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BACKGROUND OF THE INVENTION
This invention relates to the diagnosis and treatment of undesirable tissue
such as malignant tumors by certain drugs that accumulate in the
undesirable tissue.
In one class of diagnosis and treatment with photosensitizing drugs, tumors
are detected and treated by irradiating the tumors with light after the
drug accumulates in the tumor. The drugs are photosensitizing and some of
the drugs in this class are derivatives of hemoglobin.
There are several prior art techniques for such diagnosis and treatment.
For example, in "Etudes Sur Les Aspects Offerts Par Des Tumeur
Experimentales Examinee A La Lumiere De Woods", CR soc. Biol. 91:
1423-1424, 1924, Policard, the author, noted that some human and animal
tumors fluoresced when irradiated with a Wood's lamp. The red fluorescence
was attributed to porphyrins produced in the tumor. In "Untersuschungen
Uber Die Rolle Der Porphine Bei Geschwulstkranken Menschen Und Tieren", Z.
Krebsforsch 53:65-68, 1942, Auler and Banzer showed that hematoporphyrin,
a derivative of hemoglobin, would fluoresce in tumors but not in normal
tissues following systemic injection into rats.
In "Cancer Detection Therapy Affinity of Neoplastic Embryonic and
Traumatized Regenerating Tissue For Porphyrins and Metalloporphyrins",
Proc Soc Exptl Biol Med. 68: 640-641, 1948, Figge and co-workers
demonstrated that injected hematoporphyrinwould localize and fluoresce in
several types of tumors induced in mice. In "The Use of a Derivative of
Hematoporphyrin in Tumor Detection", J Natl Cancer Inst. 26:1-8, 1961,
Lipson and co-workers disclosed a crude material, prepared by acetic
acid-sulfuric acid treatment of hematoporphyrin, said material having a
superior ability to localize in tumors.
The photosensitive characteristic of tumor-selective porphyrin compounds
also make them useful in the treatment of tumors. In "Photodynamic Therapy
of Malignant Tumors", Lancet 2: 1175-1177, 1973, Diamond and co-workers
achieved tumor necrosis after lesion-bearing rats were injected with
hematoporphyrin and exposed to white light. In "Photoradiation Therapy for
the Treatment of Malignant Tumors", Cancer Res. 38: 2628-2635, 1978, and
"Photoradiation in the Treatment of Recurrent Breast Carcinoma", J Natl
Cancer Inst. 62:231-237, 1979, Dougherty and co-workers reported using the
crude Lipson hematoporphyrin derivative to accomplish photoradiation
therapy on human patients.
The crude Lipson hematoporphyrin derivative has the ability to enter a
variety of tissues and to be retained in tumor cells after it has mostly
cleared the serum. Subsequent irradiation with red light excites the crude
Lipson derivative which in turn excites oxygen molecules. The excited
oxygen molecules exist for a microsecond--long enough to attack tumor cell
walls and effect necrosis. In "Effects of Photo-Activated Porphyrins in
Cell Surface Properties", Biochem Soc Trans 5: 139-140, 1977, Kessel
explained that cross-linking of proteins in tumor cell membranes causes
leakage and eventual cell disruption.
The crude Lipson hematoporphyrin derivative has several disadvantages such
as: (1) it enters normal tissue and causes unacceptable damage to the
normal tissue when therapeutic light sufficient to treat large tumors is
applied; (2) it does not clear normal tissue sufficiently soon and thus
some patients are harmed by exposure to ordinary sunlight as much as
thirty days following treatment with the drug; and (3) it does not have an
optimum absorbance spectrum in a range that penetrates tissue most
effectively.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide novel equipment
for the localization, characterization and/or treatment of tumors and
certain other tissue.
It is a still further object of the invention to provide equipment which
may deliver radiation to neoplastic tissue, determine the depth of
penetration of the radiation and determine the amount of drug and oxygen
in the tissue.
It is a still further object of the invention to provide novel equipment
for delivering radiation to tissue, which equipment is self-monitoring.
In accordance with the above noted and other objects of the invention,
photosensitizing, undesirable-tissue-selective drugs are obtained from
phlorin or chlorin or other pyrrole-containing molecules. Generally, these
drugs are neoplastic-tissue selective including hyperproliferatic tissue
selective and tumors. These drugs are an effective in vivo photosensitizer
and have the following properties: (1) they are retained in malignant
tissue; (2) their molecules are not easily disaggregated from each other
by serum protein; (3) they are efficient in producing a photochemical
effect in vivo which is toxic to cells or tissue; (4) they absorb light at
wavelengths which penetrate tissue; (5) they are relatively non-toxic in
the absence of the photochemical effect in effective doses; (6) they are
readily cleared from normal tissues; (7) they have a triplet energy state
above 37.5 kilo-calories; (8) they are not readily oxidized; (9) they
don't readily quench required excited states; and (10) they are water
soluble.
