CA1265450A - Drug, and methods of preparing and using it for treatment of tumors - Google Patents

Abstract

DRUG, AND METHODS OF PREPARING AND USING IT
FOR TREATMENT OF TUMORS
ABSTRACT

To obtain tumor-selective, photosensitizing drugs useful in the localization of neoplastic tissue and treatment of abnormal neoplastic tissue such as tumors, one of two methods is used. In the first method, a hydrolyzed mixture of the products of reaction of hematoporphyrin with acetic acid and sulfuric acid is cycled through a microporous membrane system to exclude low molecular weight products. In the second method, drugs are synthesized or derived from other pyrrole compounds. The drugs: (1) include two covalently bound groups, each with four rings, some of which are pyrroles such as phlorins, porphyrins, chlorins, substituted pyrroles, substituted chlorins or substituted phlorins, each group being arranged in a ring structure, connected covalently to another group and have a triplet energy state above 37.5 kilocalories per mole; (2) are soluble in water, forming an aggregate of over 10,000 molecular weight in water and have an affinity for each other compared to serum protein such that 10 to 100 per cent remain self aggregated in serum serum protein; and (3) are lipophyllic and able to disaggregate and attach to cell plasma, nuclear membrane, mitochondria, lysosomes and tissue. The drug obtained by the first method has an empirical formula of approximately C68H70N8O11 or C68H66N8O11Na4. Neoplastic tissue retains the drug after it has cleared normal tissues and illumination results in necrosis.

Moreover, other photosensitizing materials may be combined with a carrier that enters undesirable and cells of the reticular endothelial system such as macrophages. These photosensitizing materials: (1) must have a triplet energy state above 3.5 kilocalories per mole; (2) cannot be easily oxidized; and (3) not physically quench any required energy state.
Preferably, this photosensitizing material should be lipophlic.

Description

DRUG, ~ND METHODS OF PREPARING AND U5ING IT
FOR TREATMENT OF TUMORS

This invention relates to the diaanosis 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 photosensitiæing drugs, tumors are detected and treated by irradiating the tumors with li~ht a~ter the dru~
accumulates in the tumor. The dru~s are photosens;tizing and some of the drugs in this class are derivatives of hemoglobin.
There are several prior art techniques for such diagnosis and treatment. ~or 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 rea fluorescence was attributed to ~0 porphyrins produced in the tumor. In "Untersuschungen Uber Die ~olle Der Porphine Bei Geschwulstkranken Menschen Und Tieren", Z Krebsforsch 53:65-68, 1942, Auler and Banzer showed that hematoporphyrin, a derivative o~ hemo~lo~in, would fluoresce in tumors but ~, ~2Çi~

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, Fig9e and co-workers demonstrated that injected hema~opor~hyrin would localize and fluoresce in several types of tumors induced in mice. In "The Use of a Derivative of Hematoporphyrin in Tumor ~etection", J Natl Cancer Inst.
2601-8, 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 use~ul in the treatment of tumors. In "Photodynamic Therapy of Mali~nant Tumors", Lance~ 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 ~reast Carcinoma", J Natl Cancer Inst. 62:231-237, 1979, Dougherty and co-workers reported using the .

~265~5~

crude Lipson hematoporphyrin derivative to accomplish photoradiation t~erapy on human patients.
The crude Lipson hematoporphyrin deriva~ive 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 attac~ tumor cell walls an~
effect necrosis. In "Effects of Photo-Activated Porphyrins in Cell Surface Properties", 8iochem 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 L,ipson 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 su~iciently 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 ha~e an optimum absorbance spectrum in a range that penetrates tissue most effectively.

~i5~

To improve some c~f these deficiencies a composition of matter comprises a mixture of porphyrins and porphyein-like compounds having as its active ingredient at least one type of porphyrin or porphyrin-like compound with a molecule that is flllorescent, photosensitiæing, localizing in and being retained in tumor cells for a longer time than normal tissues, not beinq completely disaggregated by serum protein, and forming a high molecular weiqht aggregate of more than ln 10,000 in an aqueous medium; at least 50~ of the porphyrins or porphyrin-like compounds in said mixture being said active ingredient.
The composition of matter has, in one embodiment, molecules of the active ingredient w1th the moleculae formula:

R2 ~?3 ~``1~ ~, 3.`5 n- ~<7 ~ 3 .

126545~

in which R1 includes at least one a-tom with a valence greater than one.
Advantageously, the composition R1 in the molecular formula of the active ingredient includes an ether linkage, or R1 is a substituted ethyl ether function, or Rl is a carbon-carbon linkage, or R1 is a substituted alkyl function. Moreover, R2-R15 in the molecular formula of the active ingredient are other covalently bound chemical groups with molecular weights less than 1,000, or at least one of the ring hydrogen is methine hydrogen, or R2-RlS is an alkyl group, or R2-R15 contains a carbo~ylic acid group.
The high molecular weight aggregates may comprise molecules having two groups, each group being arranged in a ring structure and is in liquid form having a concentration of approximately 2.5 mg/cc. In one embodiment, the active ingredient has an empirical formula of approximatelY C68H70N8oll or C68H66 11 4 The liquid form of the composition of matter includes isotonic saline solution at a pH of approximately 7.0 to 7.2. The composition of matter may have as its active ingredient, a molecule with at least one phlorin.
The phlorin may be encapsulated in a liposome and may have the molecular formula:

!

~i5~

3 f~l2 R I ~
~t ~

~S~ ~ ~ , ~ ~ ~

R ~a ~ lternatively, the active ingredient may have a molecule with at least one chlorin and said molecules may have the formula:

R I

2s~ RI~

R7 ~ S

N 71~ ~

~26~

A process for the production of composition of matter comprises the steps of forming a mixture of porphyrin or porphyrin-like compounds and separating at least some of the compound comprising the active ingredient from the other compounds. A compound is formed having the formula:

R~ 4 R ~ 7 R 1~ 3 Advantageously, the compound is formed by dehydration of hematoporphyrin to form an ether. The s~tep of separating the compound includes the step of separating the compound according to the molecular weight of its aggregate and selecting a range of molecular weights of the aggregates above 10,000 and separating according to this range of molecular weights or in which the step of separating the compound includes the step of separating certain of the aggregates of the ~ .
,~ .

