U.S. patent application number 10/620296 was filed with the patent office on 2004-03-25 for ultrasound assembly for use with light activated drugs.
Invention is credited to Anderson, James R., Lichttenegger, Gary, Tachibana, Katsuro, Tachibana, Shunro.
Application Number | 20040059313 10/620296 |
Document ID | / |
Family ID | 27462251 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040059313 |
Kind Code |
A1 |
Tachibana, Katsuro ; et
al. |
March 25, 2004 |
Ultrasound assembly for use with light activated drugs
Abstract
A kit and method for causing tissue death within a tissue site
is disclosed. The kit includes a media with a light activated drug
activatable upon exposure to a particular level of ultrasound
energy. The kit also includes a catheter with a lumen coupled with
a media delivery port through which the light activated drug can be
locally delivered to the tissue site. The ultrasound transducer is
configured to transmit the level of ultrasound energy which
activates the light activated drug with sufficient power that the
ultrasound energy can penetrate the tissue site.
Inventors: |
Tachibana, Katsuro;
(Fukuoka-shi, JP) ; Tachibana, Shunro;
(Fukuoka-shi, JP) ; Anderson, James R.; (Redmond,
WA) ; Lichttenegger, Gary; (Woodinville, WA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
27462251 |
Appl. No.: |
10/620296 |
Filed: |
July 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10620296 |
Jul 15, 2003 |
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10305865 |
Nov 26, 2002 |
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10305865 |
Nov 26, 2002 |
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09620701 |
Jul 20, 2000 |
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6527759 |
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09620701 |
Jul 20, 2000 |
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09158316 |
Sep 21, 1998 |
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6176842 |
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09158316 |
Sep 21, 1998 |
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09129980 |
Aug 5, 1998 |
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6210356 |
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09158316 |
Sep 21, 1998 |
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08972846 |
Nov 18, 1997 |
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08972846 |
Nov 18, 1997 |
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08611105 |
Mar 5, 1996 |
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Current U.S.
Class: |
604/500 ;
424/9.52; 604/20 |
Current CPC
Class: |
A61K 41/0047 20130101;
A61M 37/0092 20130101; A61B 2017/22062 20130101; A61B 2017/00853
20130101; A61N 7/00 20130101; A61B 2017/22021 20130101; A61B
17/2202 20130101; A61B 2017/22088 20130101; A61B 2017/22002
20130101 |
Class at
Publication: |
604/500 ;
604/020; 424/009.52 |
International
Class: |
A61M 031/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 5, 1995 |
JP |
P07048710 |
Sep 19, 1997 |
JP |
J970617JSO |
Claims
What is claimed is:
1. A method for releasing a therapeutic from a microbubble,
comprising: providing a microbubble with a light activated drug;
and delivering ultrasound energy to the microbubble at a frequency
and intensity which activates the light activated drug to cause a
rupture of the microbubble.
2. The method of claim 1, wherein the microbubble is a
liposome.
3. The method of claim 1, wherein the light activated drug is the
therapeutic.
4. The method of claim 1, wherein the microbubble includes the
therapeutic in addition to the light activated drug.
5. The method of claim 1, wherein the light activated drug is
coupled with a shell of the microbubble.
6. The method of claim 1, wherein the light activated drug is
enclosed within the microbubble.
7. The method of claim 1, wherein the light activated drug is
included in a media outside the microbubble.
8. A microbubble, comprising: a substrate defining a shell of the
microbubble and having a thickness permitting hydraulic transport
of the microbubble; a light activated drug activatable upon
exposure to ultrasound energy, activation of the light activated
drug causing a disruption in the shell sufficient to cause a
rupture of the microbubble; and a therapeutic releasable from the
microbubble upon rupture of the microbubble to yield a therapeutic
effect.
9. A method for releasing a thrombolytic agent into a blood vessel,
comprising: encapsulating the thrombolytic agent within a material
formed at least in part of a light activated drug; delivering the
thrombolytic agent into the blood vessel; and emitting ultrasound
energy at a frequency and intensity which activates the light
activated drug and thereby releases the thrombolytic agent from the
material.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/305,865, filed Nov. 26, 2002, which is a continuation of
U.S. application Ser. No. 09/620,701, filed Jul. 20, 2000, now U.S.
Pat. No. 6,527,759, which is a divisional of U.S. application Ser.
No. 09/158,316, filed Sep. 21, 1998, now U.S. Pat. No. 6,176,842,
which is a continuation-in-part of U.S. application Ser. No.
09/129,980, filed Aug. 5, 1998, now U.S. Pat. No. 6,210,356 and
U.S. application Ser. No. 08/972,846, filed Nov. 18, 1997, now
abandoned, which is a continuation of U.S. application Ser. No.
08/611,105, filed Mar. 5, 1996, now abandoned. This application
also claims priority to Japanese application number 9-255814, filed
Sep. 19, 1997 and Japanese application number 7-048710, filed Mar.
8, 1995. Each of the above applications is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a method and catheter for
treating biological tissues with light activated drugs, and more
particularly, to a method and catheter for treating biological
tissues by delivering a light activated drug to a biological tissue
and exposing the light activated drug to ultrasound energy.
[0004] 2. Description of the Related Art
[0005] It is frequently desirable to kill targeted biological
tissues such as tumors and atheroma. One technique for causing
targeted tissue death is called photodynamic therapy which requires
the use of light activated drugs. Light activated drugs are
inactive until exposed to light of particular wavelengths, however,
upon exposure to light of the appropriate wavelength, light
activated drugs can exhibit a cytotoxic effect on the tissues where
they are localized. It has been postulated that the cytotoxic
effect is a result of the formation of singlet oxygen on exposure
to light.
[0006] Photodynamic therapy begins with the systemic administration
of a selected light activated drug to a patient. At first, the drug
disperses throughout the body and is taken up by most tissues
within the body. After a period of time usually between 3 and 48
hours, the drug clears from most normal tissue and is retained to a
greater degree in lipid rich regions such as the liver, kidney,
tumor and atheroma. A light source, such as a fiber optic, is then
directed to a targeted tissue site which includes the light
activated drug. The tissues of the tissue site are then exposed to
light from the light source in order to activate any light
activated drugs within the tissue site. The activation of the light
activated drug causes tissue death within the tissue site.
[0007] Several difficulties can be encountered during photodynamic
therapy. For instance, since the light activated drug is typically
administered systemically, the concentration of the light activated
drug within the targeted tissue site is limited by the quantity of
light activated drug administered. The concentration of the light
activated drug within a tissue site can also be limited by the
degree of selective uptake of the light activated drug into the
tissue site. Specifically, if the targeted tissue site does not
selectively uptake the light activated drug, the concentration of
light activated drug within the tissue site can be too low for an
effective treatment.
[0008] An additional problem associated with photodynamic therapy
concerns depth of treatment. Light cannot penetrate deeply into
opaque tissues. As a result, the depth that light penetrates most
tissue sites is limited. This limited depth can prevent
photodynamic therapy from being used to treat tissues which are
located deeply in the interior of a tissue site.
[0009] There is currently a need for a method and apparatus which
can be used to cause death to tissues death deep within a tissue
site. When the method and apparatus employ light activated drugs,
the method and apparatus should be able to provide an appropriate
concentration of light activated drug within the tissue site.
SUMMARY OF THE INVENTION
[0010] An object for an embodiment of the invention is causing
tissue death within a tissue site.
[0011] Another object for an embodiment of the present invention is
locally delivering a light activated drug to a tissue site and
activating the light activated drug.
[0012] Yet another object for an embodiment of the present
invention is locally delivering a light activated drug to a tissue
site and delivering ultrasound energy to the delivered light
activated drug to activate the light activated drug.
[0013] A further object for an embodiment of the present invention
is using a catheter to locally deliver a light activated drug to a
tissue site and delivering ultrasound energy from an ultrasound
element on the catheter to activate the light activated drug.
[0014] Yet a further object for an embodiment of the present
invention is including the light activated drug in an emulsion,
locally delivering the emulsion to a tissue site and delivering
ultrasound energy to the light activated drug within the tissue
site to activate the light activated drug.
[0015] Even a further object for an embodiment of the present
invention is including the light activated drug in a liposome,
locally delivering the liposome to a tissue site and delivering
ultrasound energy to the light activated drug within the tissue
site to activate the light activated drug.
[0016] An additional object for an embodiment of the present
invention is including the light activated drug in an aqueous
solution, locally delivering the aqueous solution to a tissue site
and delivering ultrasound energy to the light activated drug within
the tissue site to activate the light activated drug.
[0017] Yet a further object for an embodiment of the present
invention is including the light activated drug in an emulsion,
systemically delivering the emulsion, providing the light activated
drug sufficient time to localize within a tissue site and
delivering ultrasound energy to the light activated drug within the
tissue site to activate the light activated drug.
[0018] Even a further object for an embodiment of the present
invention is including the light activated drug in liposomes,
systemically delivering the liposomes, providing the light
activated drug sufficient time to localize within a tissue site and
delivering ultrasound energy to the light activated drug within the
tissue site to activate the light activated drug.
[0019] An additional object for an embodiment of the present
invention is including the light activated drug in an aqueous
solution, systemically delivering the aqueous solution, providing
the light activated drug sufficient time to localize within a
tissue site and delivering ultrasound energy to the light activated
drug within the tissue site to activate the light activated
drug.
[0020] Another object for an embodiment of the present invention is
coupling a site directing molecule to a light activated drug,
locally delivering the light activated drug to a tissue site and
activating the light activated drug within the tissue site.
[0021] Yet another object for an embodiment of the invention is
providing a catheter for locally delivering a media including a
light activated drug to a tissue site. The catheter including an
ultrasound assembly configured to activate the light activated drug
within the tissue site.
[0022] A further object for an embodiment of the invention is
providing a catheter for delivering a media including a light
activated drug to a tissue site. The catheter including an
ultrasound assembly for reducing exposure of the light activated
drug to ultrasound energy until the light activated drug has been
delivered from within the catheter.
[0023] A kit for causing tissue death within a tissue site is
disclosed. The kit includes a media with a light activated drug
activatable upon exposure to a particular level of ultrasound
energy. The kit also includes a catheter with a lumen coupled with
a media delivery port through which the light activated drug can be
locally delivered to the tissue site. The ultrasound transducer is
configured to transmit the level of ultrasound energy which
activates the light activated drug with sufficient power that the
ultrasound energy can penetrate the tissue site.
[0024] A method for causing tissue death in a subdermal tissue site
is also disclosed. The method includes providing a catheter for
locally delivering a light activated drug to the subdermal tissue
site, the catheter including an ultrasound transducer. The method
also includes locally delivering the light activated drug to the
tissue site; producing ultrasound energy from the ultrasound
transducer; and directing the ultrasound energy to the subdermal
tissue site following penetration of the light activated drug into
the subdermal tissue site to activate at least a portion of the
light activated drug within the subdermal tissue site.
[0025] A method for activating a light activated drug is also
disclosed. The method includes providing a catheter with an
ultrasound transducer. The method also includes introducing the
light activated drug into a patient's body where a subdermal tissue
site absorbs at least a portion of the light activated drug;
producing ultrasound energy; directing the ultrasound energy to the
light activated containing subdermal tissue site including the
light activated drug; and activating at least a portion of the
light activated drug in the subdermal selected tissue site.
[0026] A method for releasing a therapeutic from a microbubble is
also disclosed. The method includes providing a microbubble with a
light activated drug activatable upon exposure to ultrasound
energy; and delivering ultrasound energy to the microbubble at a
frequency and intensity which activates the light activated drug to
cause a rupture of the microbubble.
[0027] A microbubble is also disclosed. The microbubble includes a
substrate defining a shell of the microbubble and having a
thickness permitting hydraulic transport of the microbubble. The
microbubble also includes a light activated drug activatable upon
exposure to ultrasound energy. Activation of the light activated
drug causes a disruption in the shell sufficient to cause a rupture
of the microbubble. The microbubble further includes a therapeutic
releasable from the microbubble upon rupture of the microbubble and
yielding a therapeutic effect upon release from the
microbubble.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A is a side view of a catheter for locally delivering
a media including a light activated drug to a tissue site.
[0029] FIG. 1B is an axial cross section of an ultrasound assembly
for use with the catheter shown in FIG. 1A.
[0030] FIG. 1C is a lateral cross section of an ultrasound assembly
for use with the catheter shown in FIG. 1A.
[0031] FIG. 2A is a side view of a catheter having an elongated
body and an ultrasound assembly which is flush with the elongated
body.
[0032] FIG. 2B is an axial cross section of the ultrasound assembly
illustrated in FIG. 2A.
[0033] FIG. 2C is a lateral cross section of the ultrasound
assembly illustrated in FIG. 2A.
[0034] FIG. 3A illustrates a catheter with a utility lumen and a
second utility lumen.
[0035] FIG. 3B is an axial cross section of the ultrasound assembly
illustrated in the catheter of FIG. 3A.
[0036] FIG. 4A is a side view of a catheter including a plurality
of ultrasound assemblies.
[0037] FIG. 4B is a cross section of an ultrasound assembly
included on a catheter with a plurality of utility lumens.
[0038] FIG. 4C is a cross section of an ultrasound assembly
included on a catheter with a plurality of utility lumens.
[0039] FIG. 5A is a side view of a catheter including a
balloon.
[0040] FIG. 5B is a cross section of a catheter with a balloon
which include an ultrasound assembly.
[0041] FIG. 6A is a side view of a catheter with a balloon
positioned distally relative to an ultrasound assembly.
[0042] FIG. 6B is a side view of a catheter with an ultrasound
assembly positioned distally relative to a balloon.
[0043] FIG. 6C is a cross section of a catheter with an ultrasound
assembly positioned at the distal end of the catheter.
[0044] FIG. 7A is a side view of a catheter with a media delivery
port positioned between an ultrasound assembly and a balloon.
[0045] FIG. 7B is a side view of a catheter with an ultrasound
assembly positioned between a media delivery port and a
balloon.
[0046] FIG. 7C is a cross section of a catheter with an ultrasound
assembly positioned at the distal end of the catheter.
[0047] FIG. 8A is a side view of a catheter including a media
delivery port and an ultrasound assembly positioned between first
and second balloons.
[0048] FIG. 8B is a side view of a catheter including a media
delivery port and an ultrasound assembly positioned between first
and second balloons.
[0049] FIG. 8C is a cross section of a balloon included on a
catheter having a first and second balloon.
[0050] FIG. 9A illustrates an ultrasound assembly positioned
adjacent to a tissue site and microbubbles delivered via a utility
lumen.
[0051] FIG. 9B illustrates an ultrasound assembly positioned
adjacent to a tissue site and a media delivered via a media
delivery port.
[0052] FIG. 9C illustrates an ultrasound assembly positioned
adjacent to a tissue site and a media delivered via a media
delivery port while a guidewire is positioned in a utility
lumen.
[0053] FIG. 9D illustrates a catheter including a balloon
positioned adjacent to a tissue site.
[0054] FIG. 9E illustrates a catheter including a balloon expanded
into contact with a tissue site.
[0055] FIG. 9F illustrates a catheter with an ultrasound assembly
outside a balloon positioned at a tissue site.
[0056] FIG. 9G illustrates the balloon of FIG. 9F expanded into
contact with a vessel so as to occlude the vessel.
[0057] FIG. 9H illustrates a catheter with an ultrasound assembly
outside a first and second balloon positioned at a tissue site.
[0058] FIG. 9I illustrates the first and second balloon of FIG. 9H
expanded into contact with a vessel so as to occlude the
vessel.
[0059] FIG. 10A is a cross section of an ultrasound assembly
according to the present invention.
[0060] FIG. 10B is a cross section of an ultrasound assembly
according to the present invention.
[0061] FIG. 10C illustrates a support member with integral
supports.
[0062] FIG. 10D illustrates a support member which is supported by
an outer coating.
[0063] FIG. 11A is a cross section of an ultrasound assembly
including two concentric ultrasound transducers in contact with one
another.
[0064] FIG. 11B is a cross section of an ultrasound assembly
including two separated and concentric ultrasound transducers.
[0065] FIG. 11C is a cross section of an ultrasound assembly
including two ultrasound transducers where a chamber is defined
between one of the ultrasound transducers and an elongated
body.
[0066] FIG. 11D is a cross section of an ultrasound assembly
including two longitudinally adjacent ultrasound transducers in
physical contact with one another.
[0067] FIG. 11E is a cross section of an ultrasound assembly
including two separated and longitudinally adjacent ultrasound
transducers.
[0068] FIG. 11F is a cross section of an ultrasound assembly
including two longitudinally adjacent ultrasound transducers with a
single chamber positioned between both ultrasound transducers and
an elongated body.
[0069] FIG. 11G is a cross section of an ultrasound assembly
including two longitudinally adjacent ultrasound transducers with
different chambers positioned between each ultrasound transducers
and an elongated body.
[0070] FIG. 11H is a cross section of an ultrasound assembly
including two longitudinally adjacent ultrasound transducers in
contact with one another and having a single chamber positioned
between each ultrasound transducers and an elongated body.
[0071] FIG. 12A is a cross section of a catheter which includes an
ultrasound assembly module which is independent of a first catheter
component and a second catheter component.
[0072] FIG. 12B illustrates the first and second catheter
components coupled with the ultrasound assembly module.
[0073] FIG. 12C is a cross section of an ultrasound assembly which
is integral with a catheter.
[0074] FIG. 13A is a cross section of an ultrasound assembly
configured to radiate ultrasound energy in a radial direction. The
lines which drive the ultrasound transducer pass through a utility
lumen in the catheter.
[0075] FIG. 13B is a cross section of an ultrasound assembly
configured to radiate ultrasound energy in a radial direction. The
lines which drive the ultrasound transducer pass through line
lumens in the catheter.
[0076] FIG. 13C is a cross section of an ultrasound assembly
configured to longitudinally radiate ultrasound energy. The distal
portion of one line travels proximally through the outer
coating.
[0077] FIG. 13D is a cross section of an ultrasound assembly
configured to longitudinally transmit ultrasound energy. The distal
portion of one line travels proximally through a line lumen in the
catheter.
[0078] FIG. 14A illustrates ultrasound transducers connected in
parallel.
[0079] FIG. 14B illustrates ultrasound transducers connected in
series.
[0080] FIG. 14C illustrates ultrasound transducers connected with a
common line.
[0081] FIG. 15 illustrates a circuit for electrically coupling
temperature sensors.
[0082] FIG. 16 illustrates a feedback control system for use with a
catheter including an ultrasound assembly.
[0083] FIGS. 17A-N illustrate pyrrole-based macrocyclic classes of
light emitting drugs.
[0084] FIGS. 17B-2 illustrates possible texaphyrin derivation
sites.
[0085] FIGS. 18A-F illustrate the formulae of preferred light
emitting drugs for use with media that includes microbubbles.
[0086] FIG. 19 illustrates a formula for a porphyrin group.
[0087] FIGS. 20A-D illustrate the formulae of four preferred forms
of the hydromonobenzoporphyrin derivatives of the green porphyrins
illustrated in formulae 3 and 4 of FIG. 18.
[0088] FIGS. 21A-B illustrate the formulae for specific examples of
pyrrole-based macrocycle derivatives and xanthene derivatives which
are preferred for inclusion in microbubbles to enhance rupture of
the microbubbles upon activation.
[0089] FIGS. 22A-I schematically summarize the synthesis of an
oligonucleotide conjugate of a texaphyrin metal complex.
[0090] FIGS. 23A-H illustrate the covalent coupling of texaphyrin
metal complexes with amine, thiol, or hydroxy linked
oligonucleotides.
[0091] FIGS. 24A-F illustrate the synthesis of diformyl monoic acid
and oligonucleotide conjugate.
[0092] FIGS. 25A-J illustrate the synthesis of a texaphyrin based
light activated drug.
[0093] FIG. 26 illustrates the formula for tin ethyl etiopurpurin
(SnEt.sub.2).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0094] The present invention relates to a method and catheter for
delivering a light activated drug to a tissue site and delivering
ultrasound energy to the light activated drug within the tissue
site. Since many light activated drugs are also activated by
ultrasound energy, the delivery of ultrasound energy to the light
activated drug activates the light activated drug within the tissue
site. Similar to activation of a light activated drug by light,
activation by ultrasound causes death of tissues within the tissue
site. The tissue death is believed to result from the release of a
singlet oxygen. Suitable tissue sites include, but are not limited
to, atheroma, cancerous tumors, thrombi and potential restenosis
sites. A potential restenosis site is a tissue site where
restenosis is likely to occur such as the portion of vessels
previously treated by balloon angioplasty. In contrast to light,
ultrasound energy can be transmitted through opaque tissues. As a
result, the ultrasound energy can be used to treat tissues which
are deeper within a tissue site than could be treated via light
activation.
[0095] One explanation for the activation of light activated drugs
via the application of ultrasound is a result of cavitation.
Cavitation is known to occur when ultrasonic energy above a certain
threshold is applied to a liquid. The mechanism of generation of
cavitation is described in Apfel, Robert E., "Sonic Effervescence:
Tutorial on Acoustic Cavitation" Journal of Acoustic Society of
America 101 (3): 1227-1237 (March 1997) and Atchley A., Crum L.,
"Ultrasound--Its Chemical, Physical and Biological Effects:
Acoustic Cavitation and Bubble Dynamics," pp. 1-64, 1988 VCH
Publishers, New York (1998).
[0096] Cavitation results when gas dissolved in a solution forms
bubbles under certain types of acoustic vibration. Cavitation can
also occur when small bubbles already present in the solution
oscillate or repeatedly enlarge and contract to become bubbles.
When the size of these cavitation bubbles reaches a size that
cannot be maintained, they suddenly collapse and release various
types of energy. The various types of energy include, but are not
limited to, mechanical energy, visible light, ultraviolet light and
other types of electromagnetic radiation. Heat, plasma, magnetic
fields, shock waves, free radicals, heat and other forms of energy
are also thought to be generated locally. The light activated drug
is believed to be activated by at least one of the various forms of
energy generated at the time of cavitation collapse.
[0097] The delivery of light activated drug to the tissue site can
be through traditional systemic administration of a media including
the light activated drug or can be performed through localized
delivery of the media. Localized delivery can be achieved through
injection into the tissue site or through other traditional
localized delivery techniques. A preferred delivery technique is
using a catheter which includes a media delivery lumen coupled with
a media delivery port. The catheter can be positioned such that the
media delivery port is within the tissue site or is adjacent to the
tissue site via traditional over-the-guidewire techniques. The
media can then be locally delivered to the tissue site through the
media delivery port.
[0098] The localized delivery of the light activated drug to the
tissue sight serves to localize the light activated drug within the
tissue site and can reduce the amount of light activated drug which
concentrates in tissues outside the tissue site. Further, localized
delivery of the light activated drug can serve to increase the
concentration of the light activated drug within the tissue site
above levels which would be achieved through systemic delivery of
the light activated drug. Alternatively, the same concentration of
light activated drug within the tissue site as would occur through
systemic administration can be achieved by introducing smaller
amounts of light activated drug into a patient's body.