This drug is an improvement over earlier drugs because of its selectivity.
This selectivity occurs in one embodiment because the drug has the ability
to remain self-associated in serum at least to some degree for a certain
period of time which is at least fifteen minutes and to bind within the
cell. It is believed that the self-association causes the drug to be
removed from normal tissue but retained in neoplastic tissue at least
partly in some cases by the endothelial cells of the tumors as well as by
the tumor cells in higher concentrations than in most normal tissue and
for longer periods of time than in most normal tissue.
In addition to selectivity, the drug must dissociate in the tissue or in
lipids before it is energized by radiation to damage the neoplastic
tissue. This combination of self-association in serum and dissociation in
lipids occurs, in one embodiment, because the individual molecules have
sufficiently higher attraction for each other than for water to form
aggregates of molecular weight greater than 10,000 and sufficient
attraction for lipids compared to each other to dissociate in tissue.
The individual molecules each include two groups bound to each other each
including four rings, some of which are pyrroles such as phlorins,
porphyrins, chlorins or substituted phlorins, pyrroles or chlorins, each
group forming a ring so that they have sufficient self-affinity to form
aggregates of molecular weight above 10,000 in water, in isotonic saline
and in the vascular system but may break down in neoplastic tissue and
attach to the cell.
Moreover, other photosensitizing materials may be combined with a carrier
that enters undesirable tissues and cells of the reticular endothelial
system such as macrophages. These photosensitizing materials: (1) must
have a triplet energy state above 37.5 kilocalories per mole; (2) cannot
be easily oxidized; and (3) not physically quench any required energy
state. Preferably, this photosensitizing material should be lipophlic.
In one embodiment, a known reagent is formed by hydrolysis of the reaction
mixture of hematoporphyrin and acetic-sulfuric acids. A suitable drug is
purified from this reagent by elimination of low molecular weight
compounds by filtration through a microporous membrane. This drug contains
porphyrins at least 50 percent of which, and preferably more than 90
percent of which have the emiprical formula of approximately C.sub.68
H.sub.70 N.sub.8 O.sub.11 or C.sub.68 H.sub.66 N.sub.8 O.sub.11 Na.sub.4.
Other derivatives may be formed from this compound and it is believed other
compounds may be formed either from other natural porphyrins or by
synthesis from other materials such as by polymerization of monomeric
pyrroles by dipyrollic intermediates, from pyrromethenes, from
pyromethanes, from pyroketones, from open chain tetrapyrrolic
intermediates, from bilanes, from oxobilanes and from bilines. They may
also be derived from natural pigments such as chlorophyll and hemogloblin.
Such suitable compounds are described more fully in Porphyrins and
Metalloporphyrins by J. E. Falk and Kevin M. Smith, 1975, Elsevier
Scientific Publishing Company, Amsterdam, N.Y. and Oxford, the disclosure
of which is incorporated herein.
Generally, the drugs are composed of groups of pyrroles or substituted
pyrroles combined in a pattern. That pattern includes as a basic grouping
a structure which is phlorin or a group of four pyrroles or combinations
of pyrroles and substituted pyrroles formed into a larger ring. Two such
rings are covalently bound to form a pair of units each having four
pyrrole groups of four groups at least some of which are pyrroles or
substituted pyrroles. The molecules preferably have an absorption spectrum
which is within the range of wavelengths between 350 nm and 1200 nm. The
absorption spectrum should be tailored to the desired penetration such as,
for example, being strong in the red or near infrared wavelengths
(600-1200 nm) for large bulking tumors and in the green or blue
wavelengths such as 488 or 514 nm for superficial undesirable tissue.
In use for therapy, the drug is caused to enter the subject, where it is
cleared from normal tissue sooner than from abnormal neoplastic tissue.
After the drug has cleared normal tissue but before it has cleared
abnormal neoplastic tissue, the abnormal neoplastic tissue may be located
by the luminescence of the drug in the abnormal neoplastic tissue. The
fluorescence may be observed with low intensity light some of which is
within the drug' absorbance spectrum or higher intensity light, a portion
of which is not in the drugs' absorbance spectrum. Similarly, the drug is
absorbed and retained by certain pathogens after it has cleared normal
tissue.