5~
1() compound from compounds having a molecular weiqht greater than a preselected value above 10,000.
More specifically, the step of separating the compound may include the steps of a~justing the pH oE
the solution to 9.5; and pass1ng the resultant impure solution through a porous membrane system to exclude low molecular weight by-products thereby effectinq purification.
Still more specifically, hematoporphyrin may be ln reacted with acetic/sulfuric acids to form a solution, the crude product may be precipitated by neutralization in sodium acetate, and dissolved with sodium hydroxide.
The acidity of the solution is adjusted to pH of 9.5 and the resultant impure solution is passed through a porous membrane system to exclude low molecular weight by-products thereby effecting purification.
Hematoporphyrin is reacted with acetic/sulfuric acids to form a solution, the crude product is precipi-tated by neutraliza~ion in sodium acetate, the crude product is dissolved with sodium hydroxide, the acidity of the solution is adjusted to pH of 9.5 and the resul-tant impure solution is passed through a porous membrane system to exclude low molecular weight by-products thereby ef~ecting purification and ad~ing sodium chloride to said impure solution to make the latter ~0 i4~i~

isotonic prior to passing it through said membrane sys-tem. Said substance in solution has a concentration of appro~imately 2.5 mg/cc and is adlusted to obtain said coneentration by addition or removal of liquid.
Advantageously, phlorin is combined with a ma-terial eapable of entering neoplastic tissue or combining the phlorin with a eompound having the formula:

R~ 3 ~lo ~1l Y ~ ~ ~ ~ _~1 ~ Rl~

~5~"~ g 2~ S

2~ 7 R 1~ ~ 13 The step of combining ineludes the step of eneapsulating the phlorin in a liposome.
A ehlorin may be prepared and may be eombined with a material eapable of entering neoplastie tissue. The step of combining may include the step of combining the chlorin with a eompound having the formula:

~26~
1 ?

~5~ ; ~S

R~ ?~1 Rl~ P~ 13 or encapsulating the chlorin in a liposome, or encapsulating the chlorin in DHE.
A process for the in vivo destruction of tumors comprises . _ _ _ _ _ the steps of injecting the composition of matter into a host having undesirable tissue; waiting for a predetermined period of time; and illuminating the undesirable tissue with light at a predetermined intensity. The substance may be used in a dosage of from about 1 to 4 mg/kg of body weight of the host, the time delay between the injection and illumination may be within a range of about 3 hours to 7 days and the intensity of illumination is at least 5mw/cm2 for an extended period of time, but no greater than 750mw/cm2 for twenty minutes.

.

The step of illuminating said host includes the step o transmitting radiation through a liqht conductor to a location adjacent to the area to be treated and transmitting the radiation t~rough a diffuser onto the area beinq treated. The process may include the steps of transmltting radiation from the area being treated back to the source of radiation through a light conductor and using said radiation to control the dosage of radiation or the step of transmitting radiation through an air filled bulb whereby heat is dissipated.
The intensity of illumination is between 0.5mw/square centimeters and one killowatt per square centimeter, whereby thermal effects are obtained.

Moreover, a molecule including a phlorin may be injected into a host or a chlorin may be injected into a host having undesirable tissue.
Apparatus for transmittinq light comprises: a transmitting ligl~t conductor; means for permitting radiation from a laser to enter said transmitting light conductor; and a transmitting head sealed against fluid and coupled to said transmitting light conductor. The transmitting llead has means for diffusing radiation and transmitting it.
The apparatus may further includ~e: a receiving light conductor; and a receiving hea~; the receiving head being adapted to receive reflected light and transmit it as a feedback signal through said receivin9 light conductor.
Advantageously~ the transmitting head is made of a material capable of passing ra~iation outwardly and backscattering radiation; the transmitting light conduc-tor has an end within the transmitting head; the transmitting head is substantially cylindrical and has a diameter less than one inch; and the walls of said transmitting head are less than one-quarter the diameter of said head in thickness ~hereby no surface in contact with blood is heated to a temperature to cause coagula-tion of blood~
The transmitting head may be cup-shaped, with a diffusing surface closing said cup and the interior being reflective. The diameter of the cup is less than one-hal~ in_h, and the transmitting conductor enters the cup. A tubular stem may surround at least a portion of the transmitting light conductor and is connected to the ~0 transmitting head at an angle to permit easy insertion near an eye ~or the application of radiation. The receiving light conductor may have a light sensor at one end for converting radiation to an electrical signal.
The apparatus includes at least one laser, sensor means for sensing radiation from said laser and means s~

responsive to said sensor means and said electrica]
signal for developing a signal re~.ated to radiation dosage.
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 o~ a drug in its methyl ester form FIG. 2 is a visible light spectrum of a drug in a water solution, FIGS. 3 and 3~ are in combination an infrared spectrum of the drug dispersed in potassium bromide;
FIG. 4 ls a carbon-].3 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 (a trademark of Waters Associates) column, showing various components ~0 of ~pD 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 D~IE;

~1 FIGS. 7 and 7A are carbon-13 nuclear magnetic resonance print-outs 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. lO is a simplified enlarged longitudinal sectional view of a portion of the system of FIG. 9;
FIG. ll 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. 12;
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. 14;

L~

FIG 17 is a sectional view of a portion of the embodiment of FIG. 14;
F`IG. 18 is a perspective simplified view, partly broken away of another embo~iment of a portion of FIG.
8;
FIG. l9 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.

ld Each of the drugs may be classified into one of two classes, which are: tl) each molecule of the drug aggregates in water ~o aggregates ~aving a combined molecular we.ight oF above lO,000; or (2) units of the drug are encapsulated in a liposome and molecules include at least one such photosensitizinq 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 ~n 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, ` 17 1~

nuclear membrane, ~itochondria, and lysosomes. While it may enter some normal tissue, qenera]ly there is a suf~icient difference in the rates of accumulation and removal between normal and undesirable t;ssue to provide selected conditions which permit treatment of undesirable tissue without excessive damage to normal tissue.
The form of drugs which aggregate must be sufEiciently lipophlic to dissociate in lipids so that n 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 wavelengtll; and (2) causes photodynamic effects. ~hus, 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 Lipson9s reagent without knowing that it existed therein, has the necessary ~0 characteristics but in the prior art was utilized in a mi~ture of porphyrins whiCII haa deleterious side effects. It was not known that the substance was an efEective agent in L,ipson's reagent or that it existed therein because of its resistance to separation by liquid chromatography.

i 5, L~

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 similar characteristics. With such a purified dosage, the porphyrins clear normal tissue adequately be~ore the neoplastic tissue in which the drug has accumulated is exposed to light.
~ This drug (DHE) appears to be ineffective if it is 1~ in aggregates cf 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 eit~ler with two groups bound together or single qroups encapsulated in a liposome. In any 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 ~hich the individual molecules include two groups, each of which includes either phlorin or rings of pyrroles or hydrogenated pyrroles, or substituted pyrroles connected ~o 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 ag9regate in aqueous suspensions. One such compound, dihematoporphyrin ethee (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 at~raction to lipids is, however, sufficiently geeat 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.
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 centrifuqation 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 filte~ should retain aggregates of molecular weight above 10,000.
The pT1 must be adjusted during filtering because it tends to be reduced as the impurities are reduced. This is done by monitoring p~l 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 ~0 drug or the aggregation of undesirable substances.
In methods of separation based on affinity, a hydrophobic packing is used having a higher af~inity for DHE than other porphyrins in hematoporphyrin derivative.
DHE is selectively removed after other porphyrins with a solvent higher than alcohol in the eluantrophic series ~21~S~.SC~

