 |
Description  |
|
|
BACKGROUND OF THE INVENTION
A variety of medical diseases are beneficially treated by therapeutic
agents which are selectively directed to the site of the disease, thereby
causing the death of the cells responsible for the disease without harming
normal cells. Thus, there is considerable emphasis in the medical
technology community focused on obtaining such site directed therapeutical
chemicals. Two such diseases where these types of chemicals would be most
advantageously applied are atherosclerosis, and cancer.
Atherosclerosis is a disease associated with occlusion of blood vessels,
arteries and the like in which fatty substances, particularly lipids, form
deposits in the vessels. Such deposits are commonly referred to as
"arteriosclerotic plaques". Generally, these plaques form as a result of
lipids being deposited in and beneath the intima of arteries and veins.
The intima is the innermost membrane lining of these vessels. Generally,
atherosclerosis involves medium and large-size vessels, with the most
commonly affected being the aorta, iliac, femoral, coronary, and cerebral
arteries. If the disease is not checked, tissues or organs that are distal
to the atherosclerotic plaque experience reduced blood flow, and thus are
adversely effected.
For the most part, atherosclerosis is treated by one of three approaches.
First, the vascular regions that are diseased are often replaced by
prosthetic or natural grafts. Grafting is a very expensive and medically
demanding procedure, and often presents significant associated risks to
the patient. The second approach is to put the atherosclerotic patient on
drugs, particularly antiarrhythmic, anticoagulant, and plasma lipid
lowering chemicals. These substances are also very expensive, and the
adverse long-term effects of taking them are not known.
A third method has been proposed for treating atherosclerosis. This is
exemplified in U.S. Pat. No. 4,512,762 which shows a photochemical process
for destroying atherosclerotic plaques involving the uptake of
hematoporphyrin into plaques coupled with lysis of the plaques following
irradiation. Unfortunately, this method has two undesirable aspects; first
hematoporphyrin sensitizes patients to subsequent exposure to sunlight.
Second hematoporphyrin is taken up to a significant extent by tissues or
cells that surround the plaques. Consequently normal tissue may be
destroyed along with the plaques upon subsequent irradiation.
There is a substantial body of literature concerning the treatment of
cancer. One regime, chemical therapy, involves administering drugs to a
patient that exert their effects primarily by interrupting DNA synthesis.
Such drugs have shown considerable promise, and are particularly effective
in various combinations when applied to a particular type of cancer. A
major drawback associated with chemical therapy, however, is that the
therapeutic agent is generally not cell-type specific for cancer cells,
but rather is taken up into the DNA of any dividing cell. Consequently,
normal cells, as well as cancer cells, are killed by this treatment. Thus,
there are severe side effects associated with chemical therapy as it is
presently practiced.
A more recent treatment for cancer is described by R. L. Lipson et al in
"The Use of a Derivative of Hematoporphyrin in Tumor Detection", J. Natl.
Cancer Inst. 26(1), p. 1-8, 1961. Hematoporphyrin is injected into a
patient experiencing a tumor burden. After injection it is taken up by the
tumor. Subsequent irradiation causes lysis of the tumor. Unfortunately,
this method has the same drawbacks as treatment of atherosclerosis with
hematoporphyrin: the patient may become sensitized to sunlight, and there
is the likelihood of destruction of normal tissue.
SUMMARY OF THE INVENTION
The present invention provides two new therapeutic methods of using a known
substance, phycocyanin, that are premised on the photochemical effects cf
the molecule when it is irradiated with a suitable wavelength of light.
One aspect of this invention involves the removal of atherosclerotic
plaques by contacting the plaques with phycocyanin coupled with
irradiation. As a result, the patient may expect to have a substantial
removal of the products that accumulated due to atherosclerosis. This
method consists of administering phycocyanin, preferably by intravenous or
intraarterial injection into the main artery or other blood vessels
afflicted with atherosclerosis or intraperitoneally. After a short period
of time, the injected hycocyanin contacts the cells that comprise the
atherosclerotic plaque. On contact, it is taken up into the membranes of
the cells in a selective fashion with little or no absorption by
surrounding healthy tissue. Upon subsequent irradiation with light,
phycocyanin undergoes a reaction causing the probable release of a free
radical, singlet oxygen. The latter reacts with and is destructive to the
cells that comprise the atherosclerotic plaque. Alternatively, phycocyanin
might prove effective by providing a means to selectively absorb laser
energy, thereby enhancing thermal abalation of the plaque by laser energy.
