United States Patent (19) 11 Patent Number: 5,030,200 Judy et al. (45) Date of Patent: "Jul. 9, 1991
54 METHOD FOR ERADICATING INFECTIOUS 4,708,715 11/1987 Troutner et al...... 604/6 BIOLOGICAL CONTAMINANTS IN BODY 4,878,891 1 1/1989 Judy et al...... 604/5 TISSUES Primary Examiner-Stephen C. Pellegrino (75) Inventors: Millard M. Judy; James L. Matthews; Assistant Examiner-Michael Rafa Joseph T. Newman; Franklin Attorney, Agent, or Firm-Johnson & Gibbs Sogandares-Bernal, all of Dallas, Tex. (57) ABSTRACT (73) Assignee: Baylor Research Foundation, Dallas, A method for externally eradicating infectious patho Tex. genic contaminants, such as enveloped viruses, bacteria, * Notice: The portion of the term of this patent trypanosomal and malarial parasites, present in body subsequent to Nov. 7, 2006 has been tissues, such as blood, blood components, semen, skin, disclaimed. and cornea, before the treated body tissues are intro 21) Appl. No.: 433,024 duced into, or transplanted onto, the body of a human or an animal. Such method includes the steps of: (1) 22) Filed: Nov. 6, 1989 admixing an effective, non-toxic amount of photoactive compound, which has a selectively for binding to the Related U.S. Application Data infectious pathogenic biological contaminants present (63) Continuation-in-part of Ser. No. 67,237, Jun. 25, 1987, therein, with the body tissues outside the body to pro Pat. No. 4,878,891. duce resulting body tissues; (2) maintaining the resulting 51 Int. Cl...... A61M 37/00 body tissues in a suitable container in which there is no (52) U.S. Cl...... 604/5; 424/529 net mass transfer, and (3) irradiating the resulting body 58) Field of Search ...... 604/4-6; tissues in the container for an effective period of time 424/101, 529-531; 128/395 with an effective level of radiation such that the radia tion penetrates the resulting body tissues and eradicates (56) References Cited the photoactive-compound-bound contaminants pres U.S. PATENT DOCUMENTS ent in the resulting body tissues and produces a decon 4,613,322 9/1986. Edelson ...... 354/321 taminated body tissue suitable for introducing into, or 4,614, 190 9/1986 Stanco et al...... 28/395 transplanting onto, the body of a human or animal. 4,649,151 3/1987 Dougherty et al...... 514/410 4,684,521 8/1987 Edelson ...... 424/10 66 Claims, 7 Drawing Sheets
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SYRINGE WITH BODY FLUID ATTACHED TO ATTACHED TO INFUSION PUMP FIG. 7 COLLECTING WIAL
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5,030,200 1 2 Localization of Pulmonary Malignancy, In Recent Re METHOD FOR ERADICATING INFECTIOUS sults in Cancer Research, Vol. 82, Springer-Verlag, Ber BIOLOGICAL CONTAMINANTS IN BODY lin-Heidelberg, 1982,90; Benson, R. D., et al, Detection TISSUES and Localization of In Situ Carcinoma of the Bladder with Hematoporphyrin Derivative. Mayo Clinic Proc., BACKGROUND OF THE INVENTION 57: 548, 1982). The present application is a continuation-in-part of Although the unique photodynamic properties of U.S. patent application Ser. No. 07/067,237 now U.S. Hpd, as well as its affinity toward tumor cells, had long Pat. No. 4,878,891, filed on Jun. 25, 1987, which is in been known, it was more than half a century later that corporated by reference as if reproduced in its entirety. O the potential of using Hpd to selectively destroy tumor 1. Field of the Invention cells was explored. The bulk of the research on the use The present invention relates generally to decontami of Hipd to selectively destroy tumor cells in human has nation of body tissues; and more particularly, but not by been reviewed by Dougherty et al. (Dougherty, T. J. et way of limitation, to external eradication of infectious al., Photoradiation Therapy-clinical and Drug Ad pathogenic biological contaminants from blood or 15 vances. In Porphyrin Photosensitization, D. Kessel and T. blood products prior to intravenous injection of such J. Dougherty, Eds., Plenum Press, N.Y. PP. 3-13, blood or blood products into a patient's body. 1983). The present invention also relates to external eradica The emphasis on using Hpd as the photoactivating or tion of infectious pathogenic biological contaminants light-activating compound in photoradiation of tumors from skin, cornea or semen prior to transplanting or 20 is based on two important properties of Hpd. Firstly, as introducing of such tissues into the recipient's body. judged by fluorescence, Hpd is preferentially accumu 2. Brief Description of the Prior Art lated, and retained to a higher degree in malignant tu One of the many problems that plagues the use of mors than in surrounding normal tissue or benign tu blood transfusions is the transmission of agents causing mors. Secondly, when properly photoactivated, Hpd infectious disease. A number of these infectious agents 25 causes the destruction of cells and tissue in which it are of serious clinical importance in that such agents are not only dangerous to the recipient patients, but can resides. The generally accepted mechanism of cell kill also pose a danger to physicians, and other hospital by Hpd is that when activated by appropriate light, the