-.beta.gal virus had 100% gene expression in mice bladder. A 10
min. instillation for this formulation method, however, did not
achieve an acceptable transduction rate. It was also found that
gene transduction could be achieved with a 10 min. virus
instillation after DDM pretreatment.
[0206] This study illustrates that DDM can be formulated with
Ad-.beta.gal virus in mice bladder model. Further, with a DDM
pretreatment, the time for virus instillation can be decreased to
10 min. from 45 min.
Effects of Different Diluents on the Infectivity of Mice Bladder
Urothelium by Adenovirus
[0207] Species: Female Balb/c Mouse (Taconic Laboratory)-2/group
[0208] Study Design: To test the effects of different diluents on
Adenovirus infectivity of mice urothelium followed DDM pretreatment
(QQ5 minQ). The virus would be diluted 100 fold with the diluents
prior to instillation into mice bladder for 45 min.
Dose/Route:
[0209] 1.times.10.sup.9 vp/dose of Ad.CMV.LacZ (Lot #1351.122)
[0210] Dodecyl-.beta.-D-Maltoside (Lot #100K5308).
[0211] Diluents (A): 0.9% Sodium Chloride Injection Solution
(Baxter Lot#1A1322); (B): 2.5% Dextrose and 0.45% Sodium Chloride
Injection Solution (Baxter Lot# C529040); and (C): Plasma-lyte A
Injection Solution pH 7.4 (Baxter Lot# C558106); and ARCA buffer.
[0212] Endpoints: Bladders were harvested 48 hrs post virus
infection with 0.1 ml whole organ fixative (2% Neutral buffered
formalin, 2% glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4).
[0213] Results: There were no significant differences in
Ad-.beta.gal gene expression levels between mice bladder receiving
virus diluted with different diluents. All the animals had >90%
in Ad-.beta.gal gene expression in mice bladder. Adenovirus Dose
Titration with Different Chemical Agents Pretreatment on SD Rat
Bladder Epithelium [0214] Species: Female Sprague Dawley Rat
(Taconic Laboratory)-2/group [0215] Study Design: To test the virus
infectivity of rat bladder epithelium with different doses of
Adenovirus following the pretreatment with SDS or DDM. This study
also explored relationship between bladder volume and residual of
SDS or DDM for adenovirus infectivity.
Dose/Route:
[0216] 4.times.10.sup.9 and 4.times.10.sup.10 vp/dose of
Ad.CMV.LacZ (Lot#1351.122)
[0217] 0.1% Dodecyl-.beta.-D-Maltoside (Lot#100K5308) [0218] 0.1%
SDS (Integra Lot#836011 and Lot# BK14J11)
[0219] 400 .mu.l of SDS or DDM was instilled into bladder (QQ5
minQ) followed by six PBS washes, then Adenovirus for 15 min.
[0220] Endpoints: Bladders were harvested 48 hrs post virus
infection with 0.2 ml whole organ fixative (2% Neutral buffered
forrnalin, 2% glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4).
[0221] Results: There were no significant differences in
Ad-.beta.gal gene expression levels between rat bladders pretreated
with different lot of SDS and DDM. Rat bladders infected with
4.times.10.sup.10 vp adenovirus achieved >90% Ad-.beta.gal gene
expression levels whereas rat bladders infected with
4.times.10.sup.9 vp adenovirus had Ad-.beta.gal gene expression
levels range from 30% to 50%. It was found that rat bladder needed
10 times more adenovirus to achieve similar Ad-.beta.gal gene
expression levels compared with mouse bladder. SDS and DDM
pretreatment were both effective to remove GAG layer of bladder
epithelium.
Efficacy of Dodecyl-.beta.-D-Maltoside as a Pretreatment Agent
Prior to Adenovirus Infection of Mice Bladder Urothelium
[0221] [0222] Species: Female Balb/c Mouse (Taconic
Laboratory)-2/group [0223] Study Design: To test DDM from two
different suppliers as pretreatment agents to enhance the
adenovirus infection of mice bladder urothelium in a large group of
animals.