To destroy the abnormal neoplastic tissue or pathogens, a higher intensity
light having a frequency within the absorbance spectrum of the drug is
applied. A synergistic effect without substantial destruction of tissue by
heat is achieved by applying heat before, during or after the light
radiation is applied and thus the tissue should be heated above 39.5
degrees Celsius and preferably within the range of 40.5 and 45 degrees
Celsius. The increase in temperature may be achieved by transmitting light
near or in the infrared spectrum or microwaves to the tissue. The
temperature change should be within two hours before or two hours after
treatment with light.
In the alternative, higher power laser light within the absorption spectrum
of the drug causes thermal destruction of tissue which is interactive with
the photodynamic effect of the drug. This removes bulky tumors or
obstructions by vaporization or vascular occlusion such as by coagulation
of blood.
DESCRIPTION OF THE DRAWINGS
The above noted and other features of the invention will be better
understood from the following detailed description, when considered with
reference to the accompanying drawings, in which:
FIG. 1 is a mass spectrometry printout of a drug in its methyl ester form;
FIG. 2 is a visible light spectrum of a drug in a water solution;
FIGS. 3 and 3A are in combination an infrared spectrum of the drug
dispersed in potassium bromide;
FIG. 4 is a carbon-13 nuclear magnetic resonance print-out of the drug,
referenced to dimethyl sulfoxide;
FIGS. 5 and 5A are in combination a print-out from a Waters Associates
Variable Wave Length Detector used in conjunction with its U Bondpak C-18
column, showing various components of HpD including a peak formation
representative of the drug;
FIGS. 6 and 6A are in combination a print-out from a Waters Associates
Variable Wave Length Detector used in conjunction with its U Bondpak C-18
column showing various components of the drug DHE;
FIG. 7 is a carbon-13 nuclear magnetic resonance print-out of the drug,
referenced to tetramethylsilane in deuterated chloroform solvent.
Magnification spectrum is shown in the ranges from 20-30 ppm and 55-75
ppm;
FIG. 8 is a block diagram of a system useful in practicing the invention;
FIG. 9 is a block diagram of another system useful in practicing the
invention;
FIG. 10 is a simplified enlarged longitudinal sectional view of a portion
of the system of FIG. 9;
FIG. 11 is a developed view of the portion of the system of FIG. 8 that is
shown in FIG. 10;
FIG. 12 is a simplified perspective view partly broken away of another
embodiment of a portion of FIG. 9;
FIG. 13 is a perspective view partly broken away of another embodiment of a
portion of the system of FIG. 9;
FIG. 14 is a longitudinal sectional view of the embodiment of FIG. 13;
FIG. 15 is an elevational view of still another embodiment of a portion of
the system of FIG. 9;
FIG. 16 is a perspective view partly broken away of the embodiment of FIG.
15;
FIG. 17 is a sectional view of a portion of the embodiment of FIG. 15;
FIG. 18 is a perspective simplified view, partly broken away of another
embodiment of a portion of FIG. 8;
FIG. 19 is a schematic view of another portion of the embodiment of FIG. 8;
and
FIG. 20 is a block diagram of still another portion of the embodiment of
FIG. 9.
DETAILED DESCRIPTION
General Description of the Drug
Each of the drugs may be classified into one of two classes, which are: (1)
each molecule of the drug aggregates in water to aggregates having a
combined molecular weight of above 10,000; or (2) units of the drug are
encapsulated in a liposome and molecules include at least one such
photosensitizing chemical group.
The aggregates in the former class are sufficiently large and have
characteristics which cause them to be removed by the lymphatic system so
as to be excluded from most normal tissue and usually to enter and be
retained by undesirable tissue, such as tumors. Because of the absence of
a lymphatic system, the drug is not removed effectively from the tumors.
The drugs of this invention bind within the cells to plasma membrane,
nuclear membrane, mitochondria, and lysosomes. While it may enter some
normal tissue, generally there is a sufficient difference in the rates of
accumulation and removal between normal and undesirable tissue to provide
selected conditions which permit treatment of undesirable tissue without
excessive damage to normal tissue.