for reverse phase chromatography. More specifically, an inverse phase chromatographic column with packing of 5 micron spheres is used. T~F may be used as the solvent.
Of course, the drug formed from hematoporphyrin deriva~ive may be formed by other methods. In the preferred embodiment the drug is D~, which is separated from hematoporphyrin derivative. However, D~E may ke ormed other ways and other compounds may be formed by other methods including from combinations of pyrroles or subst;tuted pyrroles. For example, a drug similar to DHE may be formed using other formation bonds than the oxygen bond or rom other hematoporphyrin derivativeS
and thus not be ethers. Moreover, such compounds may be synthesized instead from other feedstocks and stil~
other compounds having the desired characteristics may be formed from other compounds such as ch1orophyllsO
A chlorin, the s~.ructure of which is not entirely known, has been combined with D~E and shown to have some efect in vivo when light in its absorbance spectrum was used. Better results have been obtaned by encapsulating the same chlorin in liposome prepared using the method described by Dr. Eric Mayhew, "Handbook of Liposome Technologyn, Vol II, CRC Press, ed. G.
Gregoriodis. ~ molar ratio of 1:4:5 of egg phosphatidyl, glycerol, phosphatidyl choline, ~`~''1 ~i ~.~6545i~

cholesterol was used.
For treatmen~, a photosensitizing drug is injected into the subject which drug includes a plura]ity 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 (~) dissociate and attach themselves in neoplastiC
~issue. The drug is then permitted to clear norma]

tissue and the neoplastic tissue is exposed to electro-magnetic radia~ion 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 ~o 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 ~0 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 ~!

~26~
~4 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 24S0 M~lz; or (3) by any other suitable means. The temperature is preferablY
increased du~ing the application of radiation within the absorption spectrum oE 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 1~ 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 obstruc~ions by vaporization or vascular occlusion such as by coagulation of blood.
In the preferred embodiment, the drug DHE is a water soluble, high molecular weight material derived by trea~ing hematoporphyrin hydrochloride with acetic and sulfuric acids followed by appropriate hydrolysis and filtering to separate the drug based on its large ~0 size. Its failure to pass through a filter, such as the ~illiPore Pellicon 10,000 molecular weight filter pack, indicates a molecular weight in excess o~ ten thousand and thus aggregated DHE.
Mass spectrome~ry of the new drug shows in FIG.
1 especially strong peaks at mass numbes of 149, 219, :

5~.5~3 591, 609 and characteristic b~t smaller peaks at 1200, 1218, 1290, 1809. Spectrophotometry of the new orange-red colored drug in aqueous sol~tion reveals in FIG. 2 well-defined peaks at approximately 505, 537, 565 and 615 millimicrons. Infrared spectrophotometry of the new drug ~ispersed in potassium bromide, reveals in FIG. 3 a broad peak associated with hydrogen stretchin~, 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 C34H35_3~N40~_6Na2, there being some uncertainty in hydrogen and oxygen due to traces of water which cannot be removed rom the drug. A carbon-13 nuclear magnetic resonance study Oe the drug in completely deutera~ed dimethylsulfoxide shows in FIG, 4 peaks at approximately 9.0 ppm for -CH3 18.9 ppm for -CH2 , 24.7 ppm for CH3 CHOH, 3~.5 ppm for -CH2 , 62 ppm for C~3 CHOH, 94.5 ppm for =C ~methine), 130-145 ppm for ring C, and 171.7 ppm for C = O, all ppm being relative to dimethyl sulfoxide resonance a~ about 37.5 ppm. Additional vinyl peaks at approximately 118 and 127 ppm may be representative of the new drug or possibly a ~6~4~3 contaminant.
When the unfiltered reaction product was eluted from a U Bondpak C-]8 (txademark of Waters Associates, Milford~ Mass., U.S.A.~ column using first, successiveJy methanol, water and acetic acid (20 5 1) and then using tetrahydrofuran and water (4:1), four components were ound. Three by-products were identified as hemato-porphyrin, hydroxyethylviny]deuteroporphyrin and proto-porphyrin 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 benzenemethanol-water (60:40:]5) as elutent.
The fourth component shown in FIG. 5 was the biologically active drug of the invention. Chromato-graphy shows in FIG~ S that exclusion of the above-identified impurities using the MilliPore*Pel]icon cassette system fitted with a lO,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 l. This linkage may occur through *Trade Mark 31 2~54SI~

hydroxyethylvinyl groups in position 3- or 8- as numbered in formula l. 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 hal 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 -l- 13-(l-hydroxylethyl) deutero-porphyrin -8-yl] ethyl e~her~ as shown in formula l.
Other structured isomers may be named: l-[3- (l- hydro-xyethyl) deu~eroporphyrin -8-yl] -l'- 18- (l-hydroxy-ethyl) deuteroporphyrin -3-yl~ ethyl ether, or l- 18-(l-hydroxyethyl) deuteroporphyrin -3-yl~ -l' 13- (l-hydro-xyethyl) deuteroporphyrin -8-yl] ethyl ether, and Bis -l- 18- 1l-hydroxye~hyl) 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 l 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.

~6~ 5~

A carbon-13 nuclear magnetic resonance study oF
the drug in deuteeated chloroform re~eeenced to tetramethysilane reveals in FIG. 7 two 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 an~1 65.3 ppm respectively in FIG. 7 but newly-~eveloped peaks at ~7.9 ppm and 68.4 ppm in FIG. 7 represent resonances oe CH3 and H-C-O~I bonded ~rom position 3-in FIG. 7, respectively. These newly-developed resonances substantia~e the molecular formula depicted in formula 1.
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 chlorin 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 ~0 response in animals although not as satis~actory as D~E.
The exact structure oE that chlorin is not known but its spertrum 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 2~

C~ N~ 3 H3C ~q~)lCo~ H(N~) ~C(cq~ o c~\\ /~ )zC~U~

~\~ CR3 H3~< =

C~ C~ {-~ C~3 C~3 ~H3 f~r~u~ I

lOaC(C~ C~3 I~C~C~ CO~H(NI~

~N~ G~C;tl~ C~7 C~ ~N~ 02,~

J~ ~Lc~3 Nz~ ~/ C~1~3 ~ C. ~'3~
U_J~,U U~H
C~l3 Il-C OH H-C-OI~ CH3 C~ C~l~
Frr~

~5~5 lO~C~Ikca~H3c~cH2)Lco2H (lJA~

C~ o ci~ J~æco,~

H~ C ~f H C~13 ca3 H-C~O~ H-C-o~ 1 C~l CH3 ~;rMulo~ 3 ~, chlorin in a liposome to enter cells and also by mixinq with DHE. The chlorin was bound wi~hin 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 w~ich is the group, some of the rings being pyrroles such as chlorins, phlorins, porphyrins and the like.
To prepare one form of a drug from hematoporphyrin, the porphyrin is reacted to form Compounds includinq 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 atomsO 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) lowerin~ t~e 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 fl~6~