Dose/Route:
[0224] 1.times.10.sup.10 vp/dose of Ad.CMV.LacZ (Lot #1351.122)
[0225] Dodecyl-p-D-Maltoside (Lot #018H7250 and Lot #
P21/39/092)
[0226] 100 .mu.l of DDM was instilled into bladder (QQ5 minQ)
followed by three PBS washes, then Adenovirus for 15 min. [0227]
Endpoints: Bladders were harvested 48 hrs post virus infection with
50 .mu.l whole organ fixative (2% Neutral buffered formalin, 2%
glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4). [0228]
Results: There were no significant differences in Ad-.beta.gal gene
expression levels between mice bladder pretreated with these two
different lots of DDM. In each case, >90% Ad-.beta.gal
expression was achieved. Therefore, DDM from either manufacturer is
a good candidate for a chemical enhancer for adenovirus infection
in mice bladder.
Efficacy of Dodecyl-.beta.-D-Maltoside as Chemical Enhancer Prior
to Adenovirus Infection of SW780+Luc Orthotopic Bladder Tumor Model
in Mice
[0228] [0229] Species: Female NCR nu/nu Mouse (Taconic
Laboratory)-2/group [0230] Study Design: To show DDM pretreatments
enhance Ad.Lac Z infectivity in mice bladder urothelium. To further
confirm the enhancer effect on orthotopic tumors, two mice bearing
orthotopic SW780 bladder tumors were pretreated with or without DDM
(QQ5 minQ) followed by 15 min Ad.Lac Z instillation. The Ad.Lac Z
gene expression levels were checked in these tumor cells 4 days
post Ad.Lac Z infection.
Dose/Route:
[0231] 1.times.10.sup.10 vp/dose. Ad.CMV.LacZ Lot#1351.122.
[0232] Dodecyl-.beta.-D-Maltoside (Lot#018H7250).
[0233] 100 .mu.l of DDM was instilled into bladder (QQ5 minQ)
followed by three PBS washes, then Adenovirus for 15 min. [0234]
Endpoints: Bladders were harvested 96 hrs post virus infection with
50 .mu.l whole organ fixative (2% Neutral buffered formalin, 2%
glutaraldehyde, 2 mM MgCl.sub.2, 10 mM PBS, pH 7.4). [0235]
Results: There was a significant difference in Ad-.beta.gal gene
expression levels between mice bladders pretreated with DDM and
control animals. Without DDM pretreatment, there was very low
Ad-.beta.gal gene expression levels in bladder epithelium cells and
tumor cells. With DDM pretreatment, nearly 100% of the epithelium
cells were transduced with Ad-.beta.gal gene in bladder tumors
except those dead necrotic tumor cells. It was concluded that DDM
pretreatment enhances Adenovirus infection in orthotopic tumor
cells.
Testing of DDM and SDS Subgroup Compounds Containing Different
Lengths of Alkyl Side Chain and Different Types of Sugar Molecule
as Pretreatment Agents Prior to Adenovirus Infection of Mouse
Bladder Urothelium
[0235] [0236] Species: Female Balb/c Mouse (Taconic
Laboratory)-3-2/group [0237] Study Design: Several compounds
belonging to the Dodecyl-.beta.-D-Maltoside (DDM) and Sodium
Dodecyl Sulfate (SDS) subgroups containing different lengths of
alkyl side chain and different sugar molecules were tested as
pretreatment agents to enhance the adenovirus infection of mouse
bladder epithelium. All compounds were dissolved in PBS at 0.1%
concentration.
Dose/Route:
[0238] 1.times.10.sup.9vp/dose. Ad.CMV.LacZ (Lot #1351.122) 0.1%
Dodecyl-.beta.-D-Maltoside (Lot # P21/39/092) and 0.1% SDS (Lot
#101K0036) were the positive controls for this study.
[0239] 100 .mu.l of Ad-.beta.gal virus was instilled into bladder
via intravesicle administration for 15 min followed different
chemicals pretreatment (QQ5 minQ). [0240] Endpoints: Bladders were
harvested 48 hrs post virus infection with 50 .mu.l whole organ
fixative (2% Neutral buffered fornalin, 2% glutaraldehyde, 2 mM
MgCl.sub.2, 10 mM PBS, pH 7.4). [0241] Results: For the DDM
subgroup compounds, compounds with alkyl side chain lengths longer
than C12 had the best enhancer ability. DDM subgroup compounds
having C8, C10, C12, C13 and C14 alkyl side chains were evaluated.