The form of drugs which aggregate must be sufficiently lipophlic to
dissociate in lipids so that the aggregate is broken up within the tumor
into a form which: (1) readily absorbs light within the light spectrum of
350 to 1,200 nm in wavelength; and (2) causes photodynamic effects. Thus,
the drug is soluble in water to form large aggregates in aqueous
suspension but sufficiently lipophilic to dissociate in neoplastic tissue.
At least one porphyrin utilized in the past by therapists as part of
Lipson's reagent without knowing that it existed therein, has the
necessary characteristics but in the prior art was utilized in a mixture
of porphyrins which had deleterious side effects. It was not known that
the substance was effective agent in Lipson's reagent or that it existed
therein because of its resistance to separation by liquid chromatography.
Reduced side effects are obtained from such a mixture of porphyrins when
the mixture includes more than 50% of the drug and preferably 90% or more
by weight of the porphyrins should be the drug or a drug having similr
characteristics. With such a purified dosage, the porphyrins clear normal
tissue adequately before the neoplastic tissue in which the drug has
accumulated is exposed to light.
This drug (DHE) appears to be ineffective if it is in aggregates of
molecular weight less than 10,000. Such lower molecular weight aggregates
appear to be stable. Molecular weight of the aggregate in this application
means the sum of the molecular weights of the molecules in an aggregate of
molecules. An aggregate of molecules consists of a group of molecules
bound together by forces other than covalent bonds.
Other drugs such as certain phlorins or chlorins have been used either with
two groups bound together or single groups encapsulated in a liposome. In
an drug, the drug must bind within the neoplastic tissue or release a drug
that binds within the neoplastic tissue. More specifically, the drug
includes compounds in which the individual molecules include two groups,
each of which includes either phlorin or rings of pyrroles or hydrogenated
pyrroles, or substituted pyrroles connected in such a way as to expose
planes of both rings to other drug molecules.
With this structure, the attraction between molecules is greater than the
attraction to water and thus molecules of the drug aggregate in aqueous
suspensions. One such compound, dihematoporphyrin ether (DHE), purified
from Lipson's reagent, is shown in formula 1 and another such compound,
which is a chlorin, is shown in formula 2. The chlorin shown in formula 2
may be synthesized from chlorophyll or formed as a derivative from the
compound of formula 1. The attraction to lipids is, however, sufficiently
great to cause the aggregates to dissociate in a lipid environment.
Metallo derivatives of the active compounds may be used, provided they do
not interfere with the photosensitizing property of the molecules. For
example, magnesium derivatives continue to work but copper derivatives do
not.
GENERAL DESCRLIPTION OF DRUG PREPARATION
First, for one embodiment, hematoporphyrin derivative is formed, using
prior art methods or novel methods similar to prior art methods. This
mixture contains a suitable drug. This suitable drug, when formed in the
hematoporphyrin derivative, is normally in a mixture of other undesirable
porphyrins.
To separate the effective drug from the undesirable porphyrins, the pH is
raised into a range between 6.5 and 12 and preferably 9.5 to form an
aggregate and then the material is separated. The separation may be by
filtering, by precipitation, by gel electrophoresis, by centrifugation or
by any other suitable means. For best results in filtering or other
methods such as centrifugation based on the aggregate size, the pH is
raised to 9.5 and filtering done at the high pH to remove other porphyrins
rapidly and completely. The filter should retain aggregates of molecular
weight above 10,000.
The pH must be adjusted during filtering because it tends to be reduced as
the impurities are reduced. This is done by monitoring pH and adding an
appropriate adjustor such as a base. To save time and water during
purification, the concentration is increased to the lowest possible
volume. This may, in an ideal system, be limited by solubility to prevent
precipitation of the drug or the aggregation of undesirable substances.
In methods of separation based on affinity, a hydrophobic packing is used
having a higher affinity for DHE than other porphyrins in hematoporophyrin
derivative. DHE is selectively removed after other porphyrins with a
solvent higher than alcohol in the eluantrophic series for reverse phase
chromatography. More specifically, an inverse phase chromatographic column
with packing of 5 micron spheres is used. THF may be used as the solvent.
Of course, the drug formed from hematoporphyrin derivative may be formed by
other methods. In the preferred embodiment the drug is DHE, which is
separated from hematoporphyrin derivative. However, DHE may be formed
other ways and other compounds may be formed by other methods including
from combinations of pyrroles or substituted pyrroles. For example, a drug
similar to DHE may be formed using other formation bonds than the oxygen
bond or from other hematoporphyrin derivatives and thus not be ethers.
Moreover, such compounds may be synthesized instead from other feedstocks
and still other compounds having the desired characteristics may be formed
from other compounds such as chlorophylls.