[0099] Localized delivery of the light activated drug also permits
treatment of tissue sites which do not have selective uptake of the
light activated drug. As discussed above, many light activated
drugs, such as the texaphyrins, are taken up by most tissues within
the body and later localize within lipid rich tissues. As a result,
a non-lipid rich tissue site can be treated by delivering the
ultrasound energy to the tissue site before the light activated
drug has an opportunity to localize in lipid rich tissues.
[0100] Localized delivery is also advantageous when the tissue site
is lipid rich such as in an atheroma or a tumor. The localized
delivery of the light activated drug combined with the inherent
affinity of the light activated drug for tissue site can result in
a high degree of localization of the light activated drug within
lipid rich tissue sites.
[0101] To increase localization of the light activated drug within
the tissue site, the light activated drug can be coupled with a
sight directing molecule to form a light activated drug conjugate.
The site directing molecule is chosen so the light activated drug
conjugate specifically binds with the tissue site when the light
activated drug conjugate is contacted with the tissue site under
physiological conditions of temperature and pH. The specific
binding may result from specific electrostatic, hydrophobic,
entropic, or other interactions between certain residues on the
conjugate and specific residues on the tissue site.
[0102] In one preferred embodiment, the light activated drug
includes an oligonucleotide acting as a site specific molecule
coupled with a texaphyrin. The oligonucleotide can have an affinity
for a targeted site on a DNA strand. For instance, the
oligonucleotide can be designed to have complementary Watson-Crick
base pairing with the targeted DNA site. Activation of the light
activated drug after the conjugate has bound the targeted DNA site
can cause cleavage of the DNA strand at the targeted DNA site. As a
result, the activated drug conjugate can be used for cleavage of
targeted DNA sites. The light activated conjugate can be targeted
to a site on viral DNA where activation of the light activated
conjugate causes the virus to be killed. Similarly, the light
activated conjugate can be targeted to oncogenes. Other
applications of targeted DNA cleavage include, but are not limited
to, antisense applications, specific cleavage and subsequent
recombination of DNA; destruction of viral DNA; construction of
probes for controlling gene expression at the cellular level and
for diagnosis; and cleavage of DNA in footprinting analyses, DNA
sequencing, chromosome analysis, gene isolation, recombinant DNA
manipulations, mapping of large genomes and chromosomes, in
chemotherapy and in site directing mutagenesis.
[0103] In another preferred embodiment, the light activated drug
includes a hormone. The hormone may be targeted to a particular
biological receptor which is localized at the tissue site.
[0104] The light activated drug can be included within several
media suitable for delivery into the body. Many light activated
drugs are known to have low water solubilities of less than 100
mg/L. As a result, achieving the desired concentration of light
activated drug in an aqueous solution media for systemic delivery
can often be difficult. However, localized delivery of the light
activated drug requires a lower concentration of light activated
drug within the media. As a result, when the light activated drug
is delivered locally, the light activated drug can be included in
an aqueous solution.
[0105] The media can also be an emulsion which includes a lipoid as
a hydrophobic phase dispersed in a hydrophilic phase. These
emulsions provide a media which is safe for delivery into the body
with an effective concentration of light activated drug.
[0106] The media can also include microbubbles comprised from a
substrate which forms a shell. Suitable substrates for the
microbubble include, but are not limited to, biocompatible
polymers, albumins, lipids, sugars or other substances. The light
activated drug can be enclosed within the microbubble, coupled with
the shell and/or distributed in the media outside the microbubble.
A preferred microbubble comprises a lipid substrate such as
liposome. Systemic administration of liposomes with light activated
drug has been shown to result in an increased accumulation and more
prolonged retention of light activated drugs within cultured
malignant cells and within tumors in vivo. Jori et al., Br. J.
Cancer, 48:307-309 (1983); Cozzani et al., In Porphyrins in Tumor
Phototherapy, 173-183, Plenum Press (Andreoni et al. eds. 1984). As
a result, inclusion of the light activated drug within a liposome
combined with the localized delivery of the light activated drug
can serve to enhance the localization of the light activated drug
within the tissue site.
[0107] Including a light activated drug with the microbubbles has
numerous advantages over microbubbles without light activated drug.
After administration of microbubbles to a patient, the microbubbles
often must be ruptured to achieve their therapeutic effects. One
technique for rupturing microbubbles has been to expose the
microbubbles to ultrasound energy. However, ultrasound energy of
undesirably high intensity is frequently required to break the
microbubbles. Further, the ultrasound energy frequently must be
matched to the resonant frequency of the microbubbles. As a result,
rupturing the microbubbles with ultrasound can present numerous
challenges.
[0108] Activating a light activated drug within the microbubble
and/or in the substrate of the microbubble can cause the
microbubble to rupture. Activation of the light activated drug is
believed to cause a disturbance which disrupts the shell of the
microbubble enough to cause the microbubble to rupture. This
disruption occurs when the light activated drug is coupled with the
shell of the microbubble or is entirely within the microbubble.
This disruption is also believed to occur when light activated drug
located the media outside the microbubbles is activated in
proximity of the microbubble. Accordingly, including a sufficient
concentration light activated drug in the media outside the
microbubble and activating a portion of that light activated drug
can also cause rupture of the microbubbles. As a result,
microbubbles can be ruptured by activating light activated drugs
and without matching the ultrasound frequency to the resonant
frequency of the microbubble. However, a more efficient rupturing
of microbubbles can be achieved by delivering a level of ultrasound
energy which is appropriate to activate the light activated drug
and which is matched to the resonant frequency of the microbubble.
Further, the cavitation threshold can require an ultrasound
intensity which is lower than the intensity required to rupture
microbubbles without light activated drugs. As a result, including
light activated drug with microbubbles can reduce the intensity of
ultrasound energy required to rupture the microbubble.
[0109] The threshold value of cavitation is also reduced in the
proximity of many light activated drugs. As a result, the light
activated drug encourages cavitation in the proximity of the light
activated drug.
[0110] The interior of the microbubbles may include a gas or may be
devoid of gas. When a gas is present, the gas can occupy any
portion of the microbubble's volume but preferably occupies
0.01-50% of the volume of the microbubble interior, more preferably
5-30% and most preferably 10-20%. When the volume of gas is less
than 0.01% of the volume, cavitation can be hindered and when the
volume of gas is greater than 50% the structural integrity of the
microbubble shell can become too weak for the microbubble to be
transported to the tissue site. Suitable gasses for the interior of
the microbubbles include, but are not limited to, biocompatible
gasses such as air, nitrogen, carbon dioxide, oxygen, argon,
fluorine, xenon, neon, helium, or combinations thereof. The
presence of tiny bubbles is known to reduce the cavitation
threshold. As a result, the presence of an appropriately sized gas
bubble in the microbubble can enhance cavitation in the proximity
of the light activated drug.
[0111] The microbubbles are preferably 0.01-100 .mu.m in diameter.
This size microbubble reduces excretion of the microbubble outside
the body and also reduces interference of the microbubble with the
flow of fluids within the body of the patient. Further, the
microbubbles preferably have a shell thickness of 0.001-50 .mu.m,
0.01-5 .mu.m and 0.1-0.5 .mu.m. This thickness provides the shells
with sufficient thickness that the microbubble can withstand enough
of the forces within the vasculature of a patient to be transported
through at least a portion of the patient's vasculature. Similarly,
the thickness can permit the microbubbles to be transported through
a lumen in an apparatus such as a catheter. However, this thickness
is also sufficiently thin that alteration of the ultrasound
activated substance upon activation is sufficient to disrupt the
shell of the microbubble and cause the microbubble to rupture.
[0112] Activating the light activated drug to rupture microbubbles
can cause the light activated drug to be released from the
microbubble so the light activated drug can penetrate the tissue
near the site of rupture. Further exposure of the light activated
drug to ultrasound can activate the light activated drug within the
tissue and cause death of the tissue as described above.
[0113] The microbubble can include a therapeutic in addition to the
light activated drug. Activation of the light activated drug can
serve to rupture the microbubble and release the therapeutic from
the microbubble. As a result, the therapeutic is released in
proximity to a tissue site by rupturing the microbubble in
proximity to the tissue site. This is advantageous when the
therapeutic can be detrimental when administered systemically. For
instance, a therapeutic such as cisplatin is known to kill
cancerous tissues but is also known to kill other tissues
throughout the body. As a result, systemic administration of
cisplatin can be detrimental. However, microbubbles can serve to
protect tissues from the therapeutic agent until the therapeutic
agent is released from the carrier. For instance, when the
therapeutic is enclosed within the interior of the microbubble,
contact between the therapeutic agent and tissues outside the
carrier is reduced. As a result, the carrier increases protection
of tissues outside the carrier are protected from the therapeutic
agent until the microbubble is ruptured and the therapeutic
released.
[0114] The therapeutics may be encapsulated in the microbubbles,
included in the shell of the microbubbles or in the media outside
the microbubbles. Therapeutic, as used herein, means an agent
having beneficial effect on the patient.
[0115] Examples of therapeutics which can be included with the
microbubbles include, but are not limited to, hormone products such
as, vasopressin and oxytocin and their derivatives, glucagon and
thyroid agents as iodine products and anti-thyroid agents;
cardiovascular products as chelating agents and mercurial diuretics
and cardiac glycosides; respiratory products as xanthine
derivatives (theophylline & aminophylline); anti-infectives as
aminoglycosides, antifungals (amphotericin), penicillin and
cephalosporin antibiotics, antiviral agents as Zidovudine,
Ribavirin, Amantadine, Vidarabine, and Acyclovir, anti-helmintics,
antimalarials, and antituberculous drugs; biologicals as immune
serums, antitoxins and antivenins, rabies prophylaxis products,
bacterial vaccines, viral vaccines, toxoids; antineoplastics
asnitrosureas, nitrogen mustards, antimetabolites (fluorouracil,
hormones, asprogesings and estrogens and antiestrogens; antibiotics
as Dactinomycin; mitotic inhibitors as Etoposide and the Vinca
alkaloids, Radiopharmaceuticals as radioactive iodine and
phosphorus products; as well as Interferon, hydroxyurea,
procarbazine, Dacarbazine, Mitotane, Asparaginase and
cyclosporins.
[0116] Other suitable therapeutics include, but are not limited to:
thrombolytic agents such as urokinase; coagulants such as thrombin;
antineoplastic agents, such as platinum compounds (e.g.,
spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin,
taxol, mitomycin, ansamitocin, bleomycin, cytosine arabinoside,
arabinosyl adsnine, mercaptopolylysine, vincristine, busulfan,
chlorambucil, melphalan (e.g., PAM, L-PAM or phenylalanine
mustard), mercaptopurine, mitotane, procarbazine hydrochloride
dactinomycin (actinomycin D), daunorubicinhydrochloride,
doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin),
aminoglutethimide, estramustine phosphate sodium, flutamide,
leuprolide acetate, megestrol acetate, tamoxifen citrate,
testolactone, trilostane, amsacrine (m-AMSA), asparaginase
(L-asparaginase) Erwinaasparaginase, etoposide (VP-16), interferon
alpha-2a, interferon alpha-2b, teniposide (VM-26), vinblastine
sulfate (VLB), vincristine sulfate, bleomycin, bleomycin sulfate,
methotrexate, adriamycin, and arabinosyl; blood products such as
parenteral iron, hemin; biological response modifiers such as
muramyldipeptide, muramyltripeptide, microbial cell wall
components, lymphokines (e.g., bacterial endotoxin such as
lipopolysaccharide, macrophage activation factor), sub-units of
bacteria (such as Mycobacteria, Corynebacteria), the synthetic
dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine;
anti-fungalagents such as ketoconazole, nystatin, griseofulvin,
flucytosine (5-fc), miconazole, amphotericin B, ricin, and
beta-lactam antibiotics (e.g., sulfazecin); hormones such as growth
hormone, melanocyte stimulating hormone, estradiol, beclomethasone
dipropionate, betamethasone, betamethasone acetate and
betamethasone sodium phosphate, vetamethasonedisodiumphosphate,
vetamethasone sodium phosphate, cortisone acetate, dexamethasone,
dexamethasone acetate, dexamethasone sodium phosphate, flunsolide,
hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate,
hydrocortisone sodium phosphate, hydrocortisone sodium succinate,
methylprednisolone, methylprednisolone acetate, methylprednisolone
sodium succinate, paramethasone acetate, prednisolone,
prednisoloneacetate, prednisolone sodium phosphate, prednisolone
rebutate, prednisone, triamcinolone, triamcinolone acetonide,
triamcinolone diacetate, triamcinolone hexacetonide and
fludrocortisone acetate; vitamins such ascyanocobalamin neinoic
acid, retinoids and derivatives such as retinolpalmitate, and
alpha-tocopherol; peptides, such as manganese super oxidedimutase;
enzymes such as alkaline phosphatase; anti-allergic agents such as
amelexanox; anti-coagulation agents such as phenprocoumon and
heparin; circulatory drugs such as propranolol; metabolic
potentiators such asglutathione; antituberculars such as
para-aminosalicylic acid, isoniazid, capreomycin sulfate
cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,
rifampin, and streptomycin sulfate; antivirals such as acyclovir,
amantadine azidothymidine (AZT or Zidovudine), Ribavirin
andvidarabine monohydrate (adenine arabinoside, ara-A);
antianginals such asdiltiazem, nifedipine, verapamil, erythrityl
tetranitrate, isosorbidedinitrate, nitroglycerin (glyceryl
trinitrate) and pentaerythritoltetranitrate; anticoagulants such as
phenprocoumon, heparin; antibiotics such as dapsone,
chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin,
cephradine erythromycin, clindamycin, lincomycin, amoxicillin,
ampicillin, bacampicillin, carbenicillin, dicloxacillin,
cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin,
oxacillin, penicillin G, penicillin V, ticarcillin rifampin and
tetracycline; antiinflammatories such as difunisal, ibuprofen,
indomethacin, meclofenamate, mefenamic acid, naproxen,
oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin,
aspirin and salicylates; antiprotozoans such as chloroquine,
hydroxychloroquine, metronidazole, quinine and meglumine
antimonate; antirheumatics such as penicillamine; narcotics such as
paregoric; opiates such as codeine, heroin, methadone, morphine and
opium; cardiac glycosides such as deslanoside, digitoxin, digoxin,
digitalin and digitalis; neuromuscular blockers such as atracurium
besylate, gallamine triethiodide, hexafluorenium bromide,
metocurine iodide, pancuronium bromide, succinylcholine chloride
(suxamethonium chloride), tubocurarine chloride and vecuronium
bromide; sedatives (hypnotics) such as amobarbital, amobarbital
sodium, aprobarbital, butabarbital sodium, chloral hydrate,
ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide,
methotrimeprazine hydrochloride, methyprylon, midazolam
hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,
phenobarbital sodium, secobarbital sodium, talbutal, temazepam and
triazolam; local anesthetics such as bupivacaine hydrochloride,
chloroprocaine hydrochloride, etidocainehydrochloride, lidocaine
hydrochloride, mepivacaine hydrochloride, procainehydrochloride and
tetracaine hydrochloride; general anesthetics such asdroperidol,
etomidate, fentanyl citrate with droperidol, ketaminehydrochloride,
methohexital sodium and thiopental sodium; and radioactive
particles or ions such as strontium, iodide rhenium and
yttrium.
[0117] In certain preferred embodiments, the therapeutic is a
monoclonal antibody, such as a monoclonal antibody capable of
binding to melanoma antigen.
[0118] Other preferred therapeutics include genetic material such
as nucleic acids, RNA, and DNA, of either natural or synthetic
origin, including recombinant RNA and DNA and antisense RNA and
DNA. Types of genetic material that may be used include, for
example, genes carried on expression vectors such as plasmids,
phagemids, cosmids, yeast artificial chromosomes (YACs), and
defective or "helper" viruses, antigene nucleic acids, both single
and double stranded RNA and DNA and analogs thereof, such
asphosphorothioate and phosphorodithioate oligodeoxynucleotides.
Additionally, the genetic material may be combined, for example,
with proteins or other polymers.
[0119] Examples of genetic therapeutics that may be included in the
microbubbles include DNA encoding at least a portion of an HLAgene,
DNA encoding at least a portion of dystrophin, DNA encoding at
least a portion of CFTR, DNA encoding at least a portion of IL-2,
DNA encoding at least a portion of TNF, an antisense
oligonucleotide capable of binding the DNA encoding at least a
portion of Ras.
[0120] DNA encoding certain proteins may be used in the treatment
of many different types of diseases. For example, adenosine
deaminase may be provided to treat ADA deficiency; tumor necrosis
factor and/or interleukin-2 may be provided to treat advanced
cancers; HDL receptor may be provided to treat liver disease;
thymidine kinase may be provided to treat ovarian cancer, brain
tumors, or HIV infection; HLA-B7 may be provided to treat malignant
melanoma; interleukin-2 may be provided to treat neuroblastoma,
malignant melanoma, or kidney cancer; interleukin-4 may be provided
to treat cancer; HIV env may be provided to treat HIV infection;
antisense ras/p53 may be provided to treat lung cancer; and Factor
VIII may be provided to treat Hemophilia B. See, for example,
Science 258, 744-746.
[0121] If desired, more than one therapeutic may be included in the
media. For example, a single microbubble may contain more than one
therapeutic or microbubbles containing different therapeutics may
be co-administered. By way of example, a monoclonal antibody
capable of binding to melanoma antigen and an oligonucleotide
encoding at least a portion of IL-2 may be administered in a single
microbubble. The phrase "at least a portion of," as used herein,
means that the entire gene need not be represented by the
oligonucleotide, so long as the portion of the gene represented
provides an effective block to gene expression. Further,
microbubbles including a therapeutic can be administered before,
after, during or intermittently with the administration of
microbubbles without a therapeutic. For instance, microbubbles
without a therapeutic and microbubbles including a coagulant such
as thrombin can be administered to a patient having liver cancer.
Activating the light activated drug included with the microbubbles
serves to rupture the microbubbles and release the light activated
drug and thrombin from the microbubbles. Further activation of the
light activated drug can cause tissue death and the thrombin can
cause coagulation in and around the damaged tissues.
[0122] Prodrugs may be included in the microbubbles, and are
included within the ambit of the term therapeutic, as used herein.
Prodrugs are well known in the art and include inactive drug
precursors which, when exposed to high temperature, metabolizing
enzymes, cavitation and/or pressure, in the presence of oxygen or
otherwise, or when released from the microbubbles, will form active
drugs. Such prodrugs can be activated via the application of
ultrasound to the prodrug-containing microbubbles with the
resultant cavitation, heating, pressure, and/or release from the
microbubbles. Suitable prodrugs will be apparent to those skilled
in the art, and are described, for example, in Sinkula et al., J.
Pharm. Sci. 1975 64, 181-210, the disclosure of which is hereby
incorporated herein by reference in its entirety. Prodrugs, for
example, may comprise inactive forms of the active drugs wherein a
chemical group is present on the prodrug which renders it inactive
and/or confers solubility or some other property to the drug. In
this form, the prodrugs are generally inactive, but once the
chemical group has been cleaved from the prodrug, by heat,
cavitation, pressure, and/or by enzymes in the surrounding
environment or otherwise, the active drug is generated. Such
prodrugs are well described in the art, and comprise a wide variety
of drugs bound to chemical groups through bonds such as esters to
short, medium or long chain aliphatic carbonates, hemiesters of
organic phosphate, pyrophosphate, sulfate, amides, amino acids, azo
bonds, carbamate, phosphamide, glucosiduronate, N-acetylglucosamine
and beta-glucoside. Examples of drugs with the parent molecule and
the reversible modification or linkage are as follows:
convallatoxin with ketals, hydantoin with alkyl esters,
chlorphenesin with glycine or alanins esters, acetaminophen with
caffeine complex, acetylsalicylic acid with THAM salt,
acetylsalicylic acid with acetamidophenyl ester, naloxone with
sulfateester, 15-methylprostaglandin F sub 2 with methyl ester,
procaine with polyethylene glycol, erythromycin with alkyl esters,
clindamycin with alkylesters or phosphate esters, tetracycline with
betains salts, 7-acylaminocephalosporins with ring-substituted
acyloxybenzyl esters, nandrolone with phenylproprionate decanoate
esters, estradiol with enolether acetal, methylprednisolone with
acetate esters, testosterone with n-acetylglucosaminide
glucosiduronate (trimethylsilyl) ether, cortisol or prednisolone or
dexamethasone with 21-phosphate esters. Prodrugs may also be
designed as reversible drug derivatives and utilized as modifiers
to enhance drug transport to site-specific tissues. Examples of
parent molecules with reversible modifications or linkages to
influence transport to a site specific tissue and for enhanced
therapeutic effect include isocyanate with haloalkyl nitrosurea,
testosterone with propionateester, methotrexate
(3-5'-dichloromethotrexat- e) with dialkyl esters, cytosine
arabinoside with 5'-acylate, nitrogen mustard
(2,2'-dichloro-N-methyldiethylamine), nitrogen mustard with
aminomethyltetracycline, nitrogen mustard with cholesterol or
estradiol ordehydroepiandrosterone esters and nitrogen mustard with
azobenzene. As one skilled in the art would recognize, a particular
chemical group to modify a given drug may be selected to influence
the partitioning of the drug into either the shell or the interior
of the microbubbles. The bond selected to link the chemical group
to the drug may be selected to have the desired rate of metabolism,
e.g., hydrolysis in the case of ester bonds in the presence of
serum esterases after release from the microbubbles. Additionally,
the particular chemical group may be selected to influence the
biodistribution of the drug employed in the microbubbles, e.g.,
N,N-bis(2-chloroethyl)-phosphorodiamidicacid with cyclic
phosphoramide for ovarian adenocarcinoma. Additionally, the
prodrugs employed within the microbubbles may be designed to
contain reversible derivatives which are utilized as modifiers of
duration of activity to provide, prolong or depot action effects.