For the SDS subgroup compounds, compounds with alkyl side chain
lengths shorter than C12 had very low Ad-.beta.gal gene expression
levels. SDS subgroup compounds having C8, C10 and C12 alkyl side
chains were evaluated.
[0242] The type of sugar molecule did not appear to have a
significant effect on the efficacy of these compounds as
pretreatment enhancers with the exception of sucrose monolaurate.
While not wishing to be bound by theory, we believe that the
disaccharide chain could enhance adenovirus infectivity based on
currently available data.
Pilot Efficacy Study: CG8840 Treatment with SW780+Luc Orthotopic
Bladder Tumor Model in Mice [0243] Species: Female NCR nu/nu Mouse
(Taconic or Simonsen Laboratory)-6/group for CG8840 and 3/group for
CG7870 [0244] Study Design: To test the efficacy of bladder
specific oncolytic virus CG8840 alone with prostate specific
oncolytic virus CG7870. Two treatment regimens were used for
CG8840: 1.times.10.sup.10 and 1.times.10.sup.8 vp/dose. One
treatment regimen was used for for CG7870: 1.times.10.sup.10
vp/dose. One dose of virus would be delivered into bladder weekly
for the consecutive three weeks.
Dose/Route:
[0245] 1 million SW780+Luc cells Clone #19 (P4)
[0246] CG8840 Lot#1408.190; CG7870 (Lot #38.145)
[0247] 0.1% Dodecyl-.beta.-D-Maltoside (Lot #018H7250).
[0248] 15 mg/ml Luciferin (Xenogen cat# XR-1001) substrate solution
was made in PBS filtered with 0.2 .mu.M filters.
[0249] 120 .mu.l of 1.times.10.sup.7 cell/ml SW780+luc cells were
aliquot into each tube and kept in ice before instillation. One
aliquot for each mice bladder. [0250] Endpoints: Live image mice
bladder was performed weekly for 10 weeks. H & E stain and
Human Cytokeratin Stain were performed on those available bladder
and kidney samples. [0251] Results: There were significant
differences in reducing tumor volumes between CG8840
(1.times.10.sup.10 vp/dose) treated mice and control animals (both
CG7870 and no virus treated groups). Three out of six mice treated
with 1.times.10.sup.10 vp/dose CG8840 had completed tumor
regression after second dose instillation and kept tumor free until
the end of the study (week 10). Human cytokeratin stain showed
there were no tumor cells in those mouse bladders. Efficacy for
1.times.10.sup.8 vp/dose of CG8840 was not significant, there was a
one out of five mouse had incomplete tumor regression on week 5 and
kept tumors at bay to the end. Tumor metastasis to kidney is a
significant threat to tumor bearing mice survival. Any mice with
kidney metastasis would soon die even if they were free of bladder
tumors. CG8840 Treatment with SW780+Luc Orthotopic Bladder Tumor
Model in Mice [0252] Species: Female NCR nu/nu Mouse (Taconic
Laboratory)-10/group [0253] Study Design: This study was designed
to observe the efficacy of bladder specific oncolytic virus CG8840
treatment modality for bladder tumors. CG8840 virus was instilled
into the bladder via intravesical administration for 15 min and 30
min, respectively. To reduced kidney tumor metastasis, two
different DDM pretreatment procedures were tested during SW780
tumor cell implanting: (1) 0.1% DDM QQ5 minQ; and (2) 0.1% DDM
Q10min.
Dose/Route:
[0254] 1 million SW780+Luc cells Clone #19 (P4).
[0255] CG8840 Lot#1408.190. 0.1% Dodecyl-.beta.-D-Maltoside (Lot
#018H7250).
[0256] 15 mg/ml Luciferin (Xenogen cat# XR-1001) substrate solution
was made in PBS filtered with 0.2 .mu.M filters.
[0257] 90 .mu.l of 1.times.10.sup.7cell/ml SW780+luc cells were
aliquot into each tube and kept in ice before instillation. One
aliquot for each mice bladder. [0258] Endpoints: Live imaging of
the mice bladders was performed weekly for 8 weeks. H & E stain
and Human Cytokeratin Stain were performed on those available
bladder and kidney samples. [0259] Results: There were significant
difference in reducing tumor volumes between CG8840 (15 min. and 30
min. virus treatment) treated mice and control animals. In
particular, seven out of nine mice with 15 min. virus treatment
showed significant tumor volume regression after first virus
treatment. Five of them were tumor free till the end of the study.