A chlorin, the structure of which is not entirely known, has been combined
with DHE and shown to have some effect in vivo when light in its
absorbance spectrum was used. Better results have been obtained by
encapsulating the same chlorin in liposome prepared using the method
described by Dr. Eric Mayhew, "Handbook of Liposome Technology", Vol II,
CRC Press, ed. G. Gregoriodis, the disclosure of which is incorporated
herein. A molar ratio of 1:4:5 of egg phosphatidyl, glycerol, phosphatidyl
choline, cholesterol was used.
GENERAL DESCRIPTION OF TREATMENT
For treatment, a photosensitizing drug is injected into the subject
(sometimes referred to as host) which drug includes a plurality of
molecules that: (1) aggregate in an aqueous suspension into groups having
a molecular weight above 10,000 or are encapsulated in another material
that enters cells; and (2) dissociate and attach themselves in neoplastic
tissue. The drug is then permitted to clear normal tissue and the
neoplastic tissue is exposed to electromagnetic radiation having a power
at a value in a range of between 5 milliwatts per square centimeter and
0.75 watts per square centimeter without thermal effects in a wavelength
band of between 350 nm and 1,200 nm to destroy the vascular system and
other tissue within the neoplastic tissue that has accumulated the drug.
In treating humans or other mammals with the drug, light is irradiated on
the tissue in such a position as to uniformly illuminate the cancer
tissue. A synergistic effect is obtained by applying heat either before,
during or after the light to heat the tissue above 39.5 degrees Celsius
and preferably within the range of 40.5 to 45 degrees Celsius.
The increase in temperature, when used, may be achieved by transmitting
light: (1) some of which is near or in the infrared spectrum such as at
1060 nm wavelength from a Nd-Yag laser for heat with the light at 630 nm
for interaction with the photosensitive drug; or (2) by microwaves such as
at 2450 MHz; or (3) by any other suitable means. The temperature is
preferably increased during the application of radiation within the
absorption spectrum of the photosensitive drug but may be caused instead
immediately before or after, such as within two hours.
In the alternative, higher power laser light within the absorption spectrum
of the drug causes thermal destruction of tissue which is interactive with
the photodynamic effect of the drug. This removes bulky tumors or
obstructions by vaporization or vascular occlusion such as by coagulation
of blood.
SPECIFIC DESCRIPTION OF THE DRUG
In the preferred embodiment, the drug DHE is a water soluble, high
molecular weight material derived by treating hematoporphyrin
hydrochloride with acetic and sulfuric acids followed by appropriate
hydrolysis and filtering to separate the drug based on its large size. Its
failure to pass through a filter, such as the MilliPore Pellicon 10,000
molecular weight filter pack, indicates a molecular weight in excess of
ten thousand and thus aggregated DHE.
Mass spectrometry of the new drug shows in FIG. 1 especially strong peaks
at mass numbers of 149, 219, 591, 609 and characteristic but smaller peaks
at 1200, 1218, 1290, 1809, 1866 and 1899. Spectrophotometry of the new
orange-red colored drug in aqueous solution reveals in FIG. 2 well-defined
peaks at approximately 365, 505, 537, 565, 575 and 615 millimicrons.
Infrared spectrophotometry of the new drug dispersed in potassium bromide,
reveals in FIG. 3 a broad peak associated with hydrogen stretching, said
peak centered at approximately 3.0 microns, and a shoulder at
approximately 3.4 microns. Finer peaks are observed at approximately 6.4,
7.1, 8.1, 9.4, 12 and 15 microns.
Elemental analysis of the disodium salt derivative of the new drug shows it
to have an empirical formula of C.sub.34 H.sub.35-36 N.sub.4 O.sub.5-6
Na.sub.2, there being some uncertainty in hydrogen and oxygen due to
traces of water which cannot be removed from the drug. A carbon-13 nuclear
magnetic resonance study of the drug in completely deuterated
dimethylsulfoxide shows in FIG. 4 peaks at approximately 9.0 ppm for
--CH.sub.3 18.9 ppm for --CH.sub.2, 24.7 ppm for CH.sub.3 CHOH, 34.5 ppm
for --CH.sub.2, 62 ppm for CH.sub.3 CHOH, 94.5 ppm for .dbd.C (methine),
130-145 ppm for ring C, and 171.7 ppm for C.dbd.O, all ppm being relative
to dimethyl sulfoxide resonance at about 37.5 ppm. Additional vinyl peaks
at approximately 118 and 127 ppm may be representative of the new drug or
possibly a contaminant. There are additional absorption peaks in the
carbon-13 nuclear magnetic study at approximately 27.9 ppm and 68.4 ppm
relative to the resonance peak of tetramethylsilane in deuterated
chloroform solvent.