Irradiation can be provided using a catheter containing a suitable light
source. Other, less favored methods for irradiating plaques can similarly
be employed.
In the practice of the second method, phycocyanin can be used to destroy
malignant tumors. A property of phycocyanin that makes it particularly
uniquely suited as an anti-cancer therapeutic agent is that it is
selectably taken up in cancer cell membranes, and consequently upon
irradiation, primarily cancer cells are destroyed, with little destruction
of surrounding normal cells or tissue. Depending on the type of tumor that
is sought to be treated, the mode of treatment wherein phycocyanin is
presented to the tumor will vary considerably. For treating skin tumors,
phycocyanin can be injected into, or about the region of the tumor and
followed by subsequent irradiation. For tumors infernal to the body,
phycocyanin can be presented to the tumor via a catheter, and the same
catheter can be used to irradiate the tumor. Upon irradiation of the tumor
containing phycocyanin, singlet oxygen is produced or laser energy is
selectively absorbed, thereby causing the destruction of the tumor cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a light miscroscopic image of a crosssection through an
atherosclerotic human artery which has been exposed to a physiological
salt solution without phycocyanin, and irradiated.
FIG. 2 shows the selective uptake of phycocyanin in an atherosclerotic
plaque.
FIG. 3 shows a light micrograph of a cross-sectioned area of a human
atherosclerotic artery which has been exposed to phycocyanin in a
physiological salt solution, and subsequently irradiated with ultra-violet
light.
FIG. 4 shows a cross-section through an atherosclerotic blood vessel that
was treated with phycocyanin in a physiologically compatible salt
solution, and subsequently irradiated and stained with Eosin.
DETAILED DESCRIPTION OF THE INVENTION
The medical uses of phycocyanin are based either upon the release of
singlet oxygen upon irradiation of phycocyanin at a particular wavelength
or by the selective absorption of thermal energy. These properties of
phycocyanin are particularly suited for the destruction of atherosclerotic
plaques and malignant tumors. Each of these aspects of the instant
invention will be described separately.
It will be appreciated that the term phycocyanin refers to a protein-bound
pigment having an open-chain tetrapyrrole structure, and a blue
coloration. Phycocyanin is a member of a broader class of similar
compounds termed phycobilins. Because of the similar chemical structures
of the members of this group, it is anticipated that a large number of
molecules in the group can be substituted for phycocyanin in the instant
invention. Phycocyanin can be obtained commercially from several
commercial sources one of which is Sigma Chemical Company located in St.
Louis, Mo.
A favorable property of phycocyanin that enables it to be used successfully
to treat atherosclerosis is that it appears to be selectively concentrated
in atherosclerotic plaques. This was shown by incubating a segment of a
human atherosclerotic coronary artery obtained at autopsy with 0.1
milligrams/milliliter of phycocyanin in a suitable physiologically
compatible buffer. FIG. 2 is a light micrograph of a cross-section of the
segment upon exposure to monochromatic light at a wavelength of 577
nanometers. This is close to the peak absorption of phycocyanin, 620
nanometers. It is clearly seen that phycocyanin, represented by the dark
areas, is predominantly located in the plaque region, and only appears in
lesser amounts at the artery walls associated with the thin muscle coat.
Since atherosclerotic plaques are composed primarily of cells which are
laden with lipids and other materials, destruction of these cells by
photoactivation of phycocyanin or thermal absorption of laser energy
should cause destruction of the plaques. As mentioned above, this is
thought to be primarily due to singlet oxygen produced by phycocyanin upon
irradiation or to thermal ablation. While the Applicant does not consider
himself to be bound by this theory, it is, nonetheless, believed that
singlet oxygen is at least partially responsible for cellular destruction.
Thus, the instant invention consists of a method for photodestruction of
atherosclerotic plaques by activation of plaque-bound phycocyanin.