The CG8840 treatment with three doses of 1.times.10.sup.10 vp/dose
injected in three consecutive weeks proved very effective to
eradicate orthotopic SW780 tumors in mouse bladder. However, longer
virus instillation times (i.e., in excess of 15 min.) do not appear
to increase virus copy numbers within tumor cells.
[0260] For SW780 tumor cell implantation, 0.1% DDM pretreatment
with Q10 min. washes, less kidney tumor metastasis was observed in
mice with orthotopic tumors.
[0261] Varius additional studies were conducted to determine
adenovirus compatibility with various reagents. These studies are
described below.
Study #1--Compatibility of Ad5-LacZ with Ethanol and Urine
[0262] In this study, each sample was incubated at 37.degree. C.
for one hour before the plaque assay started. The data for this
study are shown below in Table 3.
TABLE-US-00003 TABLE 3 Adenovirus stability after incubation (EtOH,
urine) Titer Average Ratio Sample Name vp/mL pfu/mL Stdev. vp/pfu
Ad5-Lac Z in ARCA 1.30E+12 5.85E+10 1.49E+10 22.23 Ad5-Lac Z in
ARCA 7.80E+11 6.36E+07 1.68E+07 12255.34 40% EtOH Ad5-Lac Z in ARCA
1.24E+12 5.07E+10 0.00E+00 24.47 5% EtOH Ad5-Lac Z in ARCA 1.30E+10
6.26E+08 2.55E+08 20.75 1:100 Urine Ad5-Lac Z -80.degree. C.
1.30E+12 5.90E+10 9.28E+09 22.04 R-06-80 1 Hr. Hold 6.00E+11
2.34E+10 2.76E+09 25.65 R-06-81 6.00E+11 3.12E+10 8.27E+09
19.24
This data indicates that Ad-LacZ does not lose its activity after
incubation with urine, 5% ethanol, or ARCA at biological
temperature. However, 40% ethanol inactivated most of the virus.
Study #2: Compatibility of Ad5-LacZ with Ethanol
[0263] For this study, each sample was incubated at 37.degree. C.
before the plaque assay started. The data for this study are shown
below in Table 4.
TABLE-US-00004 TABLE 4 Adenovirus Stability after Incubation (EtOH,
PBS) Titer Average Ratio Sample Name vp/mL pfu/mL St. dev. vp/pfu
Ad 5 Lac Z 1.30E+12 4.34E+10 7.58E+09 30 Ad 5 Lac Z in PBS 1.30E+11
5.43E+09 4.26E+08 24 PBS/10% EtOH 1.30E+11 3.72E+09 4.26E+08 35
PBS/20% EtOH 1.30E+11 1.81E+09 2.84E+08 72 PBS/30% EtOH 1.30E+11
<1.1E+5 pfu/mL 1230-054 1.00E+11 9.55E+08 2.13E+08 105 R-06-80
6.00E+11 1.90E+10 6.20E+09 32
The above results indicate that higher concentrations of ethanol
(i.e., .gtoreq.10%) will result in partial to complete inactivation
of adenovirus. Study #3--Compatibility of Adenovirus with Selected
Enhancers
[0264] For this study, each sample was incubated at 37.degree. C.
or 25.degree. C. before the plaque assay started. The data for this
study are shown below in Table 5.
TABLE-US-00005 TABLE 5 Adenovirus Stability after Incubation with
Selected Enhancer Solutions Incub. Titer Average Ratio Solution
Temp Time vp/mL pfu/mL St. Dev. vp/pfu None 5.degree. C. none
4.23E+11 1.95E+10 0.00E+00 22 Oxychlorosene 37.degree. C. 1 hour
2.12E+11 8.82E+05 1.56E+05 240287 Dodecyl Maltoside 37.degree. C. 1
hour 2.12E+11 1.36E+10 1.38E+09 16 Polidocanol 37.degree. C. 1 hour
2.12E+11 1.22E+10 2.07E+09 17 Oxychlorosene 25.degree. C. 2 hours
2.12E+11 2.18E+08 0.00E+00 974 Dodecyl Maltoside 25.degree. C. 2
hours 2.12E+11 1.32E+10 6.89E+08 16 Polidocanol 25.degree. C. 2
hours 2.12E+11 1.02E+10 4.82E+09 21 none -70.degree. C. none
6.00E+11 2.76E+09 2.13E+08 21.7
[0265] As can be seen from the above data, dodecyl maltoside and
polidocanol are compatible with the adenovirus under the
experimental conditions while oxychlorosene can inactivate the
virus by increase in temperature and time of incubation.