~cc~ic ~'~ci~l ~p ~ }Ip ~ HpOAc ~ P (~C) 2 Sulfuric Acid FORMUII~ 4 Socli~Q
Hydroxi~le ~p ~ c ~ )2 ~ ~ Other Products FO~LA 5 H H O
Acet:ic Acid l ll P -- C ~ OH 0~ P -- C -- O -- C ~ C~l Suluric A~id 1 3 c~3 CH3 Il O ~1 }~
ll NaOH
2 P -- C -- O -- C ~- C~13 1~ O P --- C --- O -- C -- P

U13 C~l3 C~3 FOR~L~ 7 32`

~2~à~i4~

hematoporphyr;n diacetate, 30~ hematoporphyrin mono-acetate is formed from hematoporphyrin hydrochloride and hydrolyzed. These reactions may be generally expressed by equations 4 and 5, or more specifica]ly by equations 6 and 7 where P is the basic porphyrin group, the peri-pheral group of which has been acetylated as shown.
Thi~ mixture is ~ormed by: (1) adding 285 ml (mi~
liters) of acetic acid to a 1000 ml Erlenmeyer flask containing Teflon-coated (trademark of DuPont de Nenours, E.I., Co. of Wilmington, Del.,U.S.A.) magnetic stirring bax~ ~2) stixring ~he acetic acid; (3~ slowly adding 15 ml of concentrated sulfuric acid; (4~ weighing out 15.0 grams of hematoporphyrin hydrochloride (preferably ob~ained from Roussel Corporation, Paris, France) (5) adding said hematoporphyrin hydroch~oriae to the acid solution; and (6) stirring for one hour.
To further the preparation of DHE: (1) a so]ution of 150 grams of sodium acetate is prepared in 3 liters of glass-distilled water using a 4-liter glass beaker;
~0 (2) at the end of one hour, the acetate mixture is filtered, preferably through Whatman (trademark, Whatman Corporation, Bridewell Pt., Clifton, ~.J., U.S~A.) 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 ~' ~26~45~

which is preferably allowed to stand for one hour with occasional stirring; (4) the dark red precipitate is then again ~iltered, 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 p~ of 5.5-6.0 (150Q-2500 ml o~ wash water may be requ;red~; and (6) the Eilter cake is then preferably allowed to dry in air at room temperature.
To further purify the D~E: (1) the air-dried precipitate is ground, using for instance a mortar and pestle until a fine powder is obtained; (2) the powder is transferred to a 250 ml round bottom flask which is attached to a rotating evaporator and rotated under vacuum at room temperature for preferably 2~ hours; (3) twenty grams of the vacuum-dried powder is placed in a 4-liter aspirator bottle containing a magnetic stirring bar; (4) 1000 ml of 0.1 N sodium hydroxide is added thereto; (5) this solution is stirred for one hour; and ~0 (6) 1.0 N hydrochloric acid is then added dropwise unti].
the pH is 9.5.
For the separation of DHE: (1) the aspirator bottle conta;ning the solution is attached to transfer lines leading to a MilliPore Pellicon Cassette (trade-mark of Millipore Corporation, Bedford, Mass., U.S.A~) ~' ~5~

system fitted with a 10,000 molecular weight filter pack (2) the pH is maintained at 9O5 during filtra-tion, and preferably at ambient temperature; (3~ the concentration is increased until the tota] volume is 400 ml by turning off the feed water and continuing the pump; and ~4) the peristaltic feed pump is continued and the water feed solution run through the cassette system at a p~ of 9.5 and pressure of ~0-20 p.s.iag, main-taining the retentate volume at 400 ml. Pressure may be varied depending on the flow rate through the system.
The filtration process is continued until the retentate solution contains substantially only the high molecular weight, biologica~ly 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 ana~yzing the high molecular weight, biologically active product with a Bio-Gel P-10 (trademark Bio-Rad Corporation~ column obtainable for example from Bio-Rad, Richmond, Ca. or by ~0 high performance liquid chromatography using a Micro-Bondpak C-18 (trademark of Waters Associates, supra~
column with fixed variable wavelength detector obtain-able for example, from Waters Associates.
Concentrations of the product may be~ in-creased by running the cassette system without water s~

feed; and (2) aecreased by adding water. In the prefer-red embodiment, the concen~ration of the new drug in solution is approximately 2.5 mg/cc. The pH is adjusted to appro~imately 7~4 and made isotonic for bott]ing.
It is injected into the subject and approximately

3 hours to 2 days permitted to elapse before applying light, This time may di~fer in accordance with the patient and treatment but should be adequa~e to permit the drug to clear normal tissue.
In FIG. 8 there is shown a block diagram of one system fox 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 aelivery system shown generally at 14, posi~ioned 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 radia-~0 tion monitor and control system 12 for delivery through a fiber optic delivery system to a source of undesirab~e tissueO
The lightsource 10 includes different configura-tions such as a single argon laser pumping a ~ .

~s~

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 difEerent 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 1~ may have some or all of such means as part of it. The power applie~ 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 eÇ~ects, 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 application 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 ~rom the laser ~5~

system 10 through the delivery system 14 and transmits signals to the monitor sys~em 22 ;ndicating the intensity oE 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 ~ight interface system 20 are electrical. A power level control system 23 is connec~ed to the monitor system 22 and to the laser system 10 to control the laser system 10.
The monitor system 22 may have different configuracions 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 conigurations, 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 proces-sing equipment and equipment which displays the results o~ 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.

` 38 The light interface system 20 includes an opticalinterface 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 li~ht 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 detectionO Li~ht 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 liqht 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 e~bodiment, uses ~265~

the signals to control the power level control 23 in accordance with light from the beam splitter 30 indicaling 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 si9nal can be used to calibrate the output from the delivery system 14 since it indica~es loss in the delivery system. This loss is a constant fraction of delivered light. The delivery system is calibrated by measuring its output in an integrating sphere in a manner known in the art and correlatinq it with the output from detector 37. With the relationship between leakage and output power known, a reliable feedback for monitoring and control is obtained which relates to power being transmitted throuqh the light cond~ctor to the subject thus compensating for coupling losses to the light conductor. The shutter 33 is controlled by the integ~ator 27 to control the power dosage by blocking light to the delivery system 14 when the integrated power or energy reaches a predetermined dosage set into the integrator 27.

~2~
~1 The delivery system is intended to: (1) deliver the light in close peoximity to the neoplastic tissue that is to be observed or destroyed: (2) have sufficientl.y low attenua~ion to permit an adequate intensity of light; ~3) transmit received luminescent light and feedback signals and the like useful in observation and control; (4) be able to be inserted into locations propitious for irradiating light at the desired location; (S) be capable of directing light in an appropria~e pattern; (6) be sufficiently strong to avoid breaking off of parts in use; (7) have sufficient capability to resist deterioration from the heat it handles; and (~) incorporates materials with low absorption at the frequencies used in treatment so as to reduce heating.
In FIG. 9 there is shown a block diagram of a combination of radiation monitor and treatment system having a laser system lOA, a monitoring and radiation control system shown qenerally at 12A and a delivery system shown generally at 14A, positioned to radiate a tumor on a bronchial wall 16A of a subject. The laser system 10~ generally radiates light of the desired frequenc~ throagh the monitoring and radiation control system 12A for~delivery through a fiber optic delivery system to the cancer on the bronchial wall 16A.