When the unfiltered reaction product was eluted from a Waters Associates' U
Bandpak C-18 column using first, successively methanol, water and acetic
acid (20:5:1) and then using tetrahydrofuran and water (4:1), four
components were found. Three by-products were identified as
hematoporphyrin, hydroxyethylvinyldeuteroporphyrin and protoporphyrin by
comparison with standards on thin layer chromatography, with Rf values of
approximately 0.19, 0.23, and 0.39 respectively (FIG. 5) using Brinkman
SIL silica plates and benzene-methanol-water (60:40:15) as elutent.
The fourth component shown in FIG. 5 was the biologically active drug of
the invention. Chromatography shows in FIG. 6 that exclusion of the
aboveidentified impurities using the MilliPore Pellicon cassette system
fitted with a 10,000 molecular weight filter pack, has occurred, during
processing of the drug of the invention.
In formula 1, DHE, which is a biologically active drug of this invention,
is probably an aggregate of ether molecules formed between two
hematoporphyrin molecules by linkage of the hydroxyethylvinyl groups as
shown in formula 1. This linkage may occur through hydroxyethylvinyl
groups in position 3- or 8- as numbered in formula 1. Linkage may be
achieved at position 3- in both halves of the ether, at position 8- in
both halves of the ether or through position 3- in one half of the ether
and in position 8- in the other half of the ether.
These structures may be named as derivatives of ethyl ether, i.e.:
Bis-1-[3-(1-hydroxylethyl) deuteroporphyrin-8-yl] ethyl ether, as shown in
formula 1. Other structured isomers may be named: 1-[3-(1-hydroxyethyl)
deuteroporphyrin-8-yl]-1'-[8-(1-hydroxyethyl) deuteroporphyrin-3-yl] ethyl
ether, or 1-[8-(1-hydroxyethyl) deutoeroporphyrin-3-yl]-1'
[3-(1-hydroxyethyl) deuteroporphyrin-8-yl] ethyl ether, and
Bis-1-[8-(1-hydroxyethyl) deuteroporphyrin-3-yl] ethyl ether.
One or both hydroxyethyl groups at positions 3- or 8-, not used in ether
formation, may dehydrate to form vinyl groups. Although experiments have
not been conducted, experience indicates that ethers as shown in formula 1
might be substituted with various combinations of hydrogen, alkyl groups,
carboxylic acid groups and alcohol-containing groups at various locations
of the structure. In addition, many possible optical isomers of these
structures exist.
A carbon-13 nuclear magnetic resonance study of the drug in deuterated
chloroform referenced to tetramethylsilane reveals in FIG. 7 to additional
absorbances not previously apparent in FIG. 4. Peaks at 24.7 ppm and 62
ppm in FIG. 4 have shifted to 25.9 ppm and 65.3 ppm respectively in FIG. 7
but newly-developed peaks at 27.9 ppm and 68.4 ppm in FIG. 7 represent
resonances for CH.sub.3 and H--C--OH bonded from position 3- in FIG. 7,
respectively. These newly-developed resonances substantiate the molecular
formula depicted in formula 1.
##STR1##
Although DHE is the preferred embodiment, other photosensitizing compounds
and delivery systems having the desired ability to enter neoplastic tissue
and bind to cells have been prepared and still others are possible. For
example, the compound in formula 2, which is a chlorine and the compound
in formula 3, which is a phlorin probably will show a response.
A chlorin has been tested and shown to have a response in animals although
not as satisfactory as DHE. The exact structure of that chlorin is not
known but its spectrum shows it to be a chlorin. This chlorin does not
have delivery characteristics because it includes only one chlorin group
rather than two groups. Delivery into tumors was accomplished by
encapsulating the chlorin in a liposome to enter cells and also by mixing
with DHE. The chlorin was bound within the cell, was irradiated and a
response observed. For proper delivery, the compounds must either be
encapsulated or have two covalently bound groups, each group including
four rings forming a larger ring which is the group, some of the rings
being pyrroles such as chlorins, phlorins, porphyrins and the like.