A variety of procedures are available to effect delivery and irradiation of
phycocyanin in atherosclerotic arteries. U.S. Pat Nos. 4,336,809 and
4,512,762 present two conceivably usable methods, and both of these
patents are hereby incorporated by reference. The former patent describes
a device for delivering laser light of a particular wavelength to a
diseased site treated with hematoporphyrin. Hematoporphyrin is known to be
cytotoxic to cells when irradiated with a suitable wavelength of light.
Thus, the system shown in that patent application can be beneficially
applied to the uses described herein for phycocyanin.
U.S. Pat. No. 4,512,762 describes two methods whereby phycocyanin can be
delivered and subsequently irradiated to effect treatment at a particular
site. The first method is somewhat similar to U.S. Pat. No. 4,336,809, in
that it involves irradiating hematoporphyrin with a dye laser wherein the
light emitted is presented via a balloon catheter to tissue containing
hematoporphyrin. A variety of suitable balloon catheters are well known to
those skilled in the art. The second method shown in U.S. Pat. No.
4,512,762 is the use of "liquid-light" to effectively irradiate
phycocyanin. It is anticipated that there is a variety of chemiluminescent
liquids that when injected into the bloodstream to a patient will have few
or no side-effects, yet will provide sufficient light to irradiated
phycocyanin. U.S. Pat. No. 4,512,762 utilizes peroxyoxylate manufactured
by American Cyanamide to irradiate hematoporphyrin. It is likely that
similar chemicals can be utilized in the instant invention.
It will be appreciated that a major advantage associated with "liquid
light" is that it can be injected into the patient without knowing
precisely where the atherosclerotic plaques reside. That is, once this
substance is injected, it will pass throughout the bloodstream, causing it
to come into contact with plaques wherever they may have formed. A further
advantageous application of this method is that it avoids painful and
sometimes dangerous catherization procedures that are necessarily employed
when laser light is delivered via an optical delivery system. Regardless
of the type of system used to irradiate phycocyanin, the wavelengths of
light suitable for this purpose are in the range of 375 nm, 485-518 nm,
600 nm, 620 nm, or 647 nm. The total energy delivered at these wavelengths
can vary depending on the size of the plaque being treated. Of course, it
is possible to vary the wavelength and thereby avoid possible adverse
heating effects to surrounding tissue arising from prolonged irradiation.
The favorable properties of phycocyanin observed upon irradiation at a
suitable wavelength of light can be efficaciously applied to the treatment
of tumors as well as to the treatment of atherosclerotic plaques. The
manner in which the chemical is delivered to the site of the tumor can be
essentially similar to the manner in which it is utilized to treat
atherosclerotic plaques as described above. It will be appreciated that
virtually any type of tumor can be treated by either method.
It will be further appreciated that phycocyanin is uniquely suited to
destroy blood borne metastasis using "liquid light" to effectively
irradiate phycocyanin bound to the tumor cells. For instance, phycocyanin
in a suitable physiologically compatible solution can be injected into the
vascular tree of a patient who is carrying a metastatic tumor whereupon it
will contact and bind to any blood borne tumor cells. Upon irradiation by
"liquid light", injected along with or after injection of phycocyanin, the
metastatic cells will be destroyed.
The concentration of phycocyanin which will produce optimal effects when
applied to the treatment of atherosclerotic plaques or tumors, will vary
depending on the size and location of the disease in the body of the
patient. For a particular use, the most efficacious concentration will be
determined empirically merely by injecting different concentrations of
phycocyanin and subsequently irradiating it, and then following the course
to the patient.
It will be appreciated by those skilled in the art that there are various
ways of practicing the instant invention. Thus, the following examples are
presented in the spirit of demonstrating representative applications; by
no means should they be construed as limiting the invention to these
particular applications.
EXAMPLE I
Destruction of Atherosclerotic Plaques with Phycocyanin
Atherosclerotic arteries isolated from a post mortem human within 5 hours
after death were perfused with an oxygenated Krebs/Ringer bicarbonate
solution containing about eight micromoles of phycocyanin for about four
days. Subsequently, the tissue was irradiated for 10 minutes on four
separate days using a 15-watt Sylvania 15T8-A1 black light florescent
bulb, which emits maximally at a wavelength of 375 nm. As a control,
atherosclerotic arteries were similarly treated, except phycocyanin was
omitted from the solution.