~2~
~2 The monitoring and radiation control system 12A

includes a light intee~ace system 20A and a monitor system 22A. The light interface system 20A transmits light from the laser system lOA through the delivery system 14A an~ transmits signals to the monitor system 22A indicating the intensity of light transmitted to the delivery system 14A. It also receives feedback light from the delivery system 14A and transmits a signal representing that light to the monitor system 22A. The 1() signals between the monitor system 22A and the light interface system 20A are electrical.

The light interEace system ~OA includes an optical interface 2~A, a ~ilter 26A and a sensor 28A. The optical interace 24A, the filter 26A and the sensor 28A
are enclosed wi~hin a cabinet 34A for the shielding of light wi~h electrical conductors 36A connectinq the sensor 28~ to the monitor system 22A.

To transmit light from the laser system lOA to the delivery system 14A, the optical interface 24A includes ~0 a mirror 30A and a lens system 32A. The mirror 30A
includes a central aperture which passes light from the laser system lOA to the lens system 32A for transmission through the delivery system 14A to the spot of therapy.
Light is transmitted through the delivery system 14A
from the spot of therapy back to the lens system 32A for ~L~65~

transmission to the filter 26A.
The delivery system 14A includes a plurality of light conductors 40~ and a light transmission unit 42A
connected together so that the light conductors 40A
receive light from the lens system 32A, originating with the laser system lOA, and transmit lig'nt from a luminescent surface such as neoplastic tissue containing photosensitive drug back to the lens system 32A for transmission to the ilter 26A. There may optionally be ld included other types of equipment such as an endoscope 44A.
To monitor the therapy, the filter 26A is positioned between the mirror 30A and the sensor 2BA to pass a narrow band of frequencies to the sensor 29A
which converts the light to an electrical signal for transmission through the conductor 36A to the monitor system 22A. The mirroe is positioned such that light from the delivery system 14A passing through the lens system 32A is reflected by the mirror 30A throug~ the filter 26A to the sensor 28A.
The light leaving the delivery system 14A from the tumor is in a cone that radiates over an area of the mirror 30A while the mirror 30A has light from the laser system lOA forming a beam through the small central aperture therein onto the Iens 32A for transmission ~5'~

through a fiber of the light conductor bundle 40 onto the tu~or. The signals from the detector 29A may indicate the amount of illumination or the location of illumination or the generation of triplet state oxygen indicating destruction of neoplastic tissue and thus may be used for locating tumors or for indicating the amount of photodynamic derstruc~ion of neoplastic tissue.

To reduce noise, the monitor 22A controls a chopper 9~ to chop light at a suitable frequency such as 90 hz (hertz) which can be detected in the monitor system 22A
by synchronous demodulation. This is controlled by a signal on conductor 100 which originates from the chopper drive voltage. This frequency is low enough so that the half life of the fluorescence of the drug is much smaller than a hal cycle of the chopper so as not to be blocked. The frequency of chopping is selected to block ambien~ noise from room lamp sources and to reduce drift. Moreover, in the preferred embodiment, the light transmitted through the delivery system is 630 nm so as to be d;stinguished from 690 nm fluorescence ~rom the drug.
Although a delivery system 14A has been described which is suitable for treatment of a tumor on a bronchial wall, other types of delivery systems are known which transmit light for such use and other ` 44 5[3 confi~urations of delivery systems are available for other types of therapy such as for bladder or the like.
In FIG. 10 there is shown a sectional view of a transmission unit 42 for treating or locating a spot on a bronchial wall having a generally cylindrical shaped opaque casing 50, a fiber optic connecting socket 52 and an image control section 54. The opaque casing 50 is sealed and contains in one end, the fiber optic connecting socket 52 which is funnel-shaped for receiving the ends of the fiber optic light conductors into the hollow interior of the opaque casing 50. The light conduc~ors are sealed in place by any suitable means such as by adhesive, mo~ding, threading, swaging or the like.
The image control section 54 is fitted within the housing in communication with the fiber optic conductors to focus light from the fiber optic bundle in a fixed configuration through a light-passing window 56 in the opaque casing 50 onto a spot to be treated and to reflect fluorescent light passing through the window 56 from tissue back to the ends of the fiber optic conduc-tors in the fiber optic connecting socket 52.
The image control section 54 includes one or more lens 60 and one or more mirrors 62. The lens 60 and mirrors 62 are positioned with respect to the aperture ~ 45 56 so that light from the lens 60 focuses an image of the en~s of the fiber optic conductors in the connectin9 socket 52 onto the mirror 62 which reflects that image ~hrough the aperture 5~. The mirror also receives fluorescence and exciting light at fixed distances from the light passlng through the aperture 56 from the ends of the fiber optic connecting socket 52 back through the lens 60 onto light conductors as a feedback signal. In the preferred embodiment, there are three apertures to measure the attenuation coefficient o tissue, three mirrors, three lens and three light conductors forming three light paths, aligned with each other.
In FIG. 11 there is shown a developed view of a transmission unit 42 having three apertures, lens, windows~ mirrors and light conductors. The first or end aperture 56 transmits light to a surface indicated at 70 and two light receiver apertures are positioned side by side with the transmitting aperture 56 at 72 and 74 spaced from each other by distances Rl and R2 so that the eeceiver aperture 74 receives light at a distance R2 from the transmitted ligh~ and the receiver 72 receives light at a distance Rl. The receivers are used be-cause the light received by a receiver yièlds infor-mation concerningo ~1) total attenuation coefficient of the tissue at the exciting frequency; t2) drug levels at ~l2~i5a~

certain fluorescent frequencies; and (3) the eftectiveness of treatment of tissue at certain other fluorescent wavelengths.
Moreover, it has been discovered that fiber conductors against the surface of the tissue are able to receive a signal from the tissue without penetration of surface which represents the light diffused through the surface The measurement of this light can be used for dosemetry as described ~or the reading ~ead 42 of FIG.
1010 and the explanation of FIG. 11 applies equally to such receivers.
Firstly, t~e measurement of light at the wavelength emitted by the drug in tissue provides a measure of the drug concentration. Secondly, the measurement of light at the incident wavelength without drug in the tissue at points spaced from the location incident on the tissue provides a measure of the attenuation constant and thus the penetra~ion for certain intensities.
Thirdly, the measurement of certain frequencies at times 20related to energization of ~he drug and oxygen provides signals related to destruction of undesirable tissue.
The amount of certain frequencies of emitted light is related to the destruction of tissue and thus to the intensity of applied radiation, the attenuation constant in the tissue, the amount of drug, the availability of ~ ~26~;~.5~3 oxygen and the distance from the incident radiation.
Measurement of this radiation provides a general indication of activity. The fluorescent irradiance is linearly related to drug concentration with a known exciting irradiance so that a measure of drug concentration is obtainable after calibration. From this relationship the clearance of drug from tissue can be determined after injection and durinq periodic light treatment.
The depth of penetration of an adequate exciting radiation into tumor can be estimated from the attenuation coefficient of tissue and the irradiance output increased to the value necessary for the selected depth chosen. The attenuation coefficient can be measured by biopsy or from a measurement of the irradiance at the exciting frequency at a first and second location ~rom the incident exciting radiation.
This coefficient is equal to the product of two factors. The first factor is the reciprocal of the dif~erence between the distance from the incident radiation to the first point and the distance from the incident radiation to the second point. The distances are both within the tissue. The second factor is the natural log of a fraction having a numerator and a denominator. The numerator is the product of the ~%~