For example, nicotinic acid may be modified with dextran and
carboxymethlydextran esters, streptomycin with alginic acid salt,
dihydrostreptomycin with pamoate salt, cytarabine (ara-C) with
5'-adamantoats ester, ara-adenosine (ara-A) with 5-palmirate and
5'-benzoate esters, amphotericin B with methyl esters, testosterone
with 17-beta-alkyl esters, estradiol with formate ester,
prostaglandin with 2-(4-imidazolyl) ethylamine salt, dopamine with
amino acid amides, chloramphenicol with mono- and
bis(trimethylsilyl) ethers, and cycloguanil with pamoate salt. In
this form, a depot or reservoir of long-acting drug may be released
in vivo from the prodrug bearing microbubbles. In addition,
compounds which are generally thermally labile may be utilized to
create toxic free radical compounds. Compounds with azolinkages,
peroxides and disulfide linkages which decompose with high
temperature are preferred. With this form of prodrug, azo, peroxide
or disulfide bond containing compounds are activated by cavitation
and/or increased heating caused by the interaction of ultra with
the microbubbles to create cascades of free radicals from these
prodrugs entrapped therein. A wide variety of drugs or chemicals
may constitute these prodrugs, such as azo compounds, the general
structure of such compounds being R--N.dbd.N--R, wherein R is a
hydrocarbon chain, where the double bond between the two nitrogen
atoms may react to create free radical products in vivo. Exemplary
drugs or compounds which may be used to create free radical
products include azo containing compounds such as
azobenzene,2,2'-azobisisobutyronitrile, azodicarbonamide,
azolitmin, azomycin, azosemide, azosulfamide, azoxybenzene,
aztreonam, sudan II, sulfachrysoidine, sulfamidochrysoidine and
sulfasalazine, compounds containing disulfide bonds such as
sulbentine, thiamine disulfide, thiolutin, thiram, compounds
containing peroxides such as hydrogen peroxide and benzoylperoxide,
2,2'-azobisisobutyronitrile, 2,2'-azobis(2-amidopropane)
dihydrochloride, and 2,2'-azobis(2,4-dimethylvaleronitrile). A
microbubble having oxygen gas on its interior should create
extensive free radicals with cavitation. Also, metal ions from the
transition series, especially manganese, iron and copper can
increase the rate of formation of reactive oxygen intermediates
from oxygen. By including metal ions within the microbubbles, the
formation of free radicals in vivo can be increased. These metal
ions may be incorporated into the microbubbles as freesalts, as
complexes, e.g., with EDTA, DTPA, DOTA or desferrioxamine, or
asoxides of the metal ions. Additionally, derivatized complexes of
the metal ions may be bound to lipid head groups, or lipophilic
complexes of the ions may be incorporated into a lipid bilayer, for
example. When exposed to thermal stimulation, e.g., cavitation,
these metal ions then will increase the rate of formation of
reactive oxygen intermediates. Further, radiosensitizers such as
metronidazole and misonidazole may be incorporated into the
gas-filled liposomes to create free radicals on thermal
stimulation. By way of an example of the use of prodrugs, an
acylated chemical group may be bound to a drug via an ester linkage
which would readily cleave in vivo by enzymatic action in serum.
The acylated prodrug can be included in the microbubble. When the
microbubble is ruptured, the prodrug will then be exposed to the
serum. The ester linkage is then cleaved by esterases in the serum,
thereby generating the drug. Similarly, ultrasound may be utilized
not only to activate the light activated drug so as to burst the
gas-filled liposome, but also to cause thermal effects which may
increase the rate of the chemical cleavage and the release of the
active drug from the prodrug. The microbubbles may also be designed
so that there is a symmetric or an asymmetric distribution of the
therapeutic both inside and outside of the microbubble. The
particular chemical structure of the therapeutics may be selected
or modified to achieve desired solubility such that the therapeutic
may either be encapsulated within the interior of the microbubble
or couple with the shell of the microbubble. The shell-bound
therapeutic may bear one or more acyl chains such that, when the
microbubble is popped or heated or ruptured via cavitation, the
acylated therapeutic may then leave the surface and/or the
therapeutic may be cleaved from the acyl chains chemical group.
Similarly, other therapeutics may be formulated with a hydrophobic
group which is aromatic or sterol in structure to incorporate into
the surface of the microbubble.
[0123] When the microbubble is a liposome, the liposomes can be
"fast breaking". In fast breaking liposomes, the light activated
drug-liposome combination is stable in vitro but, when administered
in vivo, the light activated drug is rapidly released into the
bloodstream where it can associate with serum lipoproteins. As a
result, the localized delivery of liposomes combined with the fast
breaking nature of the liposomes can result in localization of the
light activated drug and/or the therapeutic in the tissues near the
catheter. Further, the fast breaking liposomes can prevent the
liposomes from leaving the vicinity of the catheter intact and then
concentrating in non-targeted tissues such as the liver. Delivery
of ultrasound energy from the catheter can also serve to break
apart the liposomes after they have been delivered from the
catheter.
[0124] A catheter for locally delivering a media including a light
activated drug includes an elongated body with at least one utility
lumen extending through the elongated body. The utility lumens can
be used to deliver the media including the light activated drug
locally to a tissue site and/or to receive a guidewire so the
catheter can be guided to the tissue site. The ultrasound assembly
can include an ultrasound transducer designed to transmit
ultrasound energy which activates the light activated drug.
[0125] A support member can support the ultrasound transducer
adjacent to an outer surface of the elongated body so as to define
a chamber between the ultrasound transducer and the elongated body.
The chamber can be filled with a material which creates a low
acoustic impedance to reduce the exposure of at least one utility
lumen within the elongated body to ultrasound energy delivered from
the ultrasound transducer. For instance, the chamber can be filled
with a material which absorbs, reflects or prevents transmission of
ultrasound energy through the chamber. Alternatively, the chamber
can be evacuated to reduce transmission of ultrasound energy
through the chamber. Reducing the exposure of at least one lumen to
the ultrasound energy reduces exposure of media delivered through
the at least one lumen to the ultrasound energy. As a result, the
effect of the ultrasound energy on the light activated drug is
reduced until the light activated drug has been delivered out of
the catheter. Further, ultrasound energy is known to rupture
microbubbles. As a result, when the media includes microbubbles,
the chamber reduces the opportunity for the ultrasound energy to
rupture the microbubbles within the catheter.
[0126] The support member can have ends which extend beyond the
ultrasound member. As a result, the chamber can be positioned
adjacent to the entire longitudinal length of the ultrasound
transducer and can extend beyond the ends of the ultrasound
transducer. This configuration maximizes the portion of the
ultrasound transducer which is adjacent to the chamber. Increasing
the portion of ultrasound transducer adjacent to the chamber can
reduce the amount of ultrasound energy transmitted to the utility
lumens. The ultrasound assembly can include an outer coating over
the ultrasound transducer. Temperature sensors can be positioned in
the outer coating adjacent to ultrasound transducer. The
temperature sensors feed back information regarding the temperature
adjacent to the ultrasound transducers where the thermal energy has
a reduced opportunity to dissipate. As a result, the temperature
sensors provide a measure of the temperature on the exterior
surface of the transducer.
[0127] FIGS. 1A-1B illustrates a catheter 10 for delivering a media
including a light activated drug to a tissue site. The catheter 10
includes an ultrasound assembly 12 for delivering ultrasound energy
to light activated drug within the tissue site. The catheter 10
includes an elongated body 14 with a utility lumen 16 extending
through the elongated body 14. The utility lumen 16 can receive a
guidewire (not shown) so the catheter 10 can be threaded along the
guidewire. The utility lumen 16 can also be used for the delivering
media which include a light activated drug. A fiber optic can also
be positioned in the utility lumen 16 to provide a view of the
tissue site or to provide light to the tissue site. As a result,
the catheter can also be used as an endoscope.
[0128] The ultrasound assembly 12 can also include an outer coating
18. Suitable outer coatings 18 include, but are not limited to,
polyimide, parylene and polyester. An ultrasound transducer 20 is
positioned within the outer coating 18. Suitable ultrasound
transducers 20 include, but are not limited to, PZT-4D, PZT-4,
PZT-8 and cylindrically shaped piezoceramics. When the ultrasound
transducer 20 has a cylindrical shape, the ultrasound transducer 20
can encircle the elongated body 14 as illustrated in FIG. 1C. One
or more temperature sensors 22 can be positioned in the outer
coating 18. The temperature sensors 22 can be positioned adjacent
to the ultrasound transducer 20 to provide feedback regarding the
temperature adjacent to the ultrasound transducer 20. The
temperature sensors can be in electrical communication with an
electrical coupling 24. The electrical coupling 24 can be coupled
with a feedback control system (not shown) which adjusts the level
of the ultrasound energy delivered from the ultrasound transducer
20 in response to the temperature at the temperature sensors
22.
[0129] The catheter 10 can include a perfusion lumen 25. The
perfusion lumen 25 allows fluid to flow from outside the catheter
into the utility lumen 16. Once a guidewire has been removed from
the utility lumen 16, fluid flow which is obstructed by the
ultrasound assembly can continue through the perfusion lumen 25 and
the utility lumen. As illustrated in FIGS. 2A-2B, the ultrasound
assembly 12 can be flush with the elongated body 14. Further, the
ultrasound transducer 20 and the temperature sensors 22 can be
positioned within the elongated body 14. This configuration of
elongated body 14 and ultrasound transducer 20 can eliminate the
need for the outer coating 18 illustrated in FIGS. 1A-1C.
[0130] As illustrated in FIG. 3A, the catheter 10 can also include
a media delivery port 26, a media inlet port 28 and a second
utility lumen 16A. The media inlet port 28 is designed to be
coupled with a media source (not shown). Media can be transported
from the media source and through the media delivery port 26 via
the second utility lumen 16A. As a result, a guidewire can be left
within the utility lumen 16 while media is delivered via the second
utility lumen 16A.
[0131] FIG. 4A illustrates a catheter 10 including a plurality of
ultrasound assemblies 12. FIGS. 4B-4C are cross sections of a
catheter 10 with a second utility lumen 16A coupled with the media
delivery ports 26. The second utility lumen 16A can also be coupled
with the media inlet port 28 illustrated in FIG. 4A. The media
inlet port 28 is designed to be coupled with a media source (not
shown). Media can be transported from the media source and through
the media delivery ports 26 via the second utility lumen 16A.
[0132] The catheter 10 can include a balloon 30 as illustrated in
FIG. 5A. The balloon 30 can be constructed from an impermeable
material or a permeable membrane or a selectively permeable
membrane which allows certain media to flow through the membrane
while preventing other media from flowing through the membrane.
Suitable membranous materials for the balloon 30 include, but are
not limited to cellulose, cellulose acetate, polyvinylchloride,
polyolefin, polyurethane and polysulfone. When the balloon 30 is
constructed from a permeable membrane or a selectively permeable
membrane, the membrane pore sizes are preferably 5 A-2 .mu.m, more
preferably 50 A-900 A and most preferably 100 A-300 A in
diameter.
[0133] As illustrated in FIG. 5B, an ultrasound assembly 12, a
first media delivery port 26A and a second media delivery port 26B
can be positioned within the balloon 30. The first and second media
delivery ports 26A, 26B are coupled with a second utility lumen 16A
and third utility lumen 16B. The second and third utility lumens
16A, 16B can be coupled with the same media inlet port 28 or with
independent media inlet ports 28. When the first and second media
delivery ports 26A, 26B are coupled with different media inlet
ports 28, different media can be delivered via the second and third
media delivery ports 26A, 26B. For instance, a medication media can
be delivered via the third utility lumen 16B and an expansion media
can be delivered via the second utility lumen 16A. The medication
media can include drugs or other medicaments which can provide a
therapeutic effect. The expansion media can serve to expand the
balloon 30 or wet the membrane comprising the balloon 30. Wetting
the membrane comprising the balloon 30 can cause a minimally
permeable membrane to become permeable.
[0134] The ultrasound assembly 12 can be positioned outside the
balloon 30 as illustrated in FIGS. 6A-6C. In FIG. 6A the balloon 30
is positioned distally of the ultrasound assembly 12 and in FIG. 6B
the ultrasound assembly 12 is positioned distally of the balloon
30. FIG. 6C is a cross section a catheter 10 with an ultrasound
assembly 12 positioned outside the balloon 30. The catheter
includes a second utility lumen 16A coupled with a first media
delivery port 26A. The second utility lumen 16A can be used to
deliver an expansion media and/or a medication media to the balloon
30. When the balloon 30 is constructed from a permeable membrane,
the medication media and/or the expansion media can pass through
the balloon 30. Similarly, when the balloon 30 is constructed from
a selectively permeable membrane, particular components of the
medication media and/or the expansion media can pass through the
balloon 30. Pressure can be used to drive the media or components
of the media across the balloon 30. Other means such as phoresis
can also be used to drive the media or components of the media
across the balloon 30.
[0135] As illustrated in FIG. 6C, the ultrasound assembly 12 may be
positioned at the distal end of the catheter 10. The second utility
lumen 16A can be used to deliver an expansion media and/or a
medication media to the balloon 30. The utility lumen 16 can be
used to deliver a medication media as well as to guide the catheter
10 along a guidewire.
[0136] As illustrated in FIGS. 7A-7C, the catheter 10 can include a
second media delivery port 26B positioned outside the balloon. In
FIGS. 7A-7C the ultrasound assembly 12 and the second media
delivery port 26B are positioned distally relative to a balloon 30,
however, the balloon 30 can be positioned distally relative to the
ultrasound assembly 12 and the second media delivery port 26B. In
FIG. 7A the ultrasound assembly 12 is positioned distally of the
second media delivery port 26B and in FIG. 7B the second media
delivery port 26B is positioned distally of the ultrasound assembly
12.
[0137] FIG. 7C is a cross section of the catheter 10 illustrated in
FIG. 7A. The catheter 10 includes first and second media delivery
ports 26A, 26B coupled with a second utility lumen 16A and third
utility lumen 16B. The second and third utility lumens 16A, 16B can
be coupled with independent media inlet ports 28 (not shown). The
second utility lumen 16A can be used to deliver an expansion media
and/or a medication media to the balloon 30 while the third utility
lumen 16B can be used to deliver a medication media through the
second media delivery port 26B.
[0138] As illustrated in FIGS. 8A-8B, the catheter 10 can include a
first balloon 30A and a second balloon 30B. The ultrasound assembly
12 can be positioned between the first and second balloons 30A,
30B. A second media delivery port 26B can optionally be positioned
between the first and second balloons 30A, 30B. In FIG. 8A the
second media delivery port 26B is positioned distally relative to
the ultrasound assembly and in FIG. 8B the ultrasound assembly is
positioned distally relative to the second media delivery port
26B.
[0139] FIG. 8C is a cross section of the first balloon 30A
illustrated in FIG. 8B. The catheter includes a second, third and
fourth utility lumens 16A, 16B, 16C. The second utility lumen 16A
is coupled with a first media delivery port 26A within the first
balloon. The third utility lumen 16B is coupled with the second
media delivery port 26B and the fourth utility lumen 16C is coupled
with a third media delivery port 26C in the second balloon 30B (not
shown). The second and fourth utility lumens 16A, 16C can be used
to deliver expansion media and/or medication media to the first and
second balloon 30A, 30B. The second and fourth utility lumens 16A,
16C can be coupled with the same media inlet port or with
independent media inlet ports (not shown). When the second and
fourth utility lumens are coupled with the same media inlet port,
the pressure within the first and second balloons 30A, 30B will be
similar. When the second and fourth utility lumens are coupled with
independent media inlet ports, different pressures can be created
within the first and second balloons 30A, 30B. The third utility
lumen 16B can be coupled with an independent media inlet port and
can be used to deliver a medication media via the second media
delivery port 26B.
[0140] FIGS. 9A-9I illustrate operation of various embodiments of
catheters 10 for delivering ultrasound energy to a light activated
drug within a tissue site. FIGS. 9A-9I illustrate the tissue site
32 as an atheroma in a vessel 34, however, it is contemplated that
the catheter 10 can be used with other tissue sites 32 such as a
tumor and that the catheter 10 can be positioned within the
vasculature of the tumor. In each of FIGS. 9A-9I, the catheter 10
is illustrated as being within a vessel 34. The catheter 10 can be
positioned within the vessel 34 by applying conventional
over-the-guidewire techniques and can be verified by including
radiopaque markers upon the catheter 10.
[0141] In FIG. 9A, the catheter 10 is positioned so the ultrasound
assembly 12 is adjacent to a tissue site 32 within a vessel 34.
When the catheter 10 is in position, the guidewire is removed from
the utility lumen 16 and media can be delivered via the utility
lumen 16 as illustrated by the arrows 36. In FIG. 9A, the media
includes microbubbles 38 but can alternatively be an emulsion. The
media is delivered to the tissue site 32 via the utility lumen 16
and ultrasound energy 40 is delivered from the ultrasound assembly
12. Suitable periods for delivering the ultrasound energy include.,
but are not limited to, 1 minute to three hours, 2 minutes to one
hour and 10-30 minutes.
[0142] Suitable intensities for the ultrasound energy include, but
are not limited to, 0.1-1000 W/cm.sup.2, 1-100 W/cm.sup.2 and 10-50
W/cm.sup.2. Suitable frequencies for the ultrasound energy include,
but are not lmited to, 10 kHz-100 MHz and 10 kHz-50 MHz but is
preferably 20 kHz-10 MHz. Suitable ultrasound energies also
include, but are not limited to 0.02 to 10 w/cm at a frequency of
about 20 KHz to about 10 MHz and more preferably about 0.3
W/cm.sup.2 at a frequency of about 1.3 MHz. The ultrasound energy
can be intermittently switched between a first and second frequency
to increase the efficiency of microbubble rupture and to increase
activation of the light activated drug. For instance, the
ultrasound energy can be switched between about 100 kHz and about
270 kHz in short pulses of approximately 0.001-10 seconds duration.
Similarly, the ultrasound energy can be switched between first and
second intensities. When the catheter includes a plurality of
ultrasound transducers as will be discussed below, the first and
second frequencies can be provided by different ultrasound
transducers. Similarly, the first and second intensities can be
provided by different ultrasound transducers. Further, when the
catheter includes a plurality of ultrasound transducers each
transducer can simultaneously transmit ultrasound energy with
different intensity and/or frequency.
[0143] The delivery of ultrasound energy 40 can be before, after,
during or intermittently with the delivery of the microbubbles 38.
As discussed above, the microbubbles 38 can be "fast breaking" so
they rupture upon exiting the utility lumen and being exposed to
the vessel 34. As described above, the ultrasound energy from the
ultrasound assembly 12 can cause the microbubbles 38 within the
delivered media to rupture. As will be described in more detail
below, the ultrasound assembly can be designed to reduce the
exposure of media within the catheter 10 to the ultrasound energy
from the ultrasound assembly 12. When the catheter 10 is so
designed, the number of microbubbles 38 which rupture within the
catheter is reduced and the number of microbubbles 38 which rupture
outside the catheter is increased.
[0144] Delivery of the ultrasound energy before delivery of the
light activated drug can enhance absorption of the light activated
drug into the tissue site. Delivery of the ultrasound energy a
pre-determined time after delivery of the light activated drug can
provide the light activated drug time to penetrate the tissue site.
The pre-determined time can be of sufficient duration that at least
a portion of the light activated drug penetrates into the tissue
site. The pre-determined time can also be of sufficient duration
that the light activated drug localizes within the lipid rich
tissue of the atheroma. Sufficient time between delivery of the
media and the ultrasound energy include but are not limited to, 1
minute to 48 hours, 1 minute to 3 hours, 1 to 15 minutes and 1 to 2
minutes. Once the light activated drug has penetrated the tissue
site 32, the ultrasound energy from the ultrasound assembly 12 can
activate the light activated dug within the tissue site 32 so as to
cause tissue death within the tissue site 32.
[0145] In FIG. 9B, ultrasound energy 40 is delivered from the
ultrasound transducer 20 and a media is delivered through the media
delivery port 26 as illustrated by the arrows 36. The delivery of
ultrasound energy 40 can be before, after, during or intermittently
with the delivery of the media via the media delivery port 26. As
illustrated in FIG. 9C, the guidewire 104 can remain in the utility
lumen 16 during the delivery of the media via the media delivery
ports 26. As will be discussed in further detail below, the
ultrasound assembly can be designed to reduce the transmission of
the ultrasound energy into the utility lumen. Because the
transmission of ultrasound energy 40 into the utility lumen 16 is
reduced, the change in the frequency of the ultrasound transducer
20 which is due to the presence of the guidewire in the utility
lumen 16 is also reduced.
[0146] In FIG. 9D, a catheter 10 including a balloon 30 is
positioned with the balloon adjacent to the tissue site 32. In FIG.
9E, the balloon 30 is expanded into contact with the tissue site
32. As discussed above, the catheter 10 can include a perfusion
lumen which permits a continuous flow of fluid from the vessel
through the utility lumen during the partial or full obstruction of
the vessel by the balloon. When the balloon 30 is constructed from
a membrane or a selectively permeable membrane a media can be
delivered to the tissue site 32 via the balloon 30. The media can
serve to wet the membrane or can include a drug or other medicament
which provides a therapeutic effect. Ultrasound energy 40 can be
delivered from the ultrasound assembly 12 before, after, during or
intermittently with the delivery of the media. The ultrasound
energy 40 can serve to drive the media across the membrane via
phonophoresis or can enhance the therapeutic effect of the
media.
[0147] In FIG. 9F a catheter 10 with an ultrasound assembly 12
outside a balloon 30 is positioned at the tissue site 32 so the
ultrasound assembly 12 is adjacent to the tissue site 32. A fluid
within the vessel flows past the balloon as indicated by the arrow
42. In FIG. 9G, the balloon 30 is expanded into contact with the
vessel 34. The balloon 30 can be constructed from an impermeable
material so the vessel 34 is occluded. As a result, the fluid flow
through the vessel 34 is reduced or stopped. A medication media is
delivered through the utility lumen 16 and ultrasound energy 40 is
delivered from the ultrasound assembly 12. In embodiments of the
catheter 10 including a media delivery port 26 outside of the
balloon 30 (i.e. FIGS. 7A-7C), the medication media can be
delivered via the media delivery port 26. Further, a first
medication media can be delivered via the media delivery port 26
while a second medication media can be delivered via the utility
lumen 16 or while a guidewire is positioned within the utility
lumen 16. The ultrasound energy 40 can be delivered from the
ultrasound assembly 12 before, after, during or intermittently with
the delivery of the media. The occlusion of the vessel 34 before
the delivery of the media can serve to prevent the media from being
swept from the tissue site 32 by the fluid flow. Although the
balloon 30 illustrated in FIGS. 9F-9G is positioned proximally
relative to the ultrasound assembly 12, the fluid flow through the
vessel 34 can also be reduced by expanding a single balloon 30
which is positioned distally relative to the ultrasound assembly
12.
[0148] In FIG. 9H a catheter 10 including a first balloon 30A and a
second balloon 30B is positioned at a tissue site 32 so the
ultrasound assembly 12 is positioned adjacent to the tissue site
32. A fluid within the vessel 34 flows past the balloon 30 as
indicated by the arrow 42. In FIG. 9I, the first and second
balloons 30A, 30B are expanded into contact with the vessel 34. The
first and second balloons 30A, 30B can be constructed from an
impermeable material so the vessel 34 is occluded proximally and
distally of the ultrasound assembly 12. As a result, the fluid flow
adjacent to the tissue site 32 is reduced or stopped. A medication
media is delivered through the media delivery port 26 and
ultrasound energy 40 is delivered from the ultrasound assembly 12.
The ultrasound energy 40 can be delivered from the ultrasound
assembly 12 before, after, during or intermittently with the
delivery of the media. The occlusion of the vessel 34 before the
delivery of the media can serve to prevent the media from being
swept from the tissue site 32 by the fluid flow.