At the end of the four day treatment period, both phycocyanin treated and
control arteries were fixed in formalin, embedded in wax, and sectioned
using well-known histological techniques. The results are shown in FIGS.
1, 2, 3, and 4.
FIG. 1 is a light micrograph of a human artery cross-sectioned after having
been exposed to Krebs/Ringer solution without phycocyanin. It is apparent
from the photograph that the plaque remains intact after treatment. In
contrast, FIG. 2 shows a micrograph of a cross-sectioned area of the
artery which was exposed to a solution containing phycocyanin, and
subsequently irradiated as described above. It is clear that there is
considerable destruction of the plaque in several locations along the
artery. Further, it appears that the plaque has pulled away from the
artery in several areas. FIG. 4 shows an artery that was treated with
phycocyanin, irradiated, and cross-sectioned. In addition, this artery was
stained with a viable stain, Eosin. It is more apparent when this stain is
applied that the atherosclerotic plaque has been considerably destroyed.
EXAMPLE II
Effect of Phycocyanin on Experimentally Induced Tumors
An experimentally inducible murine tumor was used as a model system with
which to study the effects of phycocyanin irradiation on tumor growth.
Several mice were innoculated with a tumorogenic dose using the mouse
myeloma cell line Sp2/0. The latter produces dermal tumors in Balb-c mice.
After tumors were apparent, phycocyanin was injected intravenously in a
balanced saline solution at a concentration of about 0.25 g/per kg.
Control mice were not injected with phycocyanin, but did receive the
saline solution. After 24 hours, both experimental and control mice were
irradiated externally with a 15T8 black light florescent bulb for one
hour. The latter is produced by Sylvania and emits maximally at 375 nm.
Animals which received phycocyanin showed a marked reduction in tumor size
within 5 days after light treatment compared to animals which received
saline only.
EXAMPLE III
Elimination of Tumor Growth With Phycocyanin
The materials and methods described in Example II were similar here with
the following exceptions. Five hours after intravenous injection of 0.25
grams of phycocyanin/kilogram of mouse weight, it was observed and skin
covering the tumor exhibited blue coloration indicating that phycocyanin
had been concentrated in the tumor. Surrounding normal skin areas were a
healthy pink color. Subsequently, the tumor was irradiated with an argon
laser at wave lengths of 488-518 nanometers delivered from a cleaved end
fiber placed about 5 centimeters external to the tumor. This generated a
2-centimeter diameter spot of light. The light intensity was adjusted to a
total energy dose of about 72 Joules/per square centimer Tumor growth and
metastasis was monitored over the following ten day period. This mode of
treatment completely inhibited tumor growth during this time. In contrast,
animals which were injected with a saline solution lacking phycocyanin
experienced aggressive tumor growth and metastasis during the ten day
period.
EXAMPLE IV
Toxicity of Phycocyanin
Studies were done to determine the LD.sub.50, or the concentration of
phycocyanin which kills 50% of the mice treated with phycocyanin, using
standard techniques well known to those skilled in the art. Approximately
0.3 gm of phycocyanin/ kg was determined to be the LD.sub.50 when the drug
was administered intravenously. Similar studies were conducted on mice
which received intraperitoneal injections of phycocyanin. The LD.sub.50
for this route of administration was determined to be about 0.5 gm/kg.
In addition to the above study, the toxicity of phycocyanin to heart tissue
was determined. The study consisted of isolating a beating rabbit heart,
and perfusing the heart with a suitable saline solution of 64 micromolar
phycocyanin for fifteen minutes. There was no effect on the viability of
the heart as measured by its contractile properties.
It will be apparent to those skilled in the art that there are various
material and method substitutions applicable to the instant invention.
Particularly, there are many devices which can be employed for irradiating
phycocyanin. The the embodiments described above are to be considered in
all respects as illustrating, but not restricting the scope of the
invention. Thus, the scope of the invention is indicated by the appended
claims rather than by the foregoing Examples, and all changes which come
within the meaning and range of equivalency of the claims are therefore
intended to be embraced by the claims.
* * * * *
|
|
 |
|
 |
Description |
|