measured irradiance at the seond point and the distance between the incident irradiation and the second point.
The d~nominator is the product of the irradiance at the first point and the distance between the incident exciting radiation al)d the first point.
One type of apparatus for measuring the coefficient of attenuation is shown in FIG. 12 having a outer sheath 130, a transmitting light conductor 132, a first light receiving conductor 134, a second light receiving conductor 136 and a spacinq wedge 138. ~his apparatus is shown broken away at 1~0 to illustrate that it may be longer than actually shown.
To measure the irradiance at the first and second points for calculation of the coefficient, the outer sheath 130 slidably confines the light conductors 132, 134 and 136. It is sized to be inserted to the tissue being measured and to accommodate the transmission of light to the tissue through conductor 132 and the measurement of irradiance through conductors 134 and 136. It may be inserted through an endoscope until the conductors 132, 134 and 136 contact the tissue.
To measure the distance between the incident radiation from conductor 132 and the first and second point at conductors 134 and 136 for calculating the coefficient of attenuation, the conductors are spaced at

4~

~2~
so fixed angles to each other in a line by sheath 138 so that the distance between their ends can be trigonometrically calcul~ted from the angle and the amount they are extended from the apex of the triangle.
The angles of the conductors are 30 degrees bet~een conductors 132 and 134 and 60 ~egrees between conductors 132 and 136. The lengths extended are measured by marks such as those shown at 140 on conductor 136 compared to the edge of the sheath 138.
Of course, the distance may be fixed, but the embodiment of FI~. 12 provides an adjustable device that may select diE~erent distances and be used for different tissue locations. The light conductors may be withdrawn for protection during insertion. With the attenuation constant known, the depth of penetration of a minimum irradiance or conversely the required irradiance for a minimu~ intensity at a given distance may be calculated.
The calculations are based on one oE three expressions.
In the first expression, the light is emitted from a source that is substantially a point source and the expression provides the treatment distance to a point of an assumed light flux density. In this expression, the length of treatment in tissue is the total length through the tissue from the point source in any direction through the treatment distance from the point ~2~

source. Thus, the length o~ treatment tllrough tissue or along any straight line through the point source extends for a length equal to twice the treatment distance in this expression. It will cover a sphere or a section of a sphere having a radius equal to this distance.
In this first expression, the assumed minimum irradiance is equal to the irradiance at the point source divided by a denominator which is a product of two ~actors: the first being t~le distance from the point source to the point of assumed minimum irradiance and the second being the natural log base raised to the power of the product of the distance and the attenuation coefficient. The attenuation coefficient is a number characteristic of the tissue and has the dimensions of the re~iprocal of length. It is the reciprocal of the distance at w~ich the irradiance is reduced by a factor of one divided by the natural log base.
In the second expression, the light is incident on ~he surface as an approximate plane ~ave. In this expression, the distance of treatment is perpendicular to sur~ace to a depth of the assumed necessary minimum irradiance. The minimum irradiance across the teeatment distance is equal to a fraction having a numerator and denominator. The numerator is the irradiance at the ~i5gc~

surface and the denominator is the natural 109 base raised to the power of the product of the maximum trea~men~ distance and the attenuation coefEicient.

In the third expression, the light emitter is a cylinder embedded in the tissue and the space irradiance varies as the modified Bessel function of the second kind of t~e 0 order, which decreases more slowly with distance than does the function for a point source described above in expression one.

In FIG. 13 there is shown a bulb-type light-emitting so~rce 42A having a light transmission fiber 80 inserted in a diffusing bulb 82 which receives light, diffuses it within the bulb and emits it with equal intensity in all directions. This bulb may be used to irradiate a laege area such as a bladder or the like.

In FIG. 14 there is shown a sectional view of the light-emitting source 42A having the light fiber 80 inserted into the diffusing bulb 82. The diffusing bulb 82 is polycarbonate, held in place by epoxy glue 85 and capable oE ~ransmitting light the~rethrough from ground surEaces 83 on the ends of the light conductor 80.

~lternatively, the surfaces 83 may be ~used as half a sphere to control the angle of irradiation or other lenses may be used. Its inner surface is coated with a reflective dlffùsing material 87, which in the preferred ` 52 ~6~i~5~

embodimellt is formed of particles o~ saphire united by epoxy to the inner sur~ace to reflect light within the difEusing bulb 8?-. However, it may be other reflective materials such as barium sulfate. Light is also forward scattered and emitted.
The diffusing bulb 82 is fluid tight, of sufficient size to avoid, during normal use, a temperature increase so great at any location as to degrade the material to the point of breaking. It is usually submerged in a 1uid or semifluid matter and at a distance so the power density is low at the first sur-face that absorbs light. Thus, this surface in contact with blood receives light having an optical power density low enough so that it remains relatively cool and blood does not coagulate on it.
In FIG. 15 there is shown a side elevational view of an eye applicator 42B, having a hollow tubular stem 90 for receiving a fiber conductor and a teflector 92 positioned to receive light from the fiber conductor and reflect it onto a particular tumor. lrhe holllow tubular stem 90 is relatively stiff and "L" shaped with a plastic cylindrical socket 89 on one end and the reflector 92 on the other end so that the reflector 92 may be inserted behind the eye with the socket 89 outside the eye to receive a light conductor.

6~

~s best shown in FIG. 16, the socket 89 is tubular to receive and hold a light condllctoe so that light may be conducted through the hollow tubular stem 90 to an aperture 93 where the stem 90 joins the reflec-tor 92. The stem 90 is less than one eighth inch in diameter, The reflector 92 includes a cylindrical reElective portion 95 covered by a transparent diffusing surface 97.
As shown in FIG. 17 t the reflector 92 is capshaped with a polished reflective surace curved to reflect light it receives from the light conductor 80A

in multiple paths to obtain an even distribution. The liqht passes through a 400 micron light conductor 80A in the stem 90 (FIGS, 15 and 16) and a 600 micron diameter quartz cylindrical lens 101 that transmits light in paths parallel to the open end of the ~eflector g2 through a wider angle than paths toward the open end.