[0149] In each of the FIGS. 9A-9I illustrated above, the media can
be systemically delivered. The catheter 10 is positioned adjacent
to the tissue site before, after or during the systemic
administration of the media. When the media includes microbubbles
which must be burst before their therapeutic effect can be
obtained, the ultrasound energy can be delivered after the
microbubbles have had sufficient time to reach the desired tissue
site in sufficient concentrations. A level of ultrasound which
ruptures the microbubbles is then delivered from the ultrasound
assembly. After rupture of the microbubbles, the delivery of
ultrasound energy can be stopped to provide the light activated
drug or other therapeutic time to penetrate the tissue site. The
delivery of the ultrasound energy can also be continuous to
maximize the number of microbubbles which are burst.
[0150] When the media is systemically delivered and the light
activated drug is included in media which does' not require an
ultrasound activated release, the behavior of the light activated
drug within the patient must be taken into consideration. As
described above, many light drugs such as the macrocycles,
initially disperse throughout the body and where they are taken up
by most tissues. After a period of time, usually between 3 and 48
hours, the drug clears from most normal tissue and is retained to a
greater degree in lipid rich regions such as the liver, kidney,
tumor and atheroma. As a result, when the tissue site is not a
lipid rich region, the ultrasound energy should be delivered to the
tissue site within 3 to 48 hours of systemically administering the
media. However, when the tissue site is lipid rich, improved
results can be achieved by waiting 3 to 48 hours after systemic
administration of the media before delivering the ultrasound
energy.
[0151] FIG. 10A provides a cross section of an ultrasound assembly
which reduces transmission of ultrasound energy from the ultrasound
transducer into the catheter. The ultrasound assembly 12 includes a
support member 44. Suitable support members 44 include, but are not
limited to, polyimide, polyester and nylon. The support member 44
can be attached to the ultrasound transducer 20. Suitable means for
attaching the ultrasound transducer 20 to the support member 44
include, but are not limited to, adhesive bonding and thermal
bonding.
[0152] The support member 44 supports the ultrasound transducer 20
at an external surface 46 of the elongated body 14 such that a
chamber 48 is defined between the ultrasound transducer 20 and the
external surface 46 of the elongated body 14. The chamber 48
preferably has a height from 0.25-10 .mu.m, more preferably from
0.50-5 .mu.m and most preferably from 0.0-1.5 .mu.m. The support
member 44 can be supported by supports 50 positioned at the ends 52
of the support member 44 as illustrated in FIG. 10A. The supports
50 can be integral with the support member 44 as illustrated in
FIG. 10C. The outer coating 18 can serve as the supports as
illustrated in FIG. 10D.
[0153] The ends 52 of the support member 44 can extend beyond the
ends 54 of the ultrasound transducer 20. The supports 50 can be
positioned beyond the ends 54 of the ultrasound transducer 20. As a
result, the chamber 48 can extend along the longitudinal length 56
of the ultrasound transducer 20, maximizing the portion of the
ultrasound transducer 20 which is adjacent to the chamber 48. The
chamber 48 can be filled with a medium which absorbs ultrasound
energy or which prevents transmission of ultrasound energy.
Suitable gaseous media for filling the chamber 48 include, but are
not limited to, helium, argon, air and nitrogen. Suitable solid
media for filling the chamber 48 include, but are not limited to,
silicon and rubber. The chamber 48 can also be evacuated. Suitable
pressures for an evacuated chamber 48 include, but are not limited
to, negative pressures to -760 mm Hg.
[0154] The ultrasound assembly can include a second ultrasound
transducer 20A as illustrated in FIGS. 11A-11H. In FIGS. 11A-11C
one ultrasound transducer encircles the other and in FIGS. 11D-11H
the ultrasound transducers are longitudinally adjacent to one
another. The ultrasound transducers 20, 20A can be in contact with
one another as illustrated in FIGS. 11A, 11E and 11H or separated
from one another as illustrated in FIGS. 11B-11D, 11F and 11G. A
single chamber 54 can be defined between the ultrasound transducers
20, 20A and the external surface 46 of the elongated body 14 as
illustrated in FIGS. 11C, 11F and 11G or a different chamber can be
defined between each of the ultrasound transducers 20, 20A and the
external surface 46. Although the ultrasound transducers 20, 20A in
FIGS. 11A-11C are illustrated as having the same longitudinal
length, the longitudinal length may be different.
[0155] In FIGS. 11A-11H, the different temperature sensors can be
positioned adjacent to different ultrasound transducers 20, 20A. As
a result, the temperature adjacent to different ultrasound
transducers 20, 20A can be detected and the level of ultrasound
energy produced by each ultrasound transducer adjusted in response
to the detected temperature.
[0156] When the ultrasound assembly includes a second transducer
20A, the transducers 20, 20A may be constructed from the same or
different materials. Both transducers 20, 20A may be configured to
radiate ultrasound energy in the same direction. Further, one
transducer may be configured to transmit ultrasound energy in a
radial direction and the other in a longitudinal direction in order
to increase the angular spectrum over which ultrasound energy can
be simultaneously transmitted. The ultrasound transducers can be
configured to transmit ultrasound energy having the same or
different characteristics. The transmission of ultrasound energy
with different characteristics allows the same ultrasound
assemblies to be used to perform different functions. For instance,
one ultrasound transducer can transmit a frequency which is
appropriate for activating a light activated drug while the second
ultrasound transducer transmits a frequency appropriate for
enhancing penetration of a therapeutic agent into the treatment
site. The transducers can be operated independently or
simultaneously. When the transducers are operated simultaneously,
the ultrasound assembly produces a waveform which is more complex
than a single ultrasound transducer. More complex waveforms can
provide advantages such as more efficient rupture of microbubbles.
It is also contemplated that the ultrasound assembly can include
three or more ultrasound transducers arranged similar to the
transducers illustrated in FIGS. 11A-11H.
[0157] The ultrasound assembly 12 can be a separate module 58 as
illustrated in FIGS. 12A-12B. In FIG. 12A, the catheter 10 includes
a first catheter component 60 a second catheter component 62 and an
ultrasound assembly module 58. The first and second catheter
components 60, 62 include component ends 64 which are complementary
to the ultrasound assembly module ends 66. The component ends 64
can be coupled with the ultrasound assembly module ends 66 as
illustrated in FIG. 12B. Suitable means for coupling the component
ends 64 and the ultrasound assembly module ends 66 include, but are
not limited to, adhesive, mechanical and thermal methods. The
ultrasound assembly 12 can be integral with the catheter 10 as
illustrated in FIG. 12C. Further, the outer coating 18 can have a
diameter which is larger than the diameter of the elongated body 14
as illustrated in FIG. 10A or can be flush with the external
surface 46 of the elongated body 14 as illustrated in FIGS.
12A-12C.
[0158] The ultrasound assembly 12 can be electrically coupled to
produce radial vibrations of the ultrasound transducer 20 as
illustrated in FIGS. 13A-13B. A first line 68 is coupled with an
outer surface 70 of the ultrasound transducer 20 while a second
line 72 is coupled with an inner surface 74 of the ultrasound
transducer 20. The first and second lines 68, 72 can pass
proximally through the utility lumen 16 as illustrated in FIG. 13A.
Alternatively, the first and second lines 68, 72 can pass
proximally through line lumens 76 within the catheter 10 as
illustrated in FIG. 13B. Suitable lines for the ultrasound
transducer 20 include, but are not limited to, copper, gold and
aluminum. Suitable frequencies for the ultrasound energy delivered
by the ultrasound transducer 20 include, but are not limited to, 20
KHz to 2 MHz.
[0159] The ultrasound assembly 12 can be electrically coupled to
produce longitudinal vibrations of the ultrasound transducer 20 as
illustrated in FIGS. 13C-13D. A first line 68 is coupled with a
first end 78 of the ultrasound transducer 20 while a second line 72
is coupled with a second end 80 of the ultrasound transducer 20.
The distal portion 82 of the second line 72 can pass through the
outer coating 18 as illustrated in FIG. 13C. Alternatively, the
distal portion 82 of the second line 72 can pass through line
lumens 76 in the catheter 10 as illustrated in FIG. 13D. As
discussed above, the first and second lines 68, 72 can pass
proximally through the utility lumen 16.
[0160] As discussed above, the catheter 10 can include a plurality
of ultrasound assemblies. When the catheter 10 includes a plurality
of ultrasound assemblies, each ultrasound transducer 20 can each be
individually powered. When the elongated body 14 includes N
ultrasound transducers 20, the elongated body 14 must include 2N
lines to individually power N ultrasound transducers 20. The
individual ultrasound transducers 20 can also be electrically
coupled in serial or in parallel as illustrated in FIGS. 14A-14B.
These arrangements permit maximum flexibility as they require only
2 lines. Each of the ultrasound transducers 20 receive power
simultaneously whether the ultrasound transducers 20 are in series
or in parallel. When the ultrasound transducers 20 are in series,
less current is required to produce the same power from each
ultrasound transducer 20 than when the ultrasound transducers 20
are connected in parallel. The reduced current allows smaller lines
to be used to provide power to the ultrasound transducers 20 and
accordingly increases the flexibility of the elongated body 14.
When the ultrasound transducers 20 are connected in parallel, an
ultrasound transducer 20 can break down and the remaining
ultrasound transducers 20 will continue to operate.
[0161] As illustrated in FIG. 14C, a common line 84 can provide
power to each ultrasound transducer 20 while each ultrasound
transducer 20 has its own return line 86. A particular ultrasound
transducer 20 can be individually activated by closing a switch 88
to complete a circuit between the common line 84 and the particular
ultrasound transducer's 20 return line 86. Once a switch 88
corresponding to a particular ultrasound transducer 20 has been
closed, the amount of power supplied to the ultrasound transducer
20 can be adjusted with the corresponding potentiometer 90.
Accordingly, an catheter 10 with N ultrasound transducers 20
requires only N+1 lines and still permits independent control of
the ultrasound transducers 20. This reduced number of lines
increases the flexibility of the catheter 10. To improve the
flexibility of the catheter 10, the individual return lines 86 can
have diameters which are smaller than the common line 84 diameter.
For instance, in an embodiment where N ultrasound transducers 20
will be powered simultaneously, the diameter of the individual
return lines 86 can be the square root of N times smaller than the
diameter of the common line 84.
[0162] As discussed above, the ultrasound assembly 12 can include
at least one temperature sensor 22. Suitable temperature sensors 22
include, but are not limited to, thermistors, thermocouples,
resistance temperature detectors (RTD)s, and fiber optic
temperature sensors 22 which use thermalchromic liquid crystals.
Suitable temperature sensor geometries include, but are not limited
to, a point, patch, stripe and a band encircling the ultrasound
transducer 20.
[0163] When the ultrasound assembly 12 includes a plurality of
temperature sensors 22, the temperature sensors 22 can be
electrically connected as illustrated in FIG. 15. Each temperature
sensor 22 can be coupled with a common line 84 and then include its
own return line 86. Accordingly, N+1 lines can be used to
independently sense the temperature at the temperature sensors 22
when N temperature sensors 22 are employed. A suitable common line
84 can be constructed from Constantine and suitable return lines 86
can be constructed from copper. The temperature at a particular
temperature sensor 22 can be determined by closing a switch 88 to
complete a circuit between the thermocouple's return line 86 and
the common line 84. When the temperature sensors 22 are
thermocouples, the temperature can be calculated from the voltage
in the circuit. To improve the flexibility of the catheter 10, the
individual return lines 86 can have diameters which are smaller
than the common line 84 diameter.
[0164] Each temperature sensor 22 can also be independently
electrically coupled. Employing N independently electrically
coupled temperature sensors 22 requires 2N lines to pass the length
of the catheter 10.
[0165] The catheter 10 flexibility can also be improved by using
fiber optic based temperature sensors 22. The flexibility can be
improved because only N fiber optics need to be employed sense the
temperature at N temperature sensors 22.
[0166] The catheter 10 can be coupled with a feedback control
system as illustrated in FIG. 16. The temperature at each
temperature sensor 22 is monitored and the output power of an
energy source adjusted accordingly. The physician can, if desired,
override the closed or open loop system.
[0167] The feedback control system includes an energy source 92,
power circuits 94 and a power calculation device 96 coupled with
each ultrasound transducer 20. A temperature measurement device 98
is coupled with the temperature sensors 22 on the catheter 10. A
processing unit 100 is coupled with the power calculation device
96, the power circuits 94 and a user interface and display 102.
[0168] In operation, the temperature at each temperature sensor 22
is determined at the temperature measurement device 98. The
processing unit 100 receives signals indicating the determined
temperatures from the temperature measurement device 98. The
determined temperatures can then be displayed to the user at the
user interface and display 102.
[0169] The processing unit 100 includes logic for generating a
temperature control signal. The temperature control signal is
proportional to the difference between the measured temperature and
a desired temperature. The desired temperature can be determined by
the user. The user can set the predetermined temperature at the
user interface and display 102.
[0170] The temperature control signal is received by the power
circuits 94. The power circuits 94 adjust the power level of the
energy supplied to the ultrasound transducers 20 from the energy
source 92. For instance, when the temperature control signal is
above a particular level, the power supplied to a particular
ultrasound transducer 20 is reduced in proportion to the magnitude
of the temperature control signal. Similarly, when the temperature
control signal is below a particular level, the power supplied to a
particular ultrasound transducer 20 is increased in proportion to
the magnitude of the temperature control signal. After each power
adjustment, the processing unit 100 monitors the temperature
sensors 22 and produces another temperature control signal which is
received by the power circuits 94.
[0171] The processing unit 100 can also include safety control
logic. The safety control logic detects when the temperature at a
temperature sensor 22 has exceeded a safety threshold. The
processing unit 100 can then provide a temperature control signal
which causes the power circuits 94 to stop the delivery of energy
from the energy source 92 to the ultrasound transducers 20.
[0172] The processing unit 100 also receives a power signal from
the power calculation device 96. The power signal can be used to
determine the power being received by each ultrasound transducer
20. The determined power can then be displayed to the user on the
user interface and display 102.
[0173] The feedback control system can maintain the tissue adjacent
to the ultrasound transducers 20 within a desired temperature range
for a selected period of time. As described above, the ultrasound
transducers 20 can be electrically connected so each ultrasound
transducer 20 can generate an independent output. The output
maintains a selected energy at each ultrasound transducer 20 for a
selected length of time.
[0174] The processing unit 100 can be a digital or analog
controller, or a computer with software. When the processing unit
100 is a computer it can include a CPU coupled through a system
bus. The user interface and display 102 can be a mouse, keyboard, a
disk drive, or other non-volatile memory systems, a display
monitor, and other peripherals, as are known in the art. Also
coupled to the bus is a program memory and a data memory.
[0175] In lieu of the series of power adjustments described above,
a profile of the power delivered to each ultrasound transducer 20
can be incorporated in the processing unit 100 and a preset amount
of energy to be delivered may also be profiled. The power delivered
to each ultrasound transducer 20 can then be adjusted according to
the profiles.
[0176] The above catheters are suitable for locally delivering a
media including a light activated drug. Suitable light activated
drugs include, but are not limited to, fluorescein, merocyanin.
However, preferred light activated drugs' include xanthene and its
derivatives and the photoreactive pyrrole-derived macrocycles and
their derivatives due to a reduced toxicity and an increased
biological affinity. Suitable photoreactive pyrrole-derived
macrocycles include, but are not limited to, naturally occurring or
synthetic porphyrins, naturally occurring or synthetic chlorins,
naturally occurring or synthetic bacteriochlorins, synthetic
isobateriochlorins, phthalocyanines, naphtalocyanines, and expanded
pyrrole-based macrocyclic systems such as porphycenes, sapphyrins,
and texaphyrins. Examples of suitable pyrrole-based macrocyclic
classes are illustrated in FIGS. 17A-N.
[0177] As described above, the derivative of the pyrrole-based
macrocycle classes can be used. For the purposes of illustrating
some of the derivatives a macrocycle class, FIGS. 17B-2 illustrates
a formula for the derivatives of texaphyrin: where M is H,
CH.sub.3, a divalent metal cation selected from the group
consisting of Ca(II), Mn(II), Co(II), Ni(II), Zn(II), Cd(II),
Hg(II), Fe(II), Sm(II), and UO(II) or a trivalent metal cation
selected from the group consisting of Mn(III), Co(III), Ni(III),
Fe(III), Ho(III), Ce(III), Y(III), In(III), Pr(III), Nd(III),
Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Er(III), Tm(III),
Yb(III), Lu(III), La(III), and U(III). Preferred metals include
Lu(III), Dy(III), Eu(III), or Gd(III). M may be H or CH.sub.3 in a
non-metalated form of texaphyrin. R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 can independently be hydrogen,
hydroxyl, alkyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, saccharide,
carboxyalkyl, carboxyamidealkyl, a site-directing molecule, or a
linker to a site-directing molecule where at least one of R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is hydroxyl,
hydroxyalkoxy, saccharide, alkoxy, carboxyalkyl, carboxyamidealkyl,
hydroxyalkyl, a site-directing molecule or a couple to a
site-directing molecule; and N is an integer less than or equal to
2.
[0178] A preferred paramagnetic metal complex is the Gd(III)
complex of
4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-(2-metho-
xyethoxy)ethoxy]ethoxy-13,20,25,26,27-pentaazapentacyclo
[20.2.1.sup.3,6.1.sup.8,11.0.sup.14,19]heptacosa-1,3,5,7,9,11
(27),12,14(19),15,17,20,22(25),23-tridecaene ("GdT2BET") and a
preferred diamagnetic metal complex is the Lu(III) complex of
4,5-diethyl-10,23dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-met-
hoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapentacyclo[20.2.1.1
.sup.3,6.1.sup.8,11.0.sup.14,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,1-
7,20,22(25),23-tridecaene ("LuT2BET").
[0179] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and R.sub.6 may
also independently be amino, carboxy, carboxamide, ester, amide
sulfonato, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, etheramide
or equivalent formulae conferring the desired properties. In a
preferred embodiment, at least one of R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 is a site-directing molecule or is a
couple to a site-directing molecule. For bulky R groups on the
benzene ring portion of the molecule such as oligonucleotides, one
skilled in the art would realize that derivatization at one
position on the benzene potion is more preferred.
[0180] Hydroxyalkyl means alkyl groups having hydroxyl groups
attached. Alkoxy means alkyl groups attached to an oxygen.
Hydroxyalkoxy means alkyl groups having ether or ester linkages,
hydroxyl groups, substituted hydroxyl groups, carboxyl groups,
substituted carboxyl groups or the like. Saccharide includes
oxidized, reduced or substituted saccharide; hexoses such as
D-glucose, D-mannose or D-galactose; pentoses such as D-ribose or
D-arabinose; ketoses such as D-ribulose or D-fructose;
disaccharides such as sucrose, lactose, or maltose; derivatives
such as acetals, amines, and phosphorylated sugars;
oligosacchrides, as well as open chain forms of various sugars, and
the like. Examples of amine-derivatized sugars are galactosamine,
glucosamine, and sialic acid. Carboxyamidealkyl means alkyl groups
with secondary or tertiary amide linkages or the like. Carboxyalkyl
means alkyl groups having hydroxyl groups, carboxyl or amide
substituted ethers, ester linkages, tertiary amide linkages removed
from the ether or the like.
[0181] For the above-described texaphyrins, hydroxyalkoxy may be
alkyl having independently hydroxy substituents and ether branches
or may be C.sub.(n-x)H.sub.((2n+1)-2x)O.sub.xO.sub.y or
OC.sub.(n-x)H.sub.((2n+1)-2- x)O.sub.xO.sup.y where n is a positive
integer from 1 to 10, x is zero or a positive integer less than or
equal to n, and y is zero or a positive integer less than or equal
to ((2n+1)-2x). The hydroxyalkoxy or saccharide may be
C.sub.nH.sub.((2n+1)-q)O.sub.yR.sup.a.sub.q,
OC.sub.nH.sub.((2n+1)-q)O.sub.yR.sup.a.sub.q or
(CH.sub.2).sub.nCO.sub.2R- .sup.a where n is a positive integer
from 1 to 10, y is zero or a positive integer less than ((2n+1)-q),
q is zero or a positive integer less than or equal to 2n+1, and
R.sup.a is independently H, alkyl, hydroxyalkyl, saccharide,
C.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z,
O.sub.2CC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z or
N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z. In this case, m
is a positive integer from 1 to 10, w is zero or a positive integer
less than or equal to m, z is zero or a positive integer less than
or equal to ((2 m+1)-2w), and R is H, alkyl, hydroxyalkyl, or
C.sub.mH.sub.((2m+1)-r)O.su- b.ZR.sup.b.sub.r where m is a positive
integer from 1 to 10, z is zero or a positive integer less than
((2m+1)-r), r is zero or a positive integer less than or equal to
2m+1, and Rb is independently H, alkyl, hydroxyalkyl, or
saccharide.
[0182] Carboxyamidealkyl may be alkyl having secondary or tertiary
amide linkages or (CH.sub.2).sub.nCONHR.sup.a,
O(CH.sub.2).sub.nCONHR.sup.a, (CH.sub.2).sub.nCON(R.sup.a)2, or
O(CH.sub.2).sub.nCON(R.sup.1)2 where n is a positive integer from 1
to 10, and R is independently H, alkyl, hydroxyalkyl, saccharide,
C.sub.(m-w)H.sub.((2n+1)-2w)O.sub.wO.sub.z,
O.sub.2CC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z,
N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z, or a
site-directing molecule. In this case, m is a positive integer from
1 to 10, w is zero or a positive integer less than or equal to
((2M+1)-2w), and R is H, alkyl, hydroxyalkyl, or
C.sub.mH.sub.((2m+1)-r)O.sub.zR.sup.b.sub.r. In this case, m is a
positive integer from 1 to 10, w is zero or a positive integer less
than or equal to m, z is zero or a positive integer less than or
equal to ((2M+1) r), r is zero or a positive integer less than or
equal to 2 m+1, and Rb is independently H, alkyl, hydroxyalkyl, or
saccharide. In a preferred embodiment, R.sup.a is an
oligonucleotide.
[0183] Carboxyalkyl may be alkyl having a carboxyl substituted
ether, an amide substituted ether or a tertiary amide removed from
an ether or C.sub.nH.sub.((2n+1)-q)O.sub.yR.sup.c.sub.q or
OC.sub.nH.sub.((2n+1)-q)O.- sub.yR.sup.c.sub.q where n is a
positive integer from 1 to 10; y is zero or a positive integer less
than ((2n+1)-q), q is zero or a positive integer less than or equal
to 2n+1, and R.sup.c is (CH.sub.2).sub.nCO.sub.2R.sup.d,
(CH.sub.2).sub.nCOHR.sup.d, (CH.sub.2).sub.nCON(R.sup.d).sub.2 or a
site-directing molecule. In this case, n is a positive integer from
1 to 10, Rd is independently H, alkyl, hydroxyalkyl, saccharide,
C.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z,
O.sub.2CC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z or
N(R)OCC.sub.(m-w)H.sub.((2m+1)-2w)O.sub.wO.sub.z. In this case, m
is a positive integer from 1 to 10, z is zero or a positive integer
less than ((2m+1)-2w), and R is H, alkyl, hydroxyalkyl, or
C.sub.mH.sub.((2m+1)-r)O- .sub.zR.sup.b.sub.r. In this case, m is a
positive integer from 1 to 10, z is zero or a positive integer less
than ((2m+1)-r), r is zero or a positive integer less than or equal
to 2M+1, and R.sup.b is independently H, alkyl, hydroxyalkyl, or
saccharide. In a preferred embodiment, R.sup.c is an
oligonucleotide.