SPECIFIC DESCRIPTION OF DRUG FORMATION
To prepare one form of a drug from hematoporphyrin, the porphyrin is
reacted to form compounds including two porphyrins covalently bound. This
reaction is a dehydration reaction to form an ether (DHE) or a
condensation reaction for a carbon-carbon linkage which may be possible or
any other possible combination of atoms. Moreover, a third linking
molecule may be used such as dihaloalykyl compound, which reacts with the
hydroxl groups on two porphyrins.
DHE is formed by: (1) lowering the pH of a hematoporphyrin compound to
react a hydroxyl group on one of two porphyrins with another porphyrin and
thus to form an ether containing the two rings of pyrroles; and (2)
removing the DHE formed by this reaction from other moieties.
In another method of forming the ether, a mixture consisting of
approximately 20% hematoporphyrin, 50% hematoporphyrin diacetate, 30%
hematoporphyrin monoacetate is formed from hematoporphyrin hydrochloride
and hydrolyzed. These reactions may be generally expressed by equations 4
and 5, l or more specifically by equations 6 and 7 where P is the basic
porphyrin group, the peripheral group of which has been acetylated as
shown.
##STR2##
This mixture is formed by: (1) adding 285 mL (milliliters) of acetic acid
to a 1000 ml Erlenmeyer flask containing Teflon-coated magnetic stirring
bar; (2) stirring the acetic acid; (3) slowly adding 15 ml of concentrated
sulfuric acid; (4) weighing out 15.0 grams of hematoporphyrin
hydrochloride (preferably obtained from Roussel Corporation, Paris,
France); (6) adding said hematoporphyrin hydrochloride to the acid
solution; and (7) stirring for one hour.
To further the preparation of DHE: (1) a solution of 150 grams of sodium
acetate is prepared in 3 liters of glass-distilled water using a 4-liter
glass beaker; (2) at the end of one hour, the acetate mixture is filtered,
preferably through Whatman No. 1 filter paper, allowing the filtrate to
drip into the 4-liter beaker of 5% sodium acetate; (3) the 5% sodium
acetate solution now contains a dark red precipitate which is preferably
allowed to stand for one hour with occasional stirring; (4) the dark red
precipitate is then again filtered, preferably using the above-identified
filter mechanism; (5) the filter cake from the filtering process is then
washed with glass-distilled water until the filtrate is at pH of 5.5-6.0
(1500-2500 ml of wash water may be required); and (6) the filter cake is
then preferably allowed to dry in air at room temperature.
To further purify the DHE, the air-dried precipitate is ground, using for
instance, a mortar and pestle until a fine powder is obtained. The powder
may then be transferred to a 250 ml round bottom flask. The flask is then
attached to a rotating evaporator and rotation under vacuum is maintained
at room temperature for preferably 24 hours.
Twenty grams of the vacuum-dried powder is then preferably placed in a
4-liter aspirator bottle which may contain a magnetic stirring bar, and
then 1000 ml of 0.1N sodium hydroxide is added thereto. This solution is
preferably stirred for one hour and 1.0N hydrochloric acid is then added
dropwise until the pH is 9.5.
For the separation of DHE, the aspirator bottle, containing the said
solution, is attached to transfer lines leading to a MilliPore Pellicon
Cassette system fitted with a 10,000 molecular weight filter pack of the
type sold by Millipore Corporation, Bedford, Mass. 01730. The pH of the
solution is maintained at 9.5 during this filtration process. It is
preferable that the temperature of the solution be ambient. The
concentration is increased until the total volume is 400 ml by turning off
the feed water and continuing the pump.
The peristallic feed pump is continued and the water feed solution is run
through the Pellicon cassette system at a pH of 9.5 and pressure of 10-20
p.s.i.g and maintaining the retentate volume at 400 ml. Pressure may be
varied depending on the flow rate through the system.
The filtrate process is continued until the retentate solution contains
substantially only the high molecular weight, biologically active product.
At this time waste monomers are generally no longer present. Exclusion of
the waste through the microporous membrane of the filter system is
confirmed by analyzing the high molecular weight, biologically active
product with a Bio-Gel P-10 column obtainable for example from Bio-Rad,
Richmond, Ca. or by high performance liquid chromatography using a
Micro-Bondpak C-18 column with fixed variable wavelength detector
obtainable for example, from Waters Associates, Milford, Ma.
Concentrations of the product may be increased by running the Pellicon
cassette system without water feed. Concentrations of the product may be
decreased by adding water. In the preferred embodiment, the concentration
of the new drug in solution is approximately 2.5 mg/cc. The pH is adjusted
to approximately 7.4 and made isotonic for bottling.