This increases multiple path reflections and even distribution of the light across the selected area, thus ~0 reducing spot intensity and covering an area.
The open end o the reflector 92 is either: (1) on the side closest to the socket 89; or (2) furthest from a reflective back 95. It functions to direct light into the eye or away from the eye onto optic nerves. In the former case, the open end is covered with the ~;5~5~

diffusing s~rface 99 parallel to and aligned with the open end of the reflector 92 to diffuse light. The open end is sealed by a light passing member 95. In the latter case, the open end faces in the opposite direction and is also sealed by a light passing member.
In FIG, 18, there is shown still another light emittinq source 42C having an emitting light conductor 144 and a receiving conductor 142. In this embodiment.
the receiving light conductor fits against the surface to receive radiation within the tissue and spaces the emitting conductor 14~ to which it is attached Çrom the surface of the tissue by a selected distance to irradiate a selected surface area of the tissue.
In FIG. 19, there is shown a schematic circuit diagram of a light feedback unit 37 (FIG. 8) having an electrical conductor 100, a transmi~ting fiber optic light conductor 106 of the bundle 40 (FIG. 8), an opaque housing 102 and an optical sensor 104. The light feedback unit 37 de~elops a signal on conductor 100 for application to the monitor system 22 (FIG. 8) related to light transmitted through the fiber optic conductor 106 through the opaque housing 102 which is an opaque inter~ace between the laser system 10 and the casing 74 for the light interface system 20 (FIG. ~).

~2Çi~

To develop a feedback signal for application to the monitot system 22 (FIG. 8) the feedback unit 37 includes an optical sensor 10~ having a lens 110, a light sensing diode 112, an amplifier 11~ and a resistor 116, The lens 110 receive5 light from the leakage spot through the fiber optic conductor 106 and transmits it ~o ehe lig~t sensing diode 112, which has its cathode electrically connected to one input of the amplifer 114 and its anode electrically connected to ground and to the other inpu~ of the amplifier 114. The eesistor 116 is a feedback resistor ~etween the cathode of the light sensing diode 112 and the output of the amplifier 114.
The conductor 100 i5 electrically connected to the output of the amplifier 114 to provide a signal related to the light intensity impinging upon the sensing diode 112. This signal may be used ~or control and monitoring purposes.
In FIG. 20 ~here is shown a block diagram of the monitor system 22A having t~le readout 25 (FIG. ~) which ~0 includes in the preferred embodiment a digital volt meter 124, a voltage control oscillator 126 and a speaker 128. The photodio~e 28 tFIG. 8) is electrically connected to readout 25 through conductor 36 and converts the current signal from the sensor to a voltage output, which voltage output represents the ~2~

amount of illumination from the treatment area. This may be further processed for use in the power control 23 if desired.
To provide a read-out of ~he amount of fluorescence resulting from a known intensity of light on a treated area, the conductor 36 is electrically connected to the digital volt meter 124 and to the voltage control oscillator 126. The digital volt meter 124 is read directly and the voltage control oscillator 101~6 generateC an alternating current voltage which is applied to the speaker 128 to provide an audible signal, the pitch of which indicates the amount of fluorescence.
Although a digital volt meter and a speaker are used for visual and audible indications to the user, other read-out techniques may be used and a signal, although not used in the preferred embodiment, may be applied to the lasers to alter intensity or frequency or both in a feedback system. The signal may also be utilized to generate a signal for visual interpretation 20on ~n oscilloscope or to be applied to data processing equipment for conversion to digital form and for further calculations. Moreover, it may be recorded on a chart or graph for analysis later.
While tests using the new drug have been perormed principally on animals, it is believed that ~2~

equivalent results will be obtained on humans, utilizing the same or less relative amount of drug to body weight. Tests have been, to a limited extent, performed on humans with endobronchial tumors to support this opinion as s~own in tables II, III, IV and V. It is belleved that the aforedescribed treatment utilizing the drug of the invention, can be used repeatedly without cumulative damage to normal tissues, providing that ~reatment is not overly aggressive. This is supported by the data of tables II, III, IV and V as well. Furthermore, recent ~ests of patients utilizing the drug DHE at doses to produce equal or better results compared to the prior art drug have resulted in markedly lower toxicity of healthy ~issue in lung cancer patients While the aforementioned animal tests utilized a dosage of the new drug of approximately 4 mg/kg of body weight, in the treatment of the tumors in humans, dosages as low as 1 mg/kg of body weight are believed efEective in utilizing the new drug. In any event dosages of the new drug of only approximately one-half of the prior art dosages are equivalently effective in accomplishing necrosis of tumors.
Also, while the aforementioned animal tests utilized illumination one day following injection of the new drug and t~le human tests 2 to 3 days, it is believed ~2~

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that a delay of up to seven days prior to illumination still will accomplish necrosis, and a time delay of three hours to three days between injection and illumi-nation is generally believed at this time preferable in hul~ans in order to achieve the best therapeutic ratio of drug in undesirable tissue to drug in normal tissue.
However, it is believed that these differ in various types of tissues The optimum therapeutic ratio can be determined by experience and measurement of fluorescence lQ and the ratio which provides destruction of the undesirable tissue with minimum change to the normal tissue is selected based on the drug level in both the undesirable and normal tissue~
Furthermore, while an intensi~y of 160 mw/cm2 for 30 minutes was utilized to activate the drug, it is believed that an intensity as high as 1 watt/cm2 for 20 minutes or as low as S mw/cm~ for an extended period of time may be utilized to accomplish necrosis. Less than 5 mw/cm2 of illumination intensity will probably have no ~0 therapeutic effect, irrespective of time of application.
More than 400mw/cm2 may cause undesirable thermal efects in some cases. For inserted cylindrical fibers, powers in the range of 50 to 500mw/cm of emitting length are used without thermal effects or above SOOmw/cm if thermal effects are~desired.

~65~

DBA2 Ha/D mice were transplanted with SMT-F
tumors. When the transplanted tumors reached 5-6mm (millimeters) in diameter, the mice were injected with a dose of 7.5 milligrams of the crude prior art Lipson derivative per kilogram of body weight for comparison purposes.
Approximately 24 hours following the injection, the tumor areas of the mice were shaved to remove the fur.
The mice were exposed to red light (600-700 mw) from an arc lamp at an intensity of 160mw (milliwatts) per square centimeter for 30 minutes~ Ten of twenty mice showed no apparent tumors seven days after treatment.
The injected drug is retained in the tumor cells longer as compared to normal tissue.
This protocol was repeated using the new drug disclosed in this invention and equivalent results were obtained but using a drug dose of approximately one-half (4 mg/kg of body weight), as compared to the prior art Lipson drug.
~0 In further tests ICR Swiss ~Albino) mice were injected with a therapeutic dose of the crude Lipson derivative (7.5 mg/kg of body weight)O Approximately 24 hours following such injection, the hind feet oE the mice were exposed ~o the same light conditions used in the aforesaid tumor response study. The damage to the ~659~

hind feet was assessed as 2.0 on an arbitrary scale where 0.0 is no damage and 5.0 is complete necrosis.
Moist desquamation was ~vident and the foot area slowly returned to normal after about 40 days. This protocol was repeated using the new drug disclosed in this appli-cation in doses of 4 mg/kg oE body weight. Only slight erythema and/or edema was noticed following treatment for a score of less than one on the a~orementioned scale of damage This condition disappeared after 48-72h (hours) with no residual effects. ThiS leads us to believe that s~in photosensitivity may no longer be a significan~ problem when using this new drug.
A summary of further tests on animals is shown in table I for mice comparing unpurified HPD and the purified DHE new drug indicating drug levels in mice.
From the foregoing description and accompanying drawings, it will be seen that the invention provides a new and novel drug, useful in the diagnosis and treatment of tumors, permitting utilization of reduced a~ounts of the drug as compared to related prior art drugs and which results in less severe side effects.
The invention also provides a novel method of producing t~e naw drug, together with a novel method of utilizing the drug in the treatment of tumors.