[0184] Exemplary texaphyrins are listed in Table 1.
1TABLE 1 Representative Substitutes for Texaphyrin Macrocycles TXP
R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6 A1
CH.sub.2(CH.sub.2).sub.2OH CH.sub.2CH.sub.3 CH.sub.2CH.sub.3
CH.sub.3 O(CH.sub.2).sub.3OH O(CH.sub.2).sub.3OH A2 " " " "
O(CH.sub.2CH.sub.2O).sub.3CH.sub.3 O(CH.sub.2CH.sub.2O).sub.3C-
H.sub.3 A3 " " " " O(CH.sub.2).sub.nCON- " linker-site- directing
molecule, n = 1-7 A4 " " " " O(CH.sub.2).sub.nCON- H linker-site-
directing molecule A5 " " " " OCH.sub.2CO-hormone " A6 " " " "
O(CH.sub.2CH.sub.2O).sub.3CH.sub.3 " A7 " " " "
OCH.sub.2CON-linker- O(CH.sub.2CH.sub.2O).sub.3CH.sub.3
site-directing molecule A8 " " " " OCH.sub.2CO-hormone " A9 " " " "
O(CH.sub.2CH.sub.2O).sub.120CH.sub.3
O(CH.sub.2CH.sub.2O).sub.3CH.sub.2-- CH.sub.2--N-imidazole A10 " "
" " saccharide H A11 " " " " OCH.sub.2CON-- "
(CH.sub.2CH.sub.2OH).sub.2 A12 " " " " CH.sub.2CON(CH.sub.3)CH-
.sub.2-- (CHOH).sub.4CH.sub.2OH A13 " COOH COOH "
CH.sub.2CON(CH.sub.3)CH.sub.2-- " (CHOH).sub.4CH.sub.2OH A14 "
COOCH.sub.2CH.sub.3 COOCH.sub.2CH.sub.3 " CH.sub.2CON(CH.sub.3)CH-
.sub.2-- " (CHOH).sub.4CH.sub.2OH A15
Ch.sub.2CH.sub.2CON(CH.sub.2CH.sub.2OH).sub.2 CH.sub.2CH.sub.2
CH.sub.2CH.sub.3 " CH.sub.2CON(CH.sub.3)CH.sub.2-- "
(CHOH).sub.4CH.sub.2OH A16 CH.sub.2CH.sub.2ON(CH.sub.3)CH.sub.2-- "
" " OCH.sub.3 OCH.sub.3 (CHOH).sub.4CH.sub.2OH A17
CH.sub.2(CH.sub.2).sub.2OH " " " O(CH.sub.2).sub.nCOOH, n = 1-7 H
A18 " " " " (CH.sub.2).sub.n--CON- " " linker-site-directing
molecule, n = 1-7 A19 " " " " YCOCH.sub.2-linker- " site-directing
molecule Y = NH, O A20 CH.sub.2CH.sub.3 CH.sub.3
CH.sub.2CH.sub.2COOH " O(CH.sub.2).sub.2CH.sub.2OH
O(CH.sub.2).sub.2CH.sub.2OH A21 " " CH.sub.2CH.sub.2CON-oligo " " "
A22 CH.sub.2(CH.sub.2).sub.2OH CH.sub.2CH.sub.3 CH.sub.2CH.sub.3 "
O(CH.sub.2).sub.3CO-histamine H
[0185] Preferred pyrrole-based macrocycles include, but are not
limited to the hydro-monobenzoporphyrins (the so-called "fi
porphyrine" or "Gp" compounds) disclosed in U.S. Pat. Nos.
4,920,143 and 4,883,790 which are incorporated herein by reference.
Typically, these compounds are poorly water-soluble (less than 1
mg/ml) or water-insoluble. Gp is preferably selected from the group
consisting of those compounds having one of the formulae A-F set
forth in FIGS. 18A-F, mixtures thereof, and the metalated and
labeled forms thereof.
[0186] In FIGS. 18A-F, R.sup.1 and R.sup.2 can be independently
selected from the group consisting of carbalkoxy (2-6C), alkyl
(1-6C) sulfonyl, aryl (6-10C), sulfonyl, aryl (6-10C), cyano, and
--CONR.sup.5CO-- wherein R.sup.5 is aryl (6-10C) or alkyl (1-6C).
Preferably, however, each of R.sup.1 and R.sup.2 is carbalkoxy
(2-6C). R.sup.3 can be independently carboxyalkyl (2-6C) or a salt,
amide, ester or acylhydrazone thereof, or is alkyl (1-6C).
Preferably R.sup.3 is --CH.sup.2CH.sup.2COOH or a salt, amide,
ester or acylhydrazone thereof.
[0187] R.sup.4 is --CHCH.sub.2; --CHOR.sup.4' wherein R.sup.4' is H
or alkyl (1-6C), optionally substituted with a hydrophilic
substituent; --CHO; --COOR.sup.4'; CH(OR.sup.4')CH.sub.3;
CH(OR.sup.4') CH.sub.2OR.sup.4'; --CH(SR.sup.4')CH.sub.3;
--CHNR.sup.4'.sub.2)CH.sub.3; --CH(CN)CH.sub.3;
--CH(COOR.sup.4')CH.sub.3; --CH(OOCR.sup.4')CH.sub.3;
--CH(halo)CH.sub.3; --CH(halo)CH.sub.2(halo); an organic group of
<12C resulting from direct or indirect derivatization of a vinyl
group; or R.sup.4 consists of 1-3 tetrapyrrole-type nuclei of the
formula -L-P, wherein -L- is selected from the group consisting of
1
[0188] and P is a second Gp, which is one of the formulae A-F (FIG.
18) but lacks R.sup.4, or another porphyrin group. When P is
another porphyrin group, P preferably has the formula illustrated
in FIG. 19: wherein each R is independently H or lower alkyl
(1-4C); two of the four bonds shown as unoccupied on adjacent rings
are joined to R.sup.3; one of the remaining bonds shown as
unoccupied is joined to R.sup.4; and the other is joined to L; with
the proviso that, if R.sup.4 is --CHCH.sub.2, both R.sup.3 groups
cannot be carbalkoxyethyl. The preparation and use of such
compounds is disclosed in U.S. Pat. Nos. 4,920,143 and 4,883,790,
which are hereby incorporated by reference.
[0189] Even more preferred for including in liposomes are light
activated drugs that are designated as benzoporphyrin derivatives
("BPD's"). BPD's are hydrolyzed forms, or partially hydrolyzed
forms, of the rearranged products of formula A-C or formula A-D,
where one or both of the protected carboxyl groups of R.sup.3 are
hydrolyzed. Particularly preferred is the compound referred to as
BPD-MA in FIGS. 20A-D, which has two equally active
regioisomers.
[0190] As described above, activating a light activated drug
included in a microbubble can enhance rupture of the microbubble.
Preferred light activated drugs for including in a microbubble to
enhance rupture of the microbubble include Hematporphyrin, Rose
Bengal, Eosin Y, Erythrocin, Rhodamine B, and PHOTOFRIN. The
formulae for these preferred light activated drugs are illustrated
in FIGS. 21A-B where Rose Bengal, Eosin Y, Erythrocin and Rhodamine
B are xanthene derivatives.
[0191] As discussed above, the light activated drug can be coupled
with a site directing molecule to form a light activated drug
conjugate. Suitable site-directing molecules include, but are not
limited to: polydeoxyribonucleotides, oligodeoxyribonucleotides,
polyribonucleotide analogs, oligoribonucleotide analogs; polyamides
including peptides having an affinity for a biological receptor and
proteins such as antibodies; steroids and steroid derivatives;
hormones such as estradiol or histamine; hormone mimics such as
morphine and further macrocycles such as sapphyrins and rubyrins.
It is understood that the terms "nucleotide", "polynucleotide", and
"oligonucleotide", as used herein and in the appended claims, refer
to both naturally occurring and synthetic nucleotides, poly- and
oligonucleotides and to analogs and derivatives thereof such as
methylphosphonates, phosphotriesters, phosphorothioates, and
phosphoramidates and the like. Deoxyribonucleotides and
ribonucleotide analogs are contemplated as site-directing
molecules.
[0192] When the site-directing molecule is an oligonucleotide, the
oligonucleotide may be derivatized at the bases, the sugars, the
end of the chains, or at the phosphate groups of the backbone to
promote in vivo stability. Modifications of the phosphate groups
are preferred in one embodiment since phosphate linkages are
sensitive to nuclease activity. Preferred derivatives are the
methylphosphonates, phosphotriesters, phosphorothioates, and
phosphoramidates. Additionally, the phosphate linkages may be
completely substituted with non-phosphate linkages such as amide
linkages. Appendages to the ends of the oligonucleotide chains also
provide exonuclease resistance. Sugar modifications may include
alkyl groups attached to an oxygen of a ribose moiety in a
ribonucleotide. In particular, the alkyl group is preferably a
methyl group and the methyl group is attached to the 2' oxygen of
the ribose. Other alkyl groups may be ethyl or propyl.
[0193] A linker may be used to couple the light activated drug with
the site directing molecule. Exemplary linkers include, but are not
limited to, amides, amine, thioether, ether, or phosphate covalent
bonds as described in the examples for attachment of
oligonucleotides. In a preferred embodiment, an oligonucleotide or
other site-directing molecules is covalently bonded to a texaphyrin
or other light activated drugs via a carbon-nitrogen,
carbon-sulfur, or a carbon-oxygen bond.
[0194] As described above, the media can be an emulsion which
includes a light activated drug. The emulsions described below are
suitable for delivery into a body since they avoid pharmaceutically
undesirable organic solvents, solubilizers, oils or emulsifiers. A
wide range of light activated drug concentrations can be used in
the emulsion. Suitable concentrations of light activated drug
within the emulsion include, but are not limited to, approximately
0.01 to 1 gram/100 ml, preferably about 0.05 to about 0.5 gram/100
ml, and approximately 0.1 g/100 ml.
[0195] The emulsion includes a lipoid as a hydrophobic component
dispersed in a hydrophilic phase. The hydrophobic component of the
emulsion comprises a pharmaceutically acceptable triglyceride, such
as an oil or fat of a vegetable or animal nature, and preferably is
selected from the group consisting of soybean oil, safflower oil,
marine oil, black current seed oil, borage oil, palm kernel oil,
cotton seed oil, corn oil, sunflower seed oil, olive oil or coconut
oil. Physical mixtures of oils and/or interesterfied mixtures can
be employed. The preferred oils are medium chain length
triglycerides having C.sub.8-C.sub.10 chain length and more
preferably saturated. The preferred triglyceride is a distillate
obtained from coconut oil. The hydrophobic content of the emulsion
is preferably approximately 5 to 50 g/100 ml, more preferably about
10 to about 30 g/100 ml and approximately 20 g/100 ml of the
emulsion.
[0196] The emulsion can also contains a stabilizer such as
phosphatides, soybean phospholipids, nonionic block copolymers of
polyoxethylene and polyoxpropylene (e.g. poloxamers), synthetic or
semi-synthetic phospholipids, and the like. The preferred
stabilizer is purified egg yolk phospholipid. The stabilizer is
usually present in the composition in amounts of about 0.1 to about
10, and preferably about 0.3 to about 3 grams/100 ml, a typical
example being about 1.5 grams/100 ml.
[0197] The emulsion can also include one or more bile acids salts
as a costablizer. The salts are pharmacologically acceptable salts
of bile acids selected from the group of cholic acid, deoxycholic
acid and gylcocholic acid, and preferably of cholic acid. The salts
are typically alkaline metal or alkaline earth metal salts and
preferably sodium, potassium, calcium or magnesium salts, and most
preferably, sodium salts. Mixtures of bile acid salts can be
employed if desired. The amount of bile acid salt employed is
usually about 0.01 to about 1.0 and preferably about 0.05 to about
0.4 grams/100 ml, a typical example being about 0.2 grams/100
ml.
[0198] Suitable pH for the emulsion includes, but is not limited to
approximately 7.5 to 9.5, and preferably approximately 8.5. The pH
can be adjusted to the desired value, if necessary, by adding a
pharmaceutically acceptable base, such as sodium hydroxide,
potassium hydroxide, calcium hydroxide, magnesium hydroxide and
ammonium hydroxide.
[0199] Water can be added to the emulsion to achieve the desired
concentration of various components within the emulsion. Further,
the emulsion can include auxiliary ingredients for regulating the
osmotic pressure to make the emulsion isotonic with the blood.
Suitable auxiliary ingredients include, but are not limited to,
auxiliary surfactants, isotonic agents, antioxidants, nutritive
agents, trace elements and vitamins. Suitable isotonic agents
include, but are not limited to, glycerin, amino acids, such as
alanine, histidine, glycine, and/or sugar alcohols, such as
xylitol, sorbitol and/or mannitol. Suitable concentrations for
isotonic agents within the emulsion include, but are not limited
to, approximately 0.2 to about 8.0 grams/100 ml and preferably
about 0.4 to about 4 grams/100 ml and most preferably 1.5 to 2.5
gram/100 ml.
[0200] Antioxidants can be used to enhance the stability of the
emulsion, a typical example being .alpha.-tocopherol. Suitable
concentrations for the antioxidants include, but are not limited to
approximately 0.005 to 0.5 grams/100 ml, approximately 0.02 to
about 0.2 grams/100 ml and most preferably approximately 0.05 to
0.15 grams/100 ml.
[0201] The emulsions can also contain auxiliary solvents, such as
an alcohol, such as ethyl alcohol or benzyl alcohol, with ethyl
alcohol being preferred. When employed, such is typically present
in amounts of about 0.1 to about 4.0, and preferably about 0.2 to
about 2 grams/100 ml, a typical example being about 1 gram/100 ml.
The ethanol is advantageous since it facilitates dissolution of
poorly water-soluble light activated drugs and especially those
that form crystals which may be very difficult to dissolve in the
hydrophobic phase. Accordingly, the ethanol must be added directly
to the hydrophobic phase during preparation to be effective. For
maximum effectiveness, the ethanol should constitute about 5% to
15% by weight of the hydrophobic phase. In particular, if ethanol
constitutes less than 5% by weight of the hydrophobic phase,
dissolution of the light activated drug can become unacceptably
slow. When the ethanol concentration exceeds 15%, large (>5
.mu.m diameter) and poorly emulsified oil droplets can form in the
emulsion. The particles in the emulsion are preferably less than
about 5.0 .mu.m in diameter, more preferably less than 2.0 .mu.m in
diameter and most preferably less than 0.5 .mu.m or below.
[0202] A typical emulsion is prepared using the following
technique. The triglyceride oil is heated to 50.degree.-70.degree.
C. while sparging with nitrogen gas. The required amounts of
stabilizer (e.g. egg yolk phospholipids), bile acid salt, alcohol
(e.g. ethanol), antioxidant (e.g. .alpha.-to-copherol) and light
activated drug are added to the triglyceride while processing for
about 5 to about 20 minutes with a high speed blender or overhead
mixer to ensure complete dissolution or uniform suspension.
[0203] In a separate vessel, the required amounts of water and
isotonic agent (e.g. -glycerin) are heated to the above temperature
(e.g. 50.degree.-70.degree.) while sparging with nitrogen gas.
Next, the aqueous phase is transferred into the prepared
hydrophobic phase and high speed blending is continued for another
5 to 10 minutes to produce a uniform but coarse preemulsion (or
premix). This premix is then transferred to a conventional high
pressure homogenizer (APV Gaulin) for emulsification at about
8,000-10,000 psi. The diameter of the dispersed oil droplets in the
finished emulsion will be less than 5 .mu.m, with a large
proportion less than 1 .mu.m. The mean diameter of these oil
droplets will be less than 1 .mu.m, preferably from 0.2 to 0.5
.mu.m. The emulsion product is then filled into borosilicate (Type
1) glass vials which are stoppered, capped and terminally heat
sterilized in a rotating steam autoclave at about 121.degree.
C.
[0204] These emulsions can withstanding autoclaving as well as
freezing at about 0.degree. to -20.degree. C. Such can be stored
for a relatively long time with minimal physical and chemical
breakdown, i.e. at least 12-18 months at 4.degree.-8.degree. C. The
vehicle composition employed is chemically inert with respect to
the incorporated pharmacologically active light activated drug.
[0205] The emulsions can exhibit very low toxicity following
intravenous administration and exhibit no venous irritation and no
pain on injection. The emulsions exhibit minimal physical and
chemical changes (e.e. formation of non-emulsified surface oil)
during controlled shake-testing on a horizontal platform. Moreover,
the oil-in-water emulsions promote desirable pharmacoldnetics and
tissue distribution of the light activated drug in vivo.
[0206] As discussed above, the light activated drug can also be
delivered to the body in a media which includes microbubbles.
Suitable substrates for the microbubble include, but are not
limited to, biocompatible polymers, albumins, lipids, sugars or
other substances. U.S. Pat. Nos. 5,701,899 and 5,578,291 teaches a
method for synthesizing microbubbles with a sugar and protein
substrate and is incorporated herein by reference. U.S. Pat. Nos.
5,665,383 and 5,665,382 teaches a method for synthesizing
microbubbles with a polymeric substrate and is incorporated herein
by reference. U.S. Pat. Nos. 5,626,833 and 5,798,091 teach methods
for synthesizing microbubbles with a surfactant substrate and are
incorporated herein by reference. A preferred microbubble has a
lipid substrate. U.S. Pat. Nos. 5,772,929 teaches methods for
synthesizing microbubbles with a lipid substrate. U.S. Pat. Nos.
5,776,429, 5,715,824 and 5,770,222 teach preferred methods for
synthesizing microbubbles with a lipid substrate and a gas interior
and are incorporated herein by reference.
[0207] Suitable microbubbles with a lipid substrate can be
liposomes. The liposomes can be unilamellar vesicles having a
single membrane bilayer or multilamellar vesicles having multiple
membrane bilayers, each bilayer being separated from the next by an
aqueous layer. A liposome bilayer is composed of two lipid
monolayers having a hydrophobic "tail" region and a hydrophilic
"head" region. The formula of the membrane bilayer is such that the
hydrophobic (nonpolar) "tails" of the lipid monolayers orient
themselves towards the center of the bilayer, while the hydrophilic
"heads" orient themselves toward the aqueous phase. Either
unilamellar or multilamellar or other types of liposomes may be
used.
[0208] A hydrophilic light activated drug can be entrapped in the
aqueous phase of the liposome before the drug is delivered into the
patient. Alternatively, if the light activated drug is lipophilic,
it may associate with the lipid bilayer. Liposomes may be used to
help "target" the light activated drug to an active site or to
solubilize hydrophobic light activated drugs. Light activated drugs
are typically hydrophobic and form stable drug-lipid complexes.
[0209] As discussed above, many light activated drugs have low
solubility in water at physiological pH's, but are also insoluble
in (1) pharmaceutically acceptable aqueous-organic co-solvents, (2)
aqueous polymeric solutions and (3) surfactant/micellar solutions.
However, such light activated drugs can still be "solubilized" in a
form suitable for delivery into a body by using a liposome
composition. For example, one example of a light activated drug
BPD-MA (See Formula A of FIG. 20) can be "solubilized" at a
concentration of about 2.0 mg/ml in aqueous solution using an
appropriate mixture of phospholipids to form encapsulating
liposomes.
[0210] Although the light activated drug can be included in many
different types of liposomes, the following description discloses
particular liposome compositions and methods for making the
liposomes which are known to be "fast breaking". In fast breaking
liposomes, the light activated drug-liposome combination is stable
in vitro but, when administered in vivo, the light activated drug
is rapidly released into the bloodstream where it can associate
with serum lipoproteins. As a result, the localized delivery of
liposomes combined with the fast breaking nature of the liposomes
can result in localization of the light activated drug in the
tissues near the catheter. Further, the fast breaking liposomes can
prevent the liposomes from leaving the vicinity of the catheter
intact and then concentrating in non-targeted tissues such as the
liver. Delivery of ultrasound energy from the catheter can also
serve to break apart the liposomes after they have been delivered
from the catheter.
[0211] Liposomes are typically formed spontaneously by adding water
to a dry lipid film. Liposomes which include light activated drugs
can include a mixture of the commonly encountered lipids
dimyristoyl phosphatidyl choline ("DMPC") and egg phosphatidyl
glycerol ("EPG"). The presence of DMPC is important because DMPC is
the major component in the composition to form liposomes which can
solubilize and encapsulate insoluble light activated drugs into a
lipid bilayer. The presence of EPG is important because the
negatively charged, polar head group of this lipid can prevent
aggregation of the liposomes.
[0212] Other phospholipids, in addition to DMPC and EPG, may also
be present. Examples of suitable additional phospholipids that may
also be incorporated into the liposomes include phosphatidyl
cholines (PCS), including mixtures of dipalmitoyl phosphatidyl
choline (DPPC) and distearoyl phosphatidyl choline (DSPC). Examples
of suitable phosphatidyl glycerols (PGs) include dimyristoyl
phosphatidyl glycerol (DMPG), DLPG and the like.
[0213] Other types of suitable lipids that may be included are
phosphatidyl ethanolamines (PEs), phosphatidic acids (PAs),
phosphatidyl serines, and phosphatidyl inositols.
[0214] The molar ratio of the light activated drug to the DMPC/EPG
phospholipid mixture can be as low as 1:7.0 or may contain a higher
proportion of phospholipid, such as 1:7.5. Preferably, this molar
ratio is 1:8 or more phospholipid, such as 1:10, 1:15, or 1:20.
This molar ratio depends upon the exact light activated drug being
used, but will assure the presence of a sufficient number of DMPC
and EPG lipid molecules to form a stable complex with many light
activated drugs. When the number of lipid molecules is not
sufficient to form a stable complex, the lipophilic phase of the
lipid bilayer becomes saturated with light activated drug
molecules. Then, any slight change in the process conditions can
force some of the previously encapsulated light activated drug to
leak out of the vesicle, onto the surface of the lipid bilayer, or
even out into the aqueous phase.
[0215] If the concentration of light activated drug is high enough,
it can actually precipitate out from the aqueous layer and promote
aggregation of the liposomes. The more unencapsulated light
activated drug that is present, the higher the degree of
aggregation. The more aggregation, the larger the mean particle
size will be, and the more difficult aseptic or sterile filtration
will be. As a result, small changes in the molar ratio can be
important in achieving proper filterability of the liposome
composition.