Study #4--Degradation of Adenovirus by Sodium Dodecyl Sulfate
(SDS)
[0266] A series of experiments were preformed on samples of
adenovirus incubated at 37.degree. C. for 15-30 minutes in various
concentrations of SDS. The resulting material,was examined by anion
exchange chromatography. It was confirmed that concentrations of
SDS above or equal to 0.025% SDS will rapidly degrade the virus.
SDS at 0.0125% did not reduce the particle quality or quantity by
AEX method after 30 minutes of incubation.
Study #5--Degradation of Adenovirus by Sodium Dodecyl Sulfate
(SDS)
[0267] In this study, adenovirus (Ad-LacZ) preparations
(1.0.times.10.sup.12 vp/ml) were mixed with solutions of SDS or DDM
to obtain a mixture of adenovirus and each of these surfactants.
Final concentrations of SDS or DDM were 0.1% and the virus had a
5.0.times.10.sup.11 vp/ml as the result of mixing. These samples
were incubated at 25.degree. C. or 37.degree. C. for one hour
before freezing. They were sent to assay services for plaque assay
analysis. The results for this study are shown below in Table
6.
TABLE-US-00006 TABLE 6 Adenovirus Stability after Incubation with
0.1% SDS or 0.1% DDM Solutions Mean Titer Sample ID Replicate Titer
PFU/mL Ad-LacZ 1351-122 1 3.70E+10 4.85E+10 2 6.00E+10 Ad-LacZ ARCA
1 Hr 25.degree. C. 1 2.05E+10 2.05E+10 Ad-LacZ ARCA 1 Hr 37.degree.
C. 1 2.15E+10 2.15E+10 Ad-LacZ DDM 1 Hr 25.degree. C. 1 3.25E+10
3.25E+10 Ad-LacZ DDM 1 Hr 37.degree. C. 1 3.55E+10 3.55E+10 Ad-LacZ
SDS 1 Hr 25.degree. C. 1 no activity no activity Ad-LacZ SDS 1 Hr
37.degree. C. 1 no activity no activity
This experiment confirmed that a relatively short exposure of
adenovirus at elevated temperatures to SDS solutions will
inactivate the virus. However, the DDM solution had no adverse
effects on adenovirus.
[0268] The following table (Table 7) summarizes the transduction
efficacy of exemplary pretreatment agents. In Table 7, transduction
efficacy is defined as follows: "0" means 0-10% transduction
efficacy; "+" means 10-25% transduction efficacy; "++" means 25-50%
transduction efficacy; "+++" means 50-75% transduction efficacy;
and "++++" means >75% transduction efficacy.
TABLE-US-00007 TABLE 7 Transduction Efficacy of Exemplary
Pretreatment Agents Pretreatment Agent Transduction Efficacy Alkyl
disaccharides n-Dodecyl-.beta.-D-Maltoside (C12) ++++
n-Dodecyl-.alpha.-D-Maltoside (C12) ++++ Sucrose monolaurate (C12)
++++ 6-Cyclohexylhexyl-b-D-Maltoside ++++
n-Tridecyl-.beta.-D-Maltoside (C13) +++
n-Tetradecyl-.beta.-D-Maltoside (C14) ++ n-Decyl-.beta.-D-Maltoside
(C10) + n-Octyl-.beta.-D-Maltoside (C8) (0) Alkyl sulfate (ionic
Alkyl) Sodium dodecyl sulfate (C12) ++++ Sodium decyl sulfate (C10)
+ Sodium octyl sulfate (C8) (0) Sodium tetradecyl sulfate (C14) (0)
Na salt of dodecyl benzenesulfonic acid + Alkyl (ether) alcohols
Polidocanol ++ Polymeric surfactants Triton X-100 + Poloxamers F68,
F127 (0) Tween 20, Tween 80 (0)
[0269] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be appreciated by one skilled in the art from
reading this disclosure that various changes in form and detail can
be made without departing from the true scope of the invention.
[0270] All publications cited herein are hereby incorporated by
reference in their entirety.
* * * * *