SPECIFIC DESCRIPTION OF TREATMENT
The photosensitizing drug is injected into the subject and approximately 3
hours to 2 days is permitted to elapse before applying light. This time
may differ in accordance with the patient and treatment but should be
adequate to permit the drug to clear normal tissue.
In FIG. 8 there is shown a block diagram of one system for irradiating
undesirable tissue having a light source 10 which may be a laser system, a
radiation monitor and control system shown generally at 12 and a delivery
system shown generally at 14, positioned to radiate a tumor. The light
source 10 generally radiates light of the desired frequency and may be a
fluorescent lamp system or a laser system of any of several types, such as
an argon laser pumping a dye laser, a krypton laser or the like. The light
passes through the radiation monitor and control system 12 for delivery
through a fiber optic delivery system to a source of undesirable tissue.
The light source 10 includes different configurations such as a single
argon laser pumping a dye laser, two parallel sets of argon lasers pumping
a dye laser, a krypton laser or a xenon laser. Laser arrangements or other
light sources are selected in accordance with the drug and the function.
For example, a diagnostic use may call for a different system than a
therapeutic treatment of a tumor. The laser system 10 may contain the
appropriate means to control frequency, duration and intensity of
radiation or the radiation control system 12 may have some or all of such
means as part of it. The power applied to the subject should be between 5
milliwatts per square centimeter and 3/4 of a watt per square centimeter
without thermal effects, and with thermal effects, 1/2 watt to a kilowatt
per square centimeter.
The energy application should be at a selected value within the range of
from 5 joules per square centimeter to 1,000 joules per square centimeter
within a time period for which there is no substantial repair, such as
less than two hours. For longer periods, when either intermittent or
continuous aplication is used, more energy may be required.
The radiation monitor and control system 12 includes a light interface
system 20, a monitor system 22 and a power level control system 23. The
light interface system 20 transmits light from the laser system 10 through
the delivery system 14 and transmits signals to the monitor system 22
indicating the intensity of light transmitted to the delivery system 14.
It also receives feedback light from the delivery system 14 and transmits
a signal representing that light to the monitor system 22. The signals
between the monitor system 22 and the light interface system 20 are
electrical. A power level control system 23 is connected to the monitor
system 22 and to the laser system 10 to control the laser system 10.
The monitor system 22 may have different configurations each with a
different complexity. In one arrangement, the manual controls for the
laser system 10 are on the monitor and control system 22 such as on the
power level control 23 in some of these configurations, feedback signals
are applied from the monitor system 22 to the power level control 23 to
control intensity and sampling rates for purposes of determining
therapeutic effects. The monitor system 22 may include data processing
equipment and equipment which displays the results of the laser system 10
and the light interface system 20 on an oscilloscope. The power level
control 23 may be considered part of the laser system by some
manufacturers but is discussed separately here for convenience.
The light interface system 20 includes an optical interface and a sensor
28. The optical interface and the sensor 28 are enclosed within a cabinet
for the shielding of light and electrical conductors 36 connect the sensor
28 to the monitor system 22.
To transmit light from the laser system 10 to the delivery system 14, the
optical interface includes a beam splitter 30 and a lens system 32 having
a shutter 33 and a lens 35. The beam splitter 30 passes light from the
laser system 10 to the lens system 32 for transmission through the
delivery system 14 to the spot of therapy and to the sensor 28 for
detection. Light is transmitted through the delivery system 14 to a
leakage detector at 37 which includes a light sensor electrically
connected to the monitor system 22 and the power level control system 23.
The delivery system 14 includes light conductors 40 and a light
transmission unit 42 connected together so that the light conductors 40
receive light from the lens system 32. There may optionally be included
other types of equipment such has an endoscope.
To monitor the therapy, the monitor system 22 includes a readout system 25,
an integrator 27 and a readout system 29. The light sensor 28 applies
signals to the readout system 25 which, in one embodiment, uses the
signals to control the power level control 23 in accordance with light
from the beam splitter 30 indicating laser output to the fibers 40 from
the laser system 10. The readout 25 also provides a visible readout
indicating power output from the laser system 10 as well as providing
signals to the power level control 23.
The leakage detector 37 applies signals to the readout 29, integrator 27
and power level control 23. This signal can be used to calibrate the
output from the delivery system 14 since it indicates loss in the delivery
system. This loss is a const | | |