[0216] Accordingly, slight increases in the lipid content can
increase significantly the filterability of the liposome
composition by increasing the ability to form and maintain small
particles. This is particularly advantageous when working with
significant volumes of 500 ml, a liter, five liters, 40 liters, or
more, as opposed to smaller batches of about 100-500 ml or less.
This volume effect is thought to occur because larger homogenizing
devices tend to provide less efficient agitation than can be
accomplished easily on a small scale. For example, a large size
Microfluidizer.TM. has a less efficient interaction chamber than
that one of a smaller size.
[0217] A molar ratio of 1.05:3:5 BPD-MA:EPG:DMPC (i.e., slightly
less phospholipid than 1:8.0 light activated drug:phospholipid) may
provide marginally acceptable filterability in small batches of up
to 500 ml. However, when larger volumes of the composition are
being made, a higher molar ratio of phospholipid provides more
assurance of reliable aseptic filterability. Moreover, the
substantial potency losses that are common in scale-up batches, due
at least in part to filterability problems, can thus be
avoided.
[0218] Any cryoprotective agent known to be useful in the art of
preparing freeze-dried formulations, such as di- or polysaccharides
or other bulking agents such as lysine, may be used. Further,
isotonic agents typically added to maintain isomolarity with body
fluids may be used. In a preferred embodiment, a di-saccharide or
polysaccharide is used and functions both as a cryoprotective agent
and as an isotonic agent.
[0219] In a particular embodiment, the particular combination of
the phospholipids, DMPC and EPG, and a disaccharide or
polysaccharide form a liposomal composition having liposomes of a
particularly narrow particle size distribution. When the process of
hydrating a lipid film is prolonged, larger liposomes tend to be
formed, or the light activated drug can even begin to precipitate.
The addition of a disaccharide or polysaccharide provides
instantaneous hydration and the large surface area for depositing a
thin film of the drug-phospholipid complex. This thin film provides
for faster hydration so that, when the liposome is initially formed
by adding the aqueous phase, the liposomes formed are of a smaller
and more uniform particle size. This provides significant
advantages in terms of manufacturing ease.
[0220] However, it is also possible that, when a saccharide is
present in the composition, it is added after dry lipid film
formation, as a part of the aqueous solution used in hydration. In
a particularly preferred embodiment, a saccharide is added to the
dry lipid film during hydration.
[0221] Disaccharides or polysaccharides are preferred to
monosaccharides for this purpose. To keep the osmotic pressure of
the liposome composition similar to that of blood, no more than
4-5% monosaccharides could be added. In contrast, about 9-10% of a
disaccharide can be used without generating an unacceptable osmotic
pressure. The higher amount of disaccharide provides for a larger
surface area, which results in smaller particle sizes being formed
during hydration of the lipid film.
[0222] Accordingly, the preferred liposomal composition comprises a
disaccharide or polysaccharide, in addition to the light activated
drug and the mixture of DMPC and EPG phospholipids. When present,
the disaccharide or polysaccharide is preferably chosen from among
the group consisting of lactose, trehalose, maltose, maltotriose,
palatinose, lactulose or sucrose, with lactose or trehalose being
preferred. Even more preferably, the liposomes comprise lactose or
trehalose.
[0223] Also, when present, the disaccharide or polysaccharide is
formulated in a preferred ratio of about 10-20 saccharide to
0.5-6.0 DMPC/EPG phospholipid mixture, respectively, even more
preferably at a ratio from about 10 to 1.5-4.0. In one embodiment,
a preferred but not limiting formulation is lactose or trehalose
and a mixture of DMPC and EPG in a concentration ratio of about 10
to 0.94-1.88 to about 0.65-1.30, respectively.
[0224] The presence of the disaccharide or polysaccharide in the
composition not only tends to yield liposomes having extremely
small and narrow particle size ranges, but also provides a liposome
composition in which light activated drugs, in a particular, may be
stably incorporated in an efficient manner, i.e., with an
encapsulation efficiency approaching 80-100%. Moreover, liposomes
made with a saccharide typically exhibit improved physical and
chemical stability, such that they can retain an incorporated light
activated drug without leakage upon prolonged storage, either as a
reconstituted liposomal or as a cryodesiccated powder.
[0225] Other optional ingredients include minor amounts of
nontoxic, auxiliary substances in the liposomal composition, such
as antioxidants, e.g., butylated hydroxytoluene, alphatocopherol
and ascorbyl palmitate; pH buggering agents, e.g., phosphates,
glycine, and the like.
[0226] Liposomes containing a light activated drug may be prepared
by combining the light activated drug and the DMPC and EPG
phospholipids (and any other optional phospholipids or excipients,
such as antioxidants) in the presence of an organic solvent.
Suitable organic solvents include any volatile organic solvent,
such as diethyl ether, acetone, methylene chloride, chloroform,
piperidine, piperidine-water mixtures, methanol, tert-butanol,
dimethyl sulfoxide, N-methyl-2-pyrrolidone, and mixtures thereof.
Preferably, the organic solvent is water-immiscible, such as
methylene chloride, but water immiscibility is not required. In any
event, the solvent chosen should not only be able to dissolve all
of the components of the lipid film, but should also not react
with, or otherwise deleteriously affect, these components to any
significant degree.
[0227] The organic solvent is then removed from the resulting
solution to form a dry lipid film by any known laboratory technique
that is not deleterious to the dry lipid film and the light
activated drug. Preferably, the solvent is removed by placing the
solution under a vacuum until the organic solvent is evaporated.
The solid residue is the dry lipid film. The thickness of the lipid
film is not critical, but usually varies from about 30 to about 45
mg/cm.sup.2, depending upon the amount of solid residual and the
total area of the glass wall of the flask. Once formed, the film
may be stored for an extended period of time, preferably not more
than 4 to 21 days, prior to hydration. While the temperature during
a lipid film storage period is also not an important factor, it is
preferably below room temperature, most preferably in the range
from about -20 to about 4.degree. C.
[0228] The dry lipid film is then dispersed in an aqueous solution,
preferably containing a disaccharide or polysaccharide, and
homogenized to form the desired particle size. Examples of useful
aqueous solutions used during the hydration step include sterile
water; a calcium- and magnesium-free, phosphate-buffered (pH
7.2-7.4) sodium chloride solution; a 9.75% w/v lactose solution; a
lactose-saline solution; 5% dextrose solution; or any other
physiologically acceptable aqueous solution of one or more
electrolytes. Preferably, however, the aqueous solution is sterile.
The volume of aqueous solution used during hydration can vary
greatly, but should not be so great as about 98% nor so small as
about 30-40%. A typical range of useful volumes would be from about
75% to about 95%, preferably about 85% to about 90%.
[0229] Upon hydration, coarse liposomes are formed that incorporate
a therapeutically effective amount of the light activated
drugs-phospholipid complex. The "therapeutically effective amount"
can vary widely, depending on the tissue to be treated and whether
it is coupled to a target-specific ligand, such as an antibody or
an immunologically active fragment. It should be noted that the
various parameters used for selective photodynamic therapy are
interrelated. Therefore, the therapeutically effective amount
should also be adjusted with respect to other parameters, for
example, fluence, irradiance, duration of the light used in
photodynamic therapy, and the time interval between administration
of the light activated drug and the therapeutic irradiation.
Generally, all of these parameters are adjusted to produce
significant damage to tissue deemed undesirable, such as
neovascular or tumor tissue, without significant damage to the
surrounding tissue, or to enable the observation of such
undesirable tissue without significant damage to the surrounding
tissue.
[0230] Typically, the therapeutically effective amount is such to
produce a dose of light activated drug within a range of from about
0.1 to about 20 mg/kg, preferably from about 0.15-2.0 mg/kg and,
even more preferably, from about 0.25 to about 0.75 mg/kg.
Preferably, the w/v concentration of the light activated drug in
the composition ranges from about 0.1 to about 8.0-10.0 g/L. Most
preferably, the concentration is about 2.0 to 2.5 g/L.
[0231] The hydration step should take place at a temperature that
does not exceed about 30.degree. C., preferably below the glass
transition temperature of the light activated drug-phospholipid
complex formed, even more preferably at room temperature or lower,
e.g., 15.degree.-20.degree. C. The glass transition temperature of
the light activated drug-lipid complex can be measure by using a
differential scanning microcalorimeter.
[0232] In accordance with the usual expectation that the aqueous
solubility of a substance should increase as higher temperatures
are used, at a temperature around the transition temperature of the
complex, the lipid membrane tends to undergo phase transition from
a "solid" gel state to a pre-transition state and, finally, to a
more "fluid" liquid crystal state. At these higher temperatures,
however, not only does fluidity increase, but the degree of phase
separation and the proportion of membrane defects also increases.
This results in an increasing degree of leakage of the light
activated drug from inside the membrane to the interface and event
out into the aqueous phase. Once a significant amount of liposome
leakage has occurred, even slight changes in the conditions such as
a small drop in temperature, can shift the equilibrium away from
aqueous "solubility" in favor of precipitation of the light
activated drug. Moreover, once the typically water-insoluble light
activated drug begins to precipitate, it is not possible to
re-encapsulate it when the lipid bilayer. The precipitate is
thought to contribute significantly to filterability problems.
[0233] In addition, the usual thickness of a lipid bilayer in the
"solid" gel state (about 47 A) decreases in the transition to the
"liquid" crystalline state to about 37 A, thus shrinking the
entrapped volume available for the light activated drugs to occupy.
The smaller "room" is not capable of containing as great a volume
of light activated drug, which can then be squeezed out of the
saturated lipid bilayer interstices. Any two or more liposomes
exuding light activated drug may aggregate together, introducing
further difficulties with respect to particle size reduction and
ease of sterile filtration. Moreover, the use of higher hydration
temperatures, such as, for example, about 35.degree. to 45.degree.
C., can also result in losses of light activated drug potency as
the light activated drug either precipitates or aggregates during
aseptic filtration.
[0234] The particle sizes of the coarse liposomes first formed in
hydration are then homogenized to a more uniform size, reduced to a
smaller size range, or both, to about 150 to 300 nm, preferably
also at a temperature that does not exceed about 30.degree. C.,
preferably below the glass transition temperature of the light
activated drug-phospholipid complex formed in the hydration step,
and even more preferably below room temperature of about 25.degree.
C. Various high-speed agitation devices may be used during the
homogenization step, such as a Microfluidizer.TM. model 110F; a
sonicator; a high-shear mixer; a homogenizer; or a standard
laboratory shaker.
[0235] It has been found that the homogenization temperature should
be at room temperature or lower, e.g., 15.degree.-20.degree. C. At
higher homogenization temperatures, such as about
32.degree.-42.degree. C., the relative filterability of the
liposome composition may improve initially due to increased
fluidity as expected, but then, unexpectedly, tends to decrease
with continuing agitation due to increasing particle size.
[0236] Preferably, a high pressure device such a Microfluidizer.TM.
is used for agitation. In microfluidization, a great amount of heat
is generated during the short-period of time during which the fluid
passes through a high pressure interaction chamber. In the
interaction chamber, two streams of fluid at a high speed collide
with each other at a 90.degree. angle. As the microfluidization
temperature increases, the fluidity of the membrane also increases,
which initially makes particle size reduction easier, as expected.
For example, filterability can increase by as much as four times
with the initial few passes through a Microfluidizer.TM. device.
The increase in the fluidity of the bilayer membrane promotes
particle size reduction, which makes filtration of the final
composition easier. In the initial several passes, this increased
fluidity mechanism advantageously dominates the process.
[0237] However, as the number of passes and the temperature both
increase, more of the hydrophobic light activated drug molecules
are squeezed out of the liposomes, increasing the tendency of the
liposomes to aggregate into larger particles. At the point at which
the aggregation of vesicles begins to dominate the process, the
sizes cannot be reduced any further. Surprisingly, particle sizes
actually then tend to grow through aggregation.
[0238] For this reason, the homogenization temperature is cooled
down to and maintained at a temperature no greater than room
temperature after the composition passes through the zone of
maximum agitation, e.g., the interaction chamber of a
Microfluidizer.TM. device. An appropriate cooling system can easily
be provided for any standard agitation device in which
homogenization is to take place, e.g., a Microfluidizer.TM., such
as by circulating cold water into an appropriate cooling jacket
around the mixing chamber or other zone of maximum turbulence.
While the pressure used in such high pressure devices is not
critical, pressures from about 10,000 to about 16,000 psi are not
uncommon.
[0239] As a last step, the compositions are preferably aseptically
filtered through a filter having an extremely small pore size,
i.e., 0.22 .mu.m. Filter pressures used during sterile filtration
can vary widely, depending on the volume of the composition, the
density, the temperature, the type of filter, the filter pore size,
and the particle size of the liposomes. However, as a guide, a
typical set of filtration conditions would be as follows:
filtration pressure of 15-25 psi; filtration load of 0.8 to 1.5
ml/cm.sup.2; and filtration temperature of about 25.degree. C.
[0240] A typical general procedure is described below with
additional exemplary detail:
[0241] (1) Sterile filtration of organic solvent through a
hydrophobic, 0.22 .mu.m filter.
[0242] (2) Addition of EPG, DMPC, light activated drug, and
excipients to the filtered organic solvent, dissolving both the
excipients and the light activated drug.
[0243] (3) Filtration of the resulting solution through a 0.22
.mu.m hydrophobic filter.
[0244] (4) Transfer of the filtrate to a rotary evaporator
apparatus, such as that commercially available under the name
Rotoevaporator.TM..
[0245] (5) Removal of the organic solvent to form a dry lipid
film.
[0246] (6) Analysis of the lipid film to determine the level of
organic solvent concentration.
[0247] (7) Preparation of a 10% lactose solution.
[0248] (8) Filtration of the lactose solution through a 0.22 .mu.m
hydrophilic filter.
[0249] (9) Hydration of the lipid film with a 10% lactose solution
to form coarse liposomes.
[0250] (10) Reduction of the particle sizes of the coarse liposomes
by passing them through a Microfluidizer.TM. three times.
[0251] (11) Determination of the reduced particle size distribution
of liposomes.
[0252] (12) Aseptic filtration of the liposome composition through
a 0.22 .mu.m hydrophilic filter. (Optionally, the solution may
first be pre-filtered with a 5.0 .mu.m prefilter.)
[0253] (13) Analysis of light activated drug potency.
[0254] (14) Filling of vials with the liposome composition.
[0255] (15) Freeze-drying.
[0256] Once formulated, the liposome composition may be
freeze-dried for long-term storage if desired. For example, BPD-MA,
a preferred light activated drug, has maintained its potency in a
cryodesiccated liposome composition for a period of at lest nine
months at room temperature, and a shelf life of at least two years
has been projected. If the composition is freeze-dried, it may be
packed in vials for subsequent reconstitution with a suitable
aqueous solution, such as sterile water or sterile water containing
a saccharide and/or other suitable excipients, prior to
administration, for example, by injection.
[0257] Preferably, liposomes that are to be freeze-dried are formed
upon the addition of an aqueous vehicle contain a disaccharide or
polysaccharide during hydration. The composition is then collected,
placed into vials, freeze-dried, and stored, ideally under
refrigeration. The freeze-dried composition can then be
reconstituted by simply adding water for injection just prior to
administration.
[0258] The liposomal composition provides liposomes of a
sufficiently small and narrow particle size that the aseptic
filtration of the composition through a 0.22 .mu.m hydrophilic
filter can be accomplished efficiently and with large volumes of
500 ml to a liter or more without significant clogging of the
filter. A particularly preferred particle size range is below about
300 run, more preferably below from about 250 nm. Most preferably,
the particle size is below about 220 nm.
[0259] Generally speaking, the concentration of the light activated
drugs in the liposome depends upon the nature of the light
activated drug used. When BPD-MA is used for example, the light
activated drug is generally incorporated in the liposomes at a
concentration of about 0.10% up to 0.5% w/v. If freeze-dried and
reconstituted, this would typically yield a reconstituted solution
of up to about 5.0 mg/ml light activated drug.
[0260] For diagnosis, the light activated drugs incorporated into
liposomes may be used along with, or may be labeled with, a
radioisotope or other detecting means. If this is the case, the
detection means depends on the nature of the label. Scintigraphic
labels such as technetium or indium can be detected using ex vivo
scanners. Specific fluorescent labels can also be used but, like
detection based on fluorescence of the light activated drugs
themselves, these labels can require prior irradiation.
[0261] The methods of preparing various light activated drugs,
light activated drug conjugates, emulsions and microbubbles are
described in greater detail in the examples below. These examples
are readily adapted to preparing analogous light activated drugs,
light activated drug conjugates, emulsions and microbubbles by
substitutions of appropriate light activated drugs, site directing
molecule, phospholipids, and other analogous components. The
following examples are being presented to describe the preferred
components, embodiments, utilities and attributes of the media. For
example, although BPD-MA is used as the light activated drugs in
the microbubble (liposome) examples, the invention is not intended
to be limited to this particular light activated drug.
[0262] Example 1 describes the synthesis of a preferred texaphyrin
derivative. Examples 2-4 describe different light activated drugs
conjugated with oligonucleotides as site directing molecules.
Examples 5 and 6 describes a synthesis of an emulsion including a
light activated drug. Example 7 describes preparation of
microbubbles which include a light activated drug.
EXAMPLE 1
Synthesis of Texaphyrin T2BET Metal Complexes
[0263] The synthesis of texaphyrins is provided in U.S. Pat. Nos.
4,935,498, 5,162,509 and 5,252,720, all incorporated by reference
herein. The present example provides the synthesis of a preferred
texaphyrin, named T2BET, having substituents containing ethoxy
groups.
[0264] Lutetium(III) acetate hydrate can be purchased from Strem
Chemicals, Inc. (Newburyport, Mass.), gadolinium(III) acetate
tetrahydrate can be purchased from Aesar/Johnson Matthey (Ward
Hill, Mass.) and LZY-54 zeolite can be purchased from UOP (Des
Plaines, III.). Acetone, glacial acetic acid, methanol, ethanol,
isopropyl alcohol, and n-heptanes can be purchased from J. T. Baker
(Phillipsburg, N.J.). Triethylamine and Amberlite 904 anion
exchange resin can be purchased from Aldrich (Milwaukee, Wisc.).
All chemicals should be ACS grade and used without further
purification.
[0265] FIGS. 22A-I illustrate the synthesis of the gadolinium (III)
complex of
4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-16,17-bis-
[2-[2-(2-methoxyethoxy) ethoxy]ethoxy]-pentaazapentacyclo
[20.2.1.1.sup.3,6.I.sup.8,
11.0.sup.14,19]heptacosa-1,3,5,7,9,11(27),12,1-
4,16,18,20,22(25),23-tridecaene which is illustrated as Formula I
of FIG. 22. The critical intermediate
1,2-bis[2-[2-(2-methoxyethoxy)ethoxy)ethoxy- ]-4,5-dinitrobenzene
(Formula E) can be prepared according to a three-step synthetic
process outlined in FIGS. 22A-I. (Note: References to "Formula A,"
"Formula B," etc. relate to FIGS. 22A, 22B, etc.)
[0266] Synthesis of triethylene glycol monomethyl ether
monotosylate, Formula B: In an oven dried 12 L three-necked
roundbottom flask, equipped with a magnetic stir bar and a 1000 mL
pressure-equalizing dropping funnel, a solution of NaOH (440.0 g,
11.0 mol) is added to 1800 mL water and the mixture is cooled to
approximately 0.degree. C. A solution of triethylene glycol
monomethyl ether, Formula A, (656.84 g, 4.0 mol) in THF (1000 mL)
is added. The clear solution is stirred vigorously at 0.degree. C.
for 15 min and a solution of tosyl chloride (915.12, 4.8 mol) in
THF (2.0 L) added dropwise over a 1 h period. The reaction mixture
is stirred for an additional 1 h at 0.degree. C., and 10% HCl (5.0
L) is added to quench the reaction (to pH 5-7). The two-phase
mixture is transferred to a 4 L separatory funnel, the organic
layer removed, and the aqueous layer extracted with t-butylmethyl
ether (3.times.250 mL). The combined organic extracts are washed
with brine (2.times.350 mL), dried (MgSO.sub.4), and evaporated
under reduced pressure to afford Formula B, 1217.6 g (95%) as a
light colored oil. This material is taken to the next step without
further purification.
[0267] Synthesis of
1,2-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzene, Formula D: In
a dry 5 L round-bottom flask equipped with an overhead stirrer,
reflux condenser, and a gas line, K.sub.2CO.sub.3 (439.47 g, 3.18
mol) and MeOH (1800 mL) are combined under an argon atmosphere. To
this well-stirred suspension, catechol, Formula C, (140.24 g, 1.21
mol) is added, and the mixture heated to reflux. Formula B (1012.68
g, 3.18 mol) is then added in one portion. The suspension is
stirred at reflux for 24 h, cooled to room temperature, and
filtered through Celite. The pad is rinsed with 500 mL of methanol
and the combined filtrates are evaporated under reduced pressure.
The resulting brown residue is taken up in 10% NaOH (800 mL), and
methylene chloride (800 mL) added with stirring. The mixture is
transferred to a 2 L separatory funnel, the organic layer removed
and the aqueous layer extracted with methylene chloride
(3.times.350 mL). The organic extracts are combined, washed with
brine (350 mL), dried (MgSO.sub.4), evaporated under reduced
pressure, and the residue dried in vacuo for several hours to yield
485.6 (95%) of 1,2-bis[2-[2-(2-methoxyethoxy)ethoxy)ethoxy]benzene
(Formula D). For Formula D: bp. 165.degree.-220.degree. C.,
(0.2-0.5 mm Hg); FAB MS, M.sup.+: m/e 402; HRMS, M.sup.+: 402.2258
(calcd. for C.sub.20H.sub.34O.sub.8, 402.2253).
[0268] Synthesis of
1,2-bis[2-[2-(2-methoxyetboxy)ethoxy]ethoxy]-4,5-dinit- robenzene,
Formula E: In an oven dried 1 L roundbottom flask Formula D (104 g,
0.26 mol) and glacial acetic acid (120 mL) are combined and cooled
to 5.degree. C. To this well stirred solution, concentrated nitric
acid (80 mL) is added dropwise over 15-20 min. The temperature of
the mixture is held below 40.degree. C. by cooling and proper
regulation of the rate of addition of the acid. After addition, the
reaction is allowed to stir for an additional 10-15 min and is then
cooled to 0.degree. C. Fuming nitric acid (260 mL) is added
dropwise over 30 min while the temperature of the solution is held
below 30.degree. C. After the addition is complete, the red colored
solution is allowed to stir at room temperature until the reaction
is complete (ca. 5 h, TLC: 95/5; CH.sub.2Cl.sub.2/MeOH) and then
poured into well stirred ice water (1500 mL). Methylene chloride
(400 mL) is added, the two-phase mixture transferred to a 2 L
separatory funnel and the organic layer removed. The aqueous layer
is extracted with CH2Cl2 (2.times.150 mL) and the combined organic
extracts washed with 10% NaOH (2.times.250 mL) and brine (250 mL),
dried (MgSO.sub.4), and concentrated under reduced pressure. The
resulting orange oil is dissolved in acetone (100 mL), and the
solution layered with n-hexanes (500 mL), and stored in the
freezer. The resulting precipitate is collected by filtration yield
101.69 g (80%) of Formula E as a yellow solid. For Formula E: mp
43.degree.-45.degree. C.; FAB MS, (M+H).sup.+: m/e 493; HRMS,
(M+H).sup.+; 493; HRMS, (M+H).sup.+: 493.2030 (calcd. for
C.sub.20H.sub.33N.sub.2O.sub.12, 493.2033).