~l2~i5 Table 1 T~5SUE J.~.VF.LS OF 31~-HPD ~D
H-DIIE (~g/g wet tissue) DBA/2 Ha MI OE , SMT-F TI~OR

Injected Dose (m~/lc~) _ Liver Kidnev Spleen l0 - Hpd 24 h 14.2 + 2 9.7 -~ 2.17.1 + 1.2 5 - DIIE 24 h l9.l + 3.3 8.3 + 2,3 8.1 + 2.9 10 - Hpd 72 h 13.8 ~ 6 7.3 + 36.1 + l.l 5 - D~F. 72 h 15 ~ 4 7.6 + 2.56.6 + 1.4 Injec~ed Dose (m~/k~ MuscleBrain 10 - Hpd 24 h l.9 ~ 0.4 0.76 ~ 0.25 0.33 + 0.15 5 - DliE 24 h2.7 + l.4 0.68 + 0.26 0.19 ~ 0.l 10 - ~Ipd 72 h2.3 0.91.2 + 0.7 0.7 + 0.4 5 - DH~ 72 }I2.3 + 0.81.9 + 0.6 0.9 + 0.6 Injec~ed ~ose (mg~kg) Skin Tumor l0 - ~Ipd 24 h3.5 + 1.23.6 + l.l S - DHE 24 h3.4 + 1.33.5 + 1.2 l0 - ~Ipd 72 h2.8 ~ l.92.3 + 1.08 5 - DIIE 72 h1.~ + 0.61.6 + 0.5 Mini~um nunlber of animals per tissue was 10, maximum 17. Tumor volume doubling is n~proxima~ely 3 days.

~2~;4~i~

The terms and expressions which have been used are used as terms of description and not of limitation and there is no intention in the use of such terms and expressions of excluding any equivalents of any of the features shown or described, or portions thereof.
Moreover, various modifications in the preferred em~odi~ent are possible within the scope of the claimed invention.

-

Claims (29)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A composition comprising porphyrins, porphyrin-like compounds or a mixture thereof which are fluorescent, photosensitizing, and capable of localizing in and being retained in tumor cells for a longer time than in normal tissues, which composition is prepared by a process which comprises:
treating hematoporphyrin derivative by raising the pH to 6.5-12 to form aggregates of aggregate weight 10 kd or greater, followed by recovering the aggregate.

2. A composition according to claim 1 in which the porphyrins or porphyrin-like compounds include a compound of the formula:

in which R1 includes at least one atom with a valence greater than one.

3. A composition according to claim 2 in which R1 includes an ether linkage.

4. A composition according to claim 2 in which R1 is a substituted ethyl ether function.

5. A composition according to claim 2 in which R1 is a carbon-carbon linkage.

6. A composition according to claim 2 in which R1 is a substituted alkyl function.

7. A composition according to claim 2, 3 or 4 in which each of R2-R15 is independently a covalently bound chemical group with a molecular weight less than 1,000.

8. A composition according to claim 2, 3 or 4 in which at least one of the ring hydrogens in the formula of claim 2 is methine hydrogen.

9. A composition according to claim 3, 4 or 5 in which at least one of R2-R15 is an alkyl group.

10. A composition according to claim 2, 3 or 5 in which at least one of R2-R15 contains a carboxylic acid group.

11. A composition according to claim 2, 3 or 5 in which said porphyrins or porphyrin-like compounds comprise molecules having two groups, each group being arranged in a ring structure.

12. A composition in accordance with claim 1, 2 or 3 in liquid form and containing the porphyrins or porphyrin-like compounds in a concentration of approximately 2.5 mg/cc.

13. A composition in accordance with claim 1, 2 or 3 wherein the porphyrin or porphyrin-like compounds have an empirical formula of approximately C68H70N8O11.

14. A composition in accordance with claim 1, 2 or 3 wherein the porphyrin or porphyrin-like compounds have an empirical formula of approximately C68H66O11Na4.

15. A composition in accordance with claim 2, 3 or 4 in a liquid form and including isotonic saline solution at a pH of approximately 7.0 to 7.2.

16. A composition according to claim 1 wherein the porphyrin or porphyrin-like compounds have at least one phlorin.

17. A composition in accordance with claim 16 in which said phlorin is encapsulated in a liposome.

18. A composition in accordance with claim 16 in which said compounds have the formula:

19. A composition according to claim 1 wherein the porphyrin or porphyrin-like compounds have at least one chlorin.

20. A composition in accordance with claim 19 in which said compounds have the formula:

21. A process to prepare a composition consisting essentially of 10 kd aggregates of porphyrins, porphyrin-like compounds or mixtures thereof and being retained in tumor cells for a longer time than normal tissues, which process comprises:
raising the pH of a hematoporphyrin derivative preparation in aqueous medium to 6.5-12 to obtain aggregates of 10 kd or greater, and recovering said aggregates from the remainder of the hematoporphyrin derivative preparation, and wherein the hematoporphyrin derivative preparation has been prepared by treating hematoporphyrin hydrochloride with a mixture of acetic acid and sulfuric acid.

22. The process of claim 21 wherein the pH is approximately 9.5.

23. The process of claim 21 wherein the separation is effected by filtering in a process which retains aggregates of molecular weight above 10,000.

24. The process of claim 23 wherein the pH range of 6.5-12 is maintained during filtration.

25. The process of claim 24 wherein the pH is maintained at approximately 9.5 during filtration.

26. The process of claim 21 wherein the separation is by affinity chromatography.

27. The process of claim 26 wherein the chromatography is on a reverse phase column with a packing of 5 micron spheres, using THF as solvent.

28. The process of claim 27 wherein the reverse phase column is a C18 column and elution is successively with methanol:water:acetic acid (20:5:1) followed by tetrahydrofuran:water (4:1), and the prophyrin mixture recovered corresponds to the fourth component eluted as shown in Figure 5.

29. The process of claim 21 wherein the hematoporphyrin derivative has been prepared by adding a first mixture of acetic acid and sulfuric acid to hematoporphyrin hydrochloride, stirring for one hour to obtain a second mixture, adding the second mixture to a solution of 5%
sodium acetate to obtain a red precipitate, and recovering the red precipitate.