[0269] Synthesis of
1,2-diamino-4,5-bis[2-[2-(2-methoxy-ethoxy)ethoxy]etho- xy],
Formula F: In an oven dried 500 mL round bottom flask, equipped
with a Claisen adapter, pressure equalizing dropping funnel, and
reflux condenser,
1,2-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-4,5-dinitrobenzen- e
(Formula E) (20 g, 0.04 mol) is dissolved in absolute ethanol (200
mL). To this clear solution, 10% palladium on carbon (4 g) is added
and the dark black suspension is heated to reflux under an argon
atmosphere. Hydrazine hydrate (20 mL) in EtOH (20 mL) is added
dropwise over 10 min to avoid bumping. The resulting brown
suspension is heated at reflux for 1.5 h at which time the reaction
mixture is colorless and TLC analysis (95/5; CH.sub.2Cl.sub.2/MeOH)
displays a low R_UV active spot corresponding to the diamine.
Therefore, the mixture is hot filtered through Celite and the pad
rinsed with absolute ethanol (50 mL). The solvent is removed under
reduced pressure and the resulting light brown oil is dried in
vacuo (in the dark) for 24 h to yield 15.55 g (89%) of
1,2-diamino-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]benzene
(Formula F). For Formula F: FAB MS, M.sup.+: in/e 432; HRMS,
M.sup.+: 432.2471 (calcd. for C.sub.20H.sub.36N.sub.2O.sub.8,
432.2482). This material is taken to the next step without further
purification.
[0270] Synthesis of
4,5-diethyl-10,23-dimethyl-9,24-bis(3-hydroxypropyl)-1-
6,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]-13,20,25,26,27-pentaazapenta-
cyclo[20.2.1.1.sup.3,6. 18, 11.0.sup.14,19]
heptacosa-3,5,8,10,12,14,16,18- ,20,22,24-undecaene, Formula H. In
an oven dried 1 L round-bottom flask,
2,5-bis[(5-formyl-3-(3-hydroxypropyl)-4-methyl-pyrrol-2yl)methyl]-3,4-die-
thylpyrrole (Formula G) (The synthesis of Formula G is provided in
U.S. Pat. No. 5,252,720, incorporated by reference herein.) (30.94)
g, 0.0644 mol) and
4,5-diamino-bis[2[2-(2-methoxyetboxy)ethoxy)ethoxy]benzene (Formula
F) (28.79 g, 0.0644 mol) are combined in absolute methanol (600 mL)
under an argon atmosphere. To this well stirred suspension, a
mixture of concentrated hydrochloric acid (6.7 m:) in absolute
methanol 200 mL is added in one portion. The mixture is gradually
heated to 50.degree. C., at which time the reaction goes from a
cloudy suspension of starting materials to a dark red homogeneous
solution as the reaction proceeded. After 3 h the reaction is
judged complete by TLC analysis and UV/visible spectroscopy
(.lambda..sub.max 369 nm). The reaction mixture is cooled to room
temperature, 60 g of activated carbon (DARCO.TM.) is added, and the
resulting suspension is stirred for 20 min. The dark suspension is
filtered through Celite to remove the carbon, the solvent
evaporated to dryness, and the crude Formula H dried in vacuo
overnight. Formula H is recrystallized from isopropyl
alcohol/n-heptane to afford 50 g (85%) of a scarlet red solid. For
Formula H: .sup.1H NMR (CD.sub.3OD): .delta. 1.11 (t, 6H,
CH.sub.2CH.sub.3), 1.76 (p, 4H, pyrr-CH.sub.2CH.sub.2CH.sub.2OH),
2.36 (s, 6H, pyrr-CH.sub.3), 2.46 (q, 4H, CH.sub.2CH.sub.3), 2.64
(t, 4H, pyrr-CH.sub.2CH.sub.2CH.sub.2OH), 3.29 [s, 6H,
(CH.sub.2CH.sub.2O).sub.3C- H.sub.3], 3.31 (t, 4H,
pyrr-CH.sub.2CH.sub.2CH.sub.2OH), 3.43-3.85 (m, 20H,
CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2O), 4.0 (s, 4H,
(pyrr).sub.2-CH.sub.2), 4.22 (t, 4H, PhOCH.sub.2CH.sub.2O), 7.45
(s, 2H, PhH), 8.36 (s, 2H, HC.dbd.N); UV/vis:
[(MeOH).lambda..sub.max nm]: 369; FAB MS, [M+H].sup.+: m/e 878.5;
HRMS, [M+H].sup.+: m/e 878.5274 (calcd. for
[C.sub.48H.sub.72N.sub.5O.sub.10].sup.+878.5279).
[0271] Synthesis of the gadolinium (HI) complex of
4,5-diethyl-10,23-dimet-
hyl-9,24-bis(3-hydroxypropyl)-16,17-bis[2-[2-(2-methoxyethoxy)ethoxy]ethox-
y]pentaazapentacyclo
[20.2.1.1.sup.3,6,1.sup.8,11,0.sup.14,19]heptacosa-1,-
3,5,7,9,11(27),12,14,16,18,20,22(25),23-tridecaene, Formula I.
Formula I is prepared according to the process outlined in FIG. 22.
In a dry 2 L three-necked round-bottom flask, Formula H (33.0 g,
0.036 mol) and gadolinium(II) acetate tetrahydrate (15.4 g, 0.038
mol) are combined in methanol (825 mL). To this well stirred red
solution, gadolinium(III) acetate tetrahydrate (15.4 g, 0.038 mol)
and triethylamine (50 mL) are added and the reaction is heated to
reflux. After 1.5 h, air is bubbled (i.e., at reflux) for 4 h into
the dark green reaction solution with aid of a gas dispersion tube
(flow rate=20 cm.sup.3/min). At this point, the reaction mixture is
carefully monitored by UV/visible spectroscopy (i.e., a spectrum is
taken every 0.5-1 h, .about.1 drop diluted in 4-5 mL MeOH). The
reaction is deemed complete by UV/Vis (In MeOH ratio: 342 nM/472
nm=0.22-0.24) after 4 h. The dark green reaction is cooled to room
temperature, filtered through Celite into a 2 L round bottom flask,
and the solvent removed under reduced pressure. The dark green
solid is suspended in acetone (1 L) and the resulting slurry is
stirred for 1 h at room temperature. The suspension is filtered to
remove the red/brown impurities (incomplete oxidation products),
the solids rinsed with acetone (200 mL), and air dried. The crude
complex (35 g) is dissolved in MeOH (600 mL), stirred vigorously
for 15 min, filtered through Celite, and transferred to a 2 L
Erlenmeyer flask. An additional 300 mL of MeOH and 90 mL water are
added to the flask, along with acetic acid washed LZY-54 zeolite
(150 g). The suspension is agitated with an overhead mechanical
stirrer for approximately 3-4 h. The zeolite extraction is deemed
complete with the absence of free Gd(III). [To test for free
gadolinium, the crude Formula I is spotted heavily onto a reverse
phase TLC plate (Whatman KC8F, 1.5.times.10 cm) and the
chromatogram developed using 10% acetic acid in methanol. The green
complex moved up the TLC plate close to the solvent front. Any free
gadolinium metal will remain at the origin under these conditions.
After developing the chromatogram, the plate is dried and the lower
1/4 of the plate stained with an Arsenazo III solution in methanol
(4 mg Arsenazo III in 10 mL methanol). A very faint blue spot
(indicative of free metal) is observed at the origin against a pink
background indicating very little free gadolinium metal.] The
zeolite is removed through a Whatman #3 filter paper and the
collected solids rinsed with MeOH (200 mL). The dark green filtrate
is loaded onto a column of Amberlite IRA-904 anion exchange resin
(30 cm length.times.2.5 cm diameter) and eluted through the resin
(ca. 10 mL/min flow rate) into a 2 L round bottom flask with 300 mL
1-butanol. The resin is rinsed with an additional 100 mL of MeOH
and the combined eluent evaporated to dryness under reduced
pressure. The green shiny solid Formula I is dried in vacuo for
several hours at 40.degree. C., to a well stirred ethanoic solution
(260 mL of Formula I at 55.degree.-60.degree. C., n-heptanes (ca.
600 mL) is added dropwise (flow=4 mL/min) from a 1 L
pressure-equalizing dropping funnel. During the course of 1.5 h
(300 mL addition) the green Formula I began to crystallize out of
the dark mixture. After complete addition, the green suspension is
cooled and stirred for 1 h at room temperature. The suspension is
filtered, the solids rinsed with acetone (250 mL), and dried in
vacuo for 24 h to afford 26 g (63%), UV/vis: [(MeOH)
.lambda..sub.max nm]: 316, 350, 415, 473, 739; FAB MS, (M-20
Ac).sup.+: m/e 1030; HRMS, (M-20 Ac).sup.+: m/e 1027.4036 (calcd.
for C.sub.48H.sub.66 .sup.155GdN.sub.5O.sub.10, 1027.4016). Anal.
calcd. for [C.sub.52H.sub.72GdN.sub.5O.sub.14] 0.5H.sub.2O: C,
53.96; H, 6.36; N, 6.05, Gd, 13.59. Found: C, 53.73; H, 6.26; N,
5.82; Gd, 13.92.
[0272] Synthesis of the Lutetium(III) Complex of Formula H: The
macrocyclic ligand Formula H is oxidatively metalated using
lutetium(III) acetate hydrate (9.75 g, 0.0230 mol) and
triethylamine (22 mL) in air-saturated methanol (1500 mL) at
reflux. After completion of the reaction (as judged by the optical
spectrum of the reaction mixture), the deep green solution is
cooled to room temperature, filtered through a pad of celite, and
the solvent removed under reduced pressure. The dark green solid is
suspended in acetone (600 mL, stirred for 30 min at room
temperature, and then filtered to wash away the red/brown
impurities (incomplete oxidation products and excess
triethylamine). The crude complex is dissolved into MeOH (300 mL,
stirred for -30 min, and then filtered through celite into a 1 L
Erlenmeyer flask. An additional 50 mL of MeOH and 50 mL of water
are added to the flask along with acetic acid washed LZY-54 zeolite
(40 g). The resulting mixture is agitated or shaken for 3 h, then
filtered to remove the zeolite. The zeolite cake is rinsed with
MeOH (100 mL and the rinse solution added to the filtrate. The
filtrate is first concentrated to 150 mL and then loaded onto a
column (30 cm length.times.2.5 cm diameter) of pretreated Amberlite
IRA-904 anion exchange resin (resin in the acetate form). The
eluent containing the bisacetate lutetium(III) texaphyrin complex
is collected concentrated to dryness under reduced pressure, and
recrystallized from anhydrous methanol/t-butylmethyl ether to
afford 11.7 g (63%) of a shiny green solid. For the complex:
UV/vis: [(MeOH) .lambda..sub.max nm (log .epsilon.)]: 354, 414, 474
(5.10), 672, 732; FAB MS, [IM-OAc.sup.-].sup.+: m/e 1106.4; HRMS,
(M-OAc.sup.-].sup.+: m/e 1106.4330 (calcd. for
[C.sub.48H.sub.66N.sub.5;O.sub.10Lu(OAc)].sup.+, 1106.4351). Anal.
calcd. for [C.sub.48H.sub.66N.sub.5O.sub.10Lu](OAc).sub-
.2H.sub.2O; C, 52.74; H, 6.30; N, 5.91. Found: C, 52.74; H, 6.18;
N, 5.84.
EXAMPLE 2
Synthesis of a T2B1 TXP Metal Complex-Oligonuleotide Conjugate
[0273] FIGS. 23A-H illustrate the synthesis of a light activated
drug conjugate. The light activated drug is a texaphyrin coupled
with an oligonucleotide which is complementary to a DNA site. As a
result, the light activated drug conjugate can bind the
complementary DNA site and will cleave the site upon activation by
ultrasound. (Note: References to "Formula A," "Formula B," etc.
relate to FIGS. 23A, 23B, etc.)
[0274] Synthesis of
4-Amino-1-[1-(ethyloxy)acetyl-2-oxy]-3-nitrobenzene (Formula B of
FIG. 19), n=1. Potassium carbonate (14.0 g, 101 mmol) and
4-amino-3-nitrophenol (Formula A) (10.0 g, 64.9 mmol) are suspended
in 150 mL dry acetonitrile. Ethyl-2-iodoacetate (10 mL, 84.5 mmol)
(or ethyl iodobutyrate may be used, in that case n=3) is added via
syringe, and the suspension is stirred at ambient temperature for
ca. 21 h. Chloroform (ca. 375 mL) is added and is used to transfer
the suspension to a separatory funnel, whereupon it is washed with
water (2xca. 100 mL). The water washes are in turn washed with
CHOl.sub.3 (ca. 100 mL) and the combined CHCl.sub.3 extracts are
washed with water (ca. 100 mL). Solvents are removed on a rotary
evaporator, and the residue is redissolved in CHCl.sub.3 (ca. 500
mL) and precipitated into hexanes (1.5 L). After standing two days,
the precipitate is filtered using a coarse fritted funnel and dried
in vacuo to provide 14.67 g (Formula B), n=1 (94.1%). TLC: Rf=0.43,
CHCl.sub.3.
[0275] Synthesis of
4-Amino-1-[1-(hydroxy)acetyl-2-oxy]-3-nitrobenzene (Formula C),
n=1. 4-Amino-1-[1-(ethyloxy)acetyl-2-oxy]-3-nitrobenzene (Formula
B), n=1, (10.00 g, 37.3 mmol) is dissolved in tetrahydrofuran (100
mL), aqueous sodium hydroxide (1M solution, 50 mL) is added and the
solution is stirred at ambient temperature for ca. 21 h.
Tetrahydrofuran is removed on a rotary evaporator, and water (100
mL) is added. The solution is washed with CHCl.sub.3 (ca. 200 mL),
the neutralized by addition of hydrochloric acid (1M solution, 50
mL). The precipitate which formed is filtered after standing a few
minutes, washed with water, and dried in vacuo to provide 8.913 g
compound (Formula C), n=1 (99.5%). TLC: Rf=0.65, 10%
methanol/CHCl.sub.3.
[0276] Synthesis of
16-[1-(Hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-
-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentacyclo
[20.2.1.1.sup.3,6.1.sup.8,11.0.sup.14,19]heptacosa-3,5,8,10,12,14(19),
15,17,20,22,24-undecaene (Formula E), n=1:
4-Amino-1-[1-(hydroxy)acetyl-2- -oxy]-3-nitrobenzene (Formula C),
n=1. (1,800 g, 8.49 mmol) is dissolved in methanol (100 mL) in a 1L
flask. Palladium on carbon (10%, 180 mg) is added, and the
atmosphere inside the flask is replaced with hydrogen at ambient
pressure. A grey precipitate is formed after ca. 3 h, and the
supernatant is clear. Methanol is removed in vacuo, taking
precautions to prevent exposure to oxygen, and the compound is
dried overnight in vacuo. Isopropyl alcohol (500 mL) and HCl (12M,
400 .mu.L) are added, and the suspension is allowed to stir for ca.
15'. 2,5-Bis[(3-hydroxypropyl-5-for-
myl-4-methylpyrrol-2-y)methyl]-3,4-diethylpyrrole (Formula D) (n=1)
(4.084 g, 8.49 mmol) is added, and the reaction stirred at room
temperature under argon for 3 hours. Hydrochloric acid is again
added (12M, 400 .mu.L) and the reaction again is allowed to stir
for an additional 3.5 h. The resulting red solution is filtered
through celite, and the filtercake is washed with isopropyl alcohol
until the filtrate is colorless. Solvent is reduced to a volume of
ca. 50 mL using a rotary evaporator, whereupon the solution is
precipitated into rapidly stirring Et.sub.2O (ca. 700 mL). Formula
E (n=1) is obtained as a red solid (5.550 g, 98.4%) upon filtering
and drying in vacuo. TLC: Rf=0.69, 20% methanol/CHCL.sub.3
(streaks, turns green on plate with I.sub.2).
[0277] Synthesis of metal complex of
16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis-
(3-hydroxypropyl)-4,5-diethyl-10,23-dimethyl-13,20,25,26,27-pentaazapentac-
yclo
[20.2.1.1.sup.3,16.1.sup.8,11.0.sup.14,19]heptacosa-1,3,5,7,9,11(27),-
12,14(19),15,17,20,22(25),23-tridecaene (Formula F), n=1:
Approximately equal molar amounts of the protonated form of the
macrocycle,
16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23--
dimethyl-13,20,25,26,27-pentaazapentacyclo[20.2.0.1.1.sup.3,6.1.sup.8,11.0-
.sup.14,19]heptacosa-3,5,8,10,12,14(19),15,17,20,22,24-undecaene
hydrochloride (Formula E), n=1, and a metal acetate pentahydrate
are combined with triethylamine in methanol, and are heated to
reflux under air for 5.5 h. The reaction is cooled to room
temperature, and stored at -20.degree. C., overnight. Solvent is
removed on a rotary evaporator, acetone is added, and the
suspension is stirred on a rotary evaporator for 2 h. The
suspension is filtered and the precipitate dried briefly in vacuo,
whereupon a solution is formed in methanol (ca. 250 mL) and water
(25 mL). The pH is adjusted to 4.0 using HCl (1M), HCl-washed
zeolite LZY54 is added (ca. 5 g) and the suspension is stirred on
the rotary evaporator for ca. 6 h. Amberlite.TM. IRA-900 ion
exchange resin (NaF treated, ca. 5 g) is added, and the suspension
is stirred for an additional hour. The suspension is filtered, the
resin is washed with methanol (ca. 100 mL), and the filtrate is
adjusted to pH 4.0 using HCl (1M). Solvents are removed on a rotary
evaporator, using ethanol (abs.) to remove traces of water. After
drying in vacuo, the compound is dissolved in methanol (25 mL) and
precipitated into rapidly stirring Et.sub.2O (300 mL). Formula F,
n=1, is obtained as a precipitate after filtering and drying in
vacuo. An analytical sample is prepared by treating 50 mg of
Formula F, n=1, dissolved in methanol (25 mL) with acetic
acid-washed zeolite, then acetic acid-washed Amberlite.TM. for ca.
1 h. After reducing methanol to a minimum volume, the solution is
precipitated into rapidly stirring Et.sub.2O (70 mL), filtered, and
dried in vacuo.
[0278] Postsynthetic modification of oligodeoxynucleotide-amine
(Formula G) with metal complex (Formula F), n=1: The metal complex
of
16-[1-(hydroxy)acetyl-2-oxy]-9,24-bis(3-hydroxypropyl)-4,5-diethyl-10,23--
dimethyl-13,20,25,26,27-pentaazapentacyclo
[20.2.1.1.sup.3,6.1.sup.8,11.0.-
sup.14,19]heptacosa-1,3,5,7,9,11(27),12,14(19),15,17,20,22(25),23-tridacae-
ne (Formula F), n=1, (about 30 mol) and N-hydroxysuccinimide (43
.mu.mol) are dried together overnight in vacuo. The compounds are
dissolved in dimethylformamide (anhydrous, 500 .mu.L) and
dicyclohexylcarbodiimide (10 mg, 48 .mu.mol) is added. The
resulting solution is stirred under argon with protection from
light for 8 h, whereupon a 110 .mu.L aliquot is added to a solution
of oligodeoxynucleotide (Formula G) (87 .mu.mol) in a volume of 350
.mu.L of 0.4M sodium bicarbonate buffer in a 1.6 mL Eppendorf tube.
After vortexing briefly, the solution is allowed to stand for 23 h
with light protection. The suspension is filtered through 0.45
.mu.m nylon microfilterfuge tubes, and the Eppendorf tube is washed
with 250 .mu.L sterile water. The combined filtrates are divided
into two Eppendorf tubes, and glycogen (20 mg/mL, 2 .mu.L) and
sodium acetate (3M, pH 5.4 30 .mu.L) are added to each tube. After
vortexing, ethanol (absolute, 1 mL) is added to each tube to
precipitate the DNA. Ethanol is decanted following centrifugation,
and the DNA is washed with an additional 1 mL aliquot of ethanol
and allowed to air dry. The pellet is dissolved in 50% formamide
gel loading buffer (20 .mu.L), denatured at 90.degree. C. for ca.
2', and loaded on a 20% denaturing polyacrylamide gel. The band
corresponding to conjugate (Formula H), n=1, is cut from the gel,
crushed and soaked in 1.times.TBE buffer (ca. 7 mL) for 1-2 days.
The suspension is filtered through nylon filters (0.45 .mu.m) and
desalted using a Sep-pak.TM. reverse phase cartridge. The conjugate
is eluted from the cartridge using 40% acetonitrile, lyophilized
overnight, and dissolved in 1 mM HEPES buffer, pH 7.0 (500 .mu.L).
The solution concentration is determined using UV/vis
spectroscopy.
EXAMPLE 3
Synthesis of Texaphyrin Metal Complexes with Amine-, Thiol- or
Hydroxy-Linked Oligonucleotides
[0279] Amides, ethers, and thioethers are representative of
linkages which may be used for coupling site-directing molecules
such as oligonucleotides to light activated drugs such as
texaphyrin metal complexes as illustrated in FIGS. 24A-F. (Note:
References to "Formula A," "Formula B," etc. relate to FIGS. 24A,
24B, etc.) Oligonucleotides or other site-directing molecules
functionalized with amines at the 5'-end, the 3'-end, or internally
at sugar or base residues are modified post-synthetically with an
activated carboxylic ester derivative of the texaphyrin complex. In
the presence of a Lewis acid such as FeBr.sub.3, a bromide
derivatized texaphyrin (for example, Formula C of FIG. 24) will
react with an hydroxyl group of an oligonucleotide to form and
ether linkage between the texaphyrin linker and the
oligonucleotide. Alternatively, oligonucleotide analogues
containing one or more thiophosphate or thiol groups are
selectively alkylated at the sulfur atom(s) with an alkyl halide
derivative of the texaphyrin complex. Oligodeoxynucleotide-complex
conjugates are designed so as to provide optimal catalytic
interaction between the targeted DNA phosphodiester backbone and
the texaphyrino.
[0280] Oligonucleotides are used to bind selectively compounds
which include the complementary nucleotide or oligo- or
polynucleotides containing substantially complementary sequences.
As used herein, a substantially complementary sequence is one in
which the nucleotides generally base pair with the complementary
nucleotide and in which there are very few base pair mismatches.
The oligonucleotide may be large enough to bind probably at least 9
nucleotides of complementary nucleic acid.
[0281] For general reviews of synthesis of DNA, RNA, and their
analogues, see Oligonucleotides and Analogues, F. Eckstein, Ed.,
1991. IRL Press, New York; Oligonucleotide Synthesis, M. J. Gait,
Ed., 1984, IRL Press Oxford, England; Caracciolo et al. (1989);
Bioconjugate Chemistry, Goodchild, J. (1990); or for phosphonate
synthesis, Matteucci, Md. et al., Nucleic Acids Res. 14:5399 (1986)
(these references are incorporated by reference herein).
[0282] In general, there are three commonly used solid phase-based
approaches to the synthesis of oligonucleotides containing
conventional 5'-3' linkages. These are the phosphoramidite method,
the phosphonate method, and the triester method.
[0283] A brief description of a current method used commercially to
synthesize oligomeric DNA is as follows: Oligomers up to ca. 100
residues in length are prepared on a commercial synthesizer, eg.,
Applied Biosystems Inc. (ABI) model 392, that uses phosphoramidite
chemistry. DNA is synthesized from the 3' to the 5' direction
through the sequential addition of highly reactive phosphorous(I)
reagents called phosphoramidites. The initial 3' residue is
covalently attached to a controlled porosity silica solid support,
which greatly facilitates manipulation of the polymer. After each
residue is coupled to the growing polymer chain, the
phosphorus(III) is oxidized to the more stable phosphorus(V) state
by a short treatment with iodine solution. Unreacted residues are
capped with acetic anhydride, the 5'-protective group is removed
with weak acid, and the cycle may be repeated to add a further
residue until the desired DNA polymer is synthesized. The full
length polymer is released from the solid support, with concomitant
removal of remaining protective groups, by exposure to base. A
common protocol uses saturated ethanolic ammonia.
[0284] The phosphonate based synthesis is conducted by the reaction
of a suitably protected nucleotide containing a phosphonate moiety
at a position to be coupled with a solid phase-derivatized
nucleotide chain having a free hydroxyl phosphonate ester linkage,
which is stable to acid. Thus, the oxidation to the phosphate or
thiophosphate can be conducted at any point during synthesis of the
oligonucleotide or after synthesis of the oligonucleotide is
complete. The phosphonates can also be converted to phosphoramidate
derivatives by reaction with a primary or secondary amine in the
presence of carbon tetrachloride.
[0285] In the triester synthesis, a protected phosphodiester
nucleotide is condensed with the free hydroxyl of a growing
nucleotide chain derivatized to a solid support in the presence of
coupling agent. The reaction yields a protected phosphate linkage
which may be treated with an oximate solution to form unprotected
oligonucleotide.
[0286] To indicate the three approaches generically, the incoming
nucleotide is regarded as having an "activated" phosphite/phosphate
group. In addition to employing commonly used solid phase synthesis
techniques, oligonucleotides may also be synthesized using solution
phase methods such as diester synthesis. The methods are workable,
but in general, less efficient for oligonucleotides of any
substantial length.
[0287] Preferred oligonucleotides resistant to in vivo hydrolysis
may contain a phosphorothioate substitution at each base (J. Org.
Chem. 55:4693-469, (1990) and Agrawal, (1990)).
Oligodeoxynucleotides or their phosphorothioate analogues may be
synthesized using an Applied Biosystem 380B DNA synthesizer
(Applied Biosystems, Inc., Foster City, Calif.).
EXAMPLE 4
Synthesis of Diformyl Monoacid Tripyrrane (FIG. 25, Formula H) and
Oligonucleotide Conjugate (FIG. 25, Formula J)
[0288] The present example provides for the synthesis of a light
activated drug conjugate. The light activated drug conjugate
includes a oligonucleotide acting as a site directing molecule
coupled with the tripyrrane portion of a texaphyrin as illustrated
in FIGS. 25A-J. (Note: References to "Formula A," "Formula B," etc.
relate to FIGS. 25A, 25B, etc.)
[0289] Synthesis of Dimethylester Dibenzylester Dipyrromethane
(Formula B): A three-neck 1000 mL round-bottom flask set with a
magnetic stirring bar, a thermometer, a heating mantle, and a
reflux condenser attached to an argon line is charged with
methylester acetoxypyrrole (Formula A) (100.00 g; 267.8 mmol), 200
proof ethyl alcohol (580 mL), and deionized water (30 mL.) The
reaction mixture is heated up and when the resulting solution
begins to reflux, 12N aq. hydrochloric acid (22 mL) is added all at
once. The flask contents are stirred under reflux for two hours.
The heating element is replaced by a 0.degree. C. bath and the
resulting thick mixture is stirred for two hours prior to placing
it in the freezer overnight.
[0290] The mixture is filtered over medium fritted glass funnel,
pressed with a rubber dam, and washed with hexanes until the
filtrate comes out colorless. The collected solids are set for
overnight high vacuum drying at 30.degree. C. to afford slightly
yellowish solids (65.85 g, 214.3 mmol, 80.0% yield.)
[0291] Synthesis of Dimethylester Diacid Dipyrromethane, Formula C:
All the glassware is oven dried. A three-neck 2000 mL round-bottom
flask set with a magnetic stirring bar, a hydrogen line, and a
vacuum line is charged with dimethylester dibenzylester
dipyrromethane (Formula B) (33.07 g, 53.80 mmol), anhydrous
tetrahydrofuran (1500 mL), and 10% palladium on charcoal (3.15 g.)
The flask is filled with dry hydrogen gas after each of several
purges of the flask atmosphere prior to stirring the reaction
suspension under a hydrogen atmosphere for 24 hours.
[0292] The solvent of the reaction suspension is removed under
reduced pressure. The resulting solids are dried under high vacuum
overnight.
[0293] The dry solids are suspended in a mixture of saturated
aqueous sodium bicarbonate (1500 mL) and ethyl alcohol (200 mL),
and stirred at its boiling point for five minutes. The hot
suspension is filtered over celite. The filtrate is cooled down to
room temperature and acidified to pH 6 with 12N aqueous
hydrochloric acid. The resulting mixture is filtered over medium
fritted glass. The collected solids are dried under high vacuum to
constant weight (21.63 g, 49.78 mmol, 92.5% yield.)
[0294] Synthesis of Methylester Dibenzylester Tripyrrane, Formula
E: A three-neck 2000 mL round-bottom flask set with a heating
mantle, a magnetic stirring bar, a thermometer, and a reflux
condenser attached to an argon line is charged with dimethyleser
diacid dipyrromethane (Formula C) (21.00 g, 48.33 mmol), ethyl
acetoxy pyrrole (Formula D) (30.50 g), p-toluenesulfonic acid
monohydrate (1.94 g), trifluoroacetic acid (39 mL), and methyl
alcohol (1350 mL.) The flask contents are heated and stirred under
reflux for two hours. The heating element is replaced with a
0.degree. C. bath and the stirring is continued for half an hour
prior to placing the resulting mixture in a freezer overnight.
[0295] The cold mixture is filtered over medium fritted glass. The
collected solids are washed with hexanes and dried under high
vacuum overnight (13.05 g, 19.25 mmol, 39.85 yield).
[0296] Synthesis of Methylester Diacid Tripyrrane, Formula F: All
the glassware is oven dried. A three-neck 500 mL round-bottom flask
set with a magnetic stirring bar, a hydrogen line, and a vacuum
line is charged with methylester dibenzylester tripyrrane (Formula
E) (12.97 g, 19.13 mmol), anhydrous tetrahydrofuran (365 mL), and
10% palladium on charcoal (1.13 g.) The flask is filled with dry
hydrogen gas after each of several purges of the flask atmosphere
prior to stirring the reaction suspension for 24 hours under a
hydrogen atmosphere at room temperature.
[0297] The reaction suspension is filtered over celite. The solvent
of the filtrate is removed under reduced pressure to obtain a foam
which is dried under high vacuum overnight (10.94 g, 21.99 mmol,
87.0% pure.)
[0298] Synthesis of Monoacid Tripyrrane, Formula H: All the
glassware is oven dried. A three-neck 500 mL round-bottom flask set
with a mechanical stirrer, a thermometer, a 0.degree. C. bath, and
an additional funnel set with an argon line is charged with
methylester diacid tripyrrane (Formula F) (10.20 g, 17.83 mmol).
Trifluoroacetic acid (32.5 mL) is dripped into the reaction flask
from the addition funnel over a 45 minute period keeping the flask
contents below 5.degree. C. The resulting reaction solution is
stirred at 0.degree. C. for 15 minutes, and then at 20.degree. C.
for three hours. Triethylorthoformate (32.5 mL) is dripped into the
flask from the addition funnel over a 20 minute period keeping the
flask contents below -25.degree. C. by means of a dry ice/ethylene
glycol bath. The reaction solution is stirred for one hour at
-25.degree. C. and then a 0.degree. C. bath is set up. Deionized
water (32.5 mL) is dripped into the reaction flask from the
addition funnel keeping the flask contents below 110.degree. C. The
resulting two phase mixture is stirred at room temperature for 75
minutes and then added 1-butanol (200 mL.) The solvents are removed
under reduced pressure. The resulting dark oil is dried under high
vacuum overnight to obtain black solids (11.64 g.)
[0299] A three-neck 2000 mL round-bottom flask set with a
thermometer, a heating mantle, a magnetic stirring bar, and a
reflux condenser attached to an argon line, is charged with the
crude methylester diformyl tripyrrane (Formula G) (11.64 g), methyl
alcohol (900 mL), deionized water (60 mL), and lithium hydroxide
monohydrate (4.7 g.) The flask contents are heated, stirred under
reflux for two hours, cooled down to room temperature, added
deionized water (250 mL), acidified with 12N aq. HCL to pH 5, and
then stirred at 0.degree. C. for one hour. The resulting mixture is
filtered over medium fritted glass funnel. The collected solids are
dried under high vacuum to constant weight prior to their
purification by column chromatography (silica gel, MeOH in
CH.sub.2Cl.sub.2, 0-10%; 3.64 g, 8.06 mmol, 45.2% yield.)
[0300] The monoacid tripyrrane (Formula H) is condensed with a
derivatized ortho-phenylene diamine to form a nonaromatic precursor
which is then oxidized to an aromatic metal complex, for example,
Formula I. An oligonucleotide amine may be reacted with the
carboxylic acid derivatized texaphyrin Formula I to form the
conjugate Formula J having the site-directing molecule on the T
(tripyrrane) portion of the molecule rather than the B (benzene)
portion.
EXAMPLE 5
[0301] The following example describes the synthesis of an emulsion
including tin ethyl etiopurpurin (SnEt.sub.2) which is illustrated
in FIG. 26.
[0302] Several emulsions are prepared as described above. In 5 ml
glass tubes, medium chain length oil known as MCT oil (Miglyol 801,
Huls America, Piscataway, N.J.) is combined with 10 mg/gm
SnEt.sub.2 plus excipients as described above. Certain emulsions
also included additional excipients in the following
concentrations: ethanol at mg/gm oil; egg phospholipids at 75 mg/gm
oil; and sodium cholate at 10 mg/gm oil. After incubating for 30
minutes at 55.degree. C., the tubes stand overnight at room
temperature (19.degree.-22.degree. C.). The tubes are centrifuged
to remove bulk precipitates, and supernatants are filtered through
0.45 .mu.m nylon membrane to remove any undissolved drug. Aliquots
of filtrate are then diluted in chloroform:isopropyl alcohol (1:1)
for spectrophotometric determination of drug concentration
(absorbance at 662 nm). Reference standards are prepared with known
concentrations of SnEt.sub.2 in the same solvent.
[0303] The concentration of SnEt.sub.2 in each of the emulsions is
illustrated in Table 2. As illustrated, the concentration of
SnEt.sub.2 in the emulsion can be more than ten times the
concentration in MCT oil alone.
2TABLE 2 Drug Solubility in Oil SnEt.sub.2 SnEt.sub.2 Excipient
Combination Concentration Concentration Added to MCT Oil mg/gm oil
Normalized MCT oil alone 0.38 1.00 + ethanol 0.28 0.74 + egg
phospholipids (EYP) 0.89 2.34 + Na cholate 1.17 3.08 + ethanol +
EYP 1.37 3.61 + EYP + Na cholate 1.77 4.66 + ethanol + Na cholate
2.20 5.79 + ethanol + EYP + Na cholate 4.92 12.95
EXAMPLE 6
[0304] This example illustrates relative efficiencies of several
bile salts. Mixtures of MCT oil, egg phospholipids, ethanol, and
SnEt.sub.2 are incubated with different bile salts, all at 4.6 mM,
under the same conditions described above. As shown in Table 3,
sodium cholate is the most efficient solubilizer. Cholic acid lacks
solubilizing action in the oil.
3TABLE 3 Sodium Cholate is the Most Efficient Co-Solubilizer for
SnEt.sub.2 SnEt.sub.2 SnEt.sub.2 Concentration Concentration Bile
compound mg/gm oil Normalized None 1.26 1.00 Na Tauracholate 1.13
0.90 Cholic acid 1.33 1.06 Na glycocholate 2.22 1.76 Na
deoxycholate 2.31 1.83 Na cholate 3.70 2.94 .sup.1 equivalent to
sodium cholate addition at 10 mg per gram oil or 0.2% w/v in a 20%
o/w emulsion .sup.1 equivalent to sodium cholate addition at 10 mg
per gram oil or 0.2% w/v in a 20% o/w emulsion
EXAMPLE 7
[0305] The following Example illustrates the preparation of
liposomes including BPD-MA (See FIG. 17) as a light activated drug.
A 100-ml batch of BPD-MA liposomes is prepared at room temperature
(about 20.degree. C.) using the following general procedure. BPDMA,
butylated hydroxytoluene ("BHT"), ascorbyl palmirate, and the
phospholipids DMPC and EPG are dissolved in methylene chloride. The
molar ratio of light activated drug: EPG:DMPC is 1.0:3.7 and has
the compositions illustrated in Table 4.
4 TABLE 4 Light activated drug 0.21 g EPG 0.68 g DMPC 1.38 g BHT
0.0002 g Ascorbic acid 6- 0.002 g palmitate Lactose NF 10 g
crystalline injectable Water for injection 100 ml
[0306] Using the above formulation, the total lipid concentration
(% w/v) is about 2.06. The resulting solution is filtered through a
0.22 .mu.m filter and then dried under vacuum using a rotary
evaporator. Drying is continued until the amount of methylene
chloride in the solid residue is no longer detectable by gas
chromatography.
[0307] A 10% lactose/water-for-injection solution is then prepared
and filtered through a 0.22 .mu.m filter. Instead of being warmed
to a temperature of about 35.degree. C., the lactose/water solution
is allowed to remain at room temperature (about 25.degree. C.) for
addition to the flask containing the solid residue of the light
activated drug/phospholipid. The solid residue is dispersed in the
10% lactose/water solution at room temperature, stirred for about
one hour, and passed through a Microfluidizer.TM. homogenizer three
to four times with the outlet temperature controlled to about
200.degree.-250.degree. C. The solution is then filtered through a
0.22 .mu.m Durapore, hydrophilic filter.
[0308] The filterability of the composition in g/cm.sup.2 is
typically greater than about 10. Moreover, the yield is about 100%
by HPLC analysis, with light activated drug potency typically being
maintained even after sterile filtration. Average particle sizes
vary from about 150 to about 300 nm (+50 nm).
EXAMPLE 8
[0309] The following Example describes the delivery of a light
activated drug to an atheroma. An emulsion is prepared having about
0.6 g SnEt.sub.2/ml of emulsion and about 20 g of MCT oil based
hydrophobic phase/ml of emulsion. The catheter illustrated in FIG.
7C is positioned in a vessel of the cardiovascular system using
over the guidewire techniques. The catheter is positioned such that
the media delivery port is adjacent to the atheroma using
radiopaque markers on the catheter and the balloon is expanded into
contact with the vessel wall. The emulsion is delivered via the
third utility lumen 16B of the catheter 10. After the delivery of
the emulsion, the ultrasound energy is delivered at about 0.3
W/cm.sup.2 at a frequency of approximately 1.3 MHz for about ten
minutes. After the delivery of ultrasound energy has concluded, the
catheter is withdrawn from the vasculature of the tumor.
EXAMPLE 9
[0310] The following Example describes the delivery of a light
activated drug to a tumor. An emulsion is prepared having
approximately 0.8 g SnEt.sub.2/ml of emulsion and approximately 30
g of MCT oil based hydrophobic phase/ml of emulsion. The catheter
10 illustrated in FIG. 3A is positioned in the vasculature of a
tumor using over the guidewire techniques. The catheter is
positioned such that the media delivery port is within the tumor
using radiopaque markers included on the catheter. The prepared
emulsion is delivered into the vasculature of the tumor via the
utility lumen 16A. After the delivery of the emulsion, the
ultrasound energy is delivered at about 0.3 W/cm.sup.2 at a
frequency of approximately 1.3 MHz for about fifteen minutes. After
the delivery of ultrasound energy has concluded, the catheter is
withdrawn from the vascular system of the patient.
EXAMPLE 10
[0311] The following Example describes the delivery of a light
activated drug to a potential restenosis site. An emulsion is
prepared having approximately 0.6 g SnEt.sub.2/ml of emulsion and
approximately 30 g of MCT oil based hydrophobic phase/ml of
emulsion. The catheter illustrated in FIG. 7C is positioned in the
vasculature of a patient using over the guidewire techniques. The
catheter is positioned such that the media delivery port is
adjacent to a portion of the vessel which was previously treated
with balloon angioplasty and the balloon is expanded into contact
with the vessel wall. The prepared emulsion is delivered into the
vasculature of the patient via the third utility lumen 16B.
Ultrasound energy is delivered from the ultrasound assembly to the
potential restenosis site at about 0.3 W/cm.sup.2 at a frequency of
approximately 1.3 MHz for about ten minutes. After the delivery of
ultrasound energy has concluded, the catheter is withdrawn from the
vascular system of the patient.
EXAMPLE 11
[0312] The following Example describes the delivery of a light
activated drug to an atheroma. Liposomes are prepared including
BPD-MA (See FIG. 17) as the light activated drug and DMPC and EPG
as the phospholipids. The molar ratio of BPDMA:EPG:DMPC is about
1:3:7. The catheter illustrated in FIG. 7C is positioned in a
vessel of the cardiovascular system using over the guidewire
techniques. The catheter is positioned such that the media delivery
port is adjacent to the atheroma using radiopaque markers included
on the catheter and the balloon is expanded into contact with the
vessel. Ultrasound energy is delivered at about 0.3 W/cm.sup.2 at a
frequency of approximately 1.3 MHz for about 20 minutes in order to
rupture the liposomes and cause tissue death within the atheroma.
After the delivery of ultrasound energy has concluded, the catheter
is withdrawn from the vascular system of the patient.
EXAMPLE 12
[0313] The following Example describes the delivery of a light
activated drug to a tumor. Liposomes are prepared including BPD-MA
(See FIG. 17) as the light activated drug and DMPC and EPG as the
phospholipids. The molar ratio of BPD-MA:EPG:DMPC is about 1:3:7.
The catheter illustrated in FIG. 8 is positioned in the vasculature
of a tumor using over the guidewire techniques. The catheter is
positioned such that the media delivery port is within the tumor
using radiopaque markers included on the catheter. Ultrasound
energy is delivered at about 0.3 W/cm.sup.2 at a frequency of
approximately 1.3 MHz for about 20 minutes in order to rupture the
liposomes and cause tissue death within the atheroma. After the
delivery of ultrasound energy is concluded, the catheter is
withdrawn from the vasculature of the tumor.
EXAMPLE 13
[0314] The following Example describes the delivery of a light
activated drug to a potential restenosis site. Liposomes are
prepared including BPD-MA (See FIG. 17) as the light activated drug
and DMPC and EPG as the phospholipids. The molar ratio of
BPD-MA:EPG:DMPC is approximately 1:3:7. The catheter illustrated in
FIG. 7C is positioned in the vasculature of a patient using over
the guidewire techniques. The catheter is positioned such that the
media delivery port is adjacent to a portion of the vasculature
which was previously treated with balloon angioplasty and the
balloon is inflated into contact with the vessel wall. Ultrasound
energy is delivered at about 0.3 W/cm.sup.2 at a frequency of
approximately 1.3 MHz for about 15 minutes in order to rupture the
liposomes and cause tissue death within the atheroma. After the
delivery of ultrasound energy is concluded, the catheter is
withdrawn from the vasculature of the patient.
EXAMPLE 14
[0315] The following Example describes the delivery of a light
activated drug to an atheroma. Liposomes are prepared including
BPD-MA (See FIG. 17) as the light activated drug and DMPC and EPG
as the phospholipids. The molar ratio of BPD-MA:EPG:DMPC is about
1:3:7. The phospholipids are systemically delivered. The catheter
illustrated in FIG. 7C is positioned in the vasculature of a
patient using over the guidewire techniques. The catheter is
positioned such that the media delivery port is adjacent to the
atheroma and the balloon is inflated into contact with the vessel
wall. Ultrasound energy is delivered at about 0.3 W/cm at a
frequency of approximately 1.3 MHz for about 15 minutes. After the
delivery of ultrasound energy is concluded, the catheter is
withdrawn from the vasculature of the patient.
EXAMPLE 15
[0316] The following Example describes the delivery of a light
activated drug to a tumor. Microbubbles are prepared including
cisplatin and photofrin according to the methods disclosed in U.S.
Pat. No. 5,770,222. The microbubbles are systemically administered.
The catheter illustrated in FIG. 1A is positioned within the
vasculature of a tumor. Ultrasound energy is delivered at about 0.3
W/cm.sup.2 at a frequency of approximately 1.3 MHz for about 15
minutes. After the delivery of ultrasound energy is concluded, the
catheter is withdrawn from the vasculature of the patient.
EXAMPLE 16
[0317] The following Example describes the delivery of a light
activated drug to a tumor. Microbubbles are prepared including
cisplatin and photofrin according to the methods disclosed in U.S.
Pat. No. 5,770,222. The catheter illustrated in FIG. 3A is
positioned within the vasculature of a tumor. The microbubbles are
delivered to the tumor via the second utility lumen 16A of the
catheter. Ultrasound energy is delivered at about 0.3 W/cm.sup.2 at
a frequency of approximately 1.3 MHz for about 15 minutes. After
the delivery of ultrasound energy is concluded, the catheter is
withdrawn from the vasculature of the patient.
EXAMPLE 17
[0318] The following Example describes the delivery of a light
activated drug to a thrombosis. Microbubbles are prepared including
heparin, photofrin and an albumin substrate. The microbubbles are
systemically administered. The catheter illustrated in FIG. 1A is
positioned adjacent to the thrombosis. Ultrasound energy is
delivered at about 0.2 W/cm.sup.2 at a frequency of approximately
1.3 MHz for about 20 minutes. After the delivery of ultrasound
energy is concluded, the catheter is withdrawn from the vasculature
of the patient.
* * * * *