personnel, handling the blood and blood products. Hpd can undergo an energy transfer process with oxy Many efforts have been made to ensure that the blood 30 gen to form a singlet oxygen, which subsequently oxi to be transfused is free of pathogenic biological contam dizes, hence kills the cells or tissues to which it has inants. So far, screening of blood donors and blood attached as a substrate. (Weishaupt, K. R., Gomer, C.J. samples is the only effective method to ensure that the and Dougherty, T. J., Identification of Singlet Oxygen blood to be transfused is not contaminated with infec as the Cytotoxic Agent in Photoinactivation of a Mu tious agents. Unfortunately despite screening tech 35 rine Tumor. Cancer Res. 36: 2326-2329, 1976). niques, infections still occur following blood transfu Despite the enormous progress and research in the SOS use of light-activated photosensitizer, such as Hpd, to U.S. Pat. No. 3,041,242 describes a process for eradi pinpoint the location of malignant tumor cells and to cation of virus contained in dried plasma wherein the eradicate them, relatively little work has been done to dried plasma is heated at an elevated temperature for a 40 determine if such a photosensitizer will behave similarly length of time followed by applying a gas lethal to toward virus, bacteria, fungi, protozoa, or other para microorganisms, under high vacuum conditions. How sites. ever, the method is not applicable to the treatment of U.S. Pat. No. 4,649,151 to Dougherty, et al., discloses human whole blood; and the viability of the dried the preparation, purification and utilization of tumor plasma would most likely be impaired during the pro 45 selective, photosensitizing drugs (i.e. mixtures of por CSS. phyrins) in the localization and treatment of neoplastic It has been known for over 30 years that hematopor tissue, such as tumors or cancers in patients and animals. phyrin derivatives accumulate in neoplastic, embryonic, A time delay of several hours to several days is required and regenerating tissues. Thus, injected hematoporphy between injection of the drug and illumination in order rin has been found to have localized and fluoresced in 50 for the drug to metabolically clear normal tissue and several types of tumor induced in mice (Figge, F. H. J., hence achieve the best therapeutic ratio of drug in Weiland G. S., Mangiollo, L. O.: Cancer Detection and tumor cells to drug in normal cells. One of the objects Therapy. Affinity of Neoplastic, Embryonic, and Trau set forth in Dougherty, et al., is to provide a drug which matized Tissues for Porphyrins and Metalloporphyrins. is selective to certain pathogens within an animal or Proc. Soc. Exp. Biol. Med. 64: 640-641, 1948). 55 within blood, blood plasma or serum or fractions The red fluorescence of hematoporphyrin has also thereof and which permits photochemical destruction been observed under ultraviolet light in various malig of the pathogens in vivo or in vitro. However, the refer nant tumors in patients who had been given large doses ence contains no teaching or suggestion of the external of crude mixtures of hematoporphyrin compounds purification of human tissues such as human blood, (hereinafter referred to as Hpd) (Rasmussen-Taxdal, D. 60 blood plasma, serum, semen, skin or cornea utilizing a S., Ward, G. E., and Figge, F. H. J.: Fluorescence of mixture of porphyrins to eradicate infectious pathogens Human Lymphatic and Cancer Tissues Following High and to provide purified and sterilized human tissues Doses of Intravenous Hematoporphyrin. Cancer 8: . such as human blood, blood plasma, human serum, se 78-81, 1955). As a result, methods have been developed men, skin or cornea which can be infused or introduced to capitalize on the unique property of Hpd as a tumor 65 into a patient's body. marker in the detection and localization of different The Dougherty, et al. patent also fails to demonstrate forms of cancer (King, E. G., et al., Hematoporphyrin the tolerance of either human blood, human blood Derivative as a Tumor Marker in the Detection and plasma, or human serum outside the body toward the 5,030,200 3 4 drug. Moreover, no evidence was provided to demon Skiles, M. M. Judy, and J. P. Newman, Photodynamic strate that either human blood, human blood plasma, Inactivation of Viruses With Hematoporphyrin Deriva human serum, or other human tissues outside the body tives, Abstract A38, American Society for Microbiol remains unchanged after the irradiation of such blood, ogy, page 7, 1985). The combined effect of light and blood plasma, serum, or other tissues containing the Photofrin IITM on Herpes simplex virus type I grown in described drug. Similarly, neither the effective range of culture can be observed in a flow cell system made up of concentrations of the described drug nor the effective loops of transparent tubing attached to a glass slide, range of radiation is disclosed for its use outside an with a 1000 W Xeon light equipped with a red filter animal or human body. serving as the light source. (F. Sogandares-Bernal, J. L. U.S. Pat. No. 4,614, 190 to Stanco, et al. discloses an O Matthews, H. Skiles, M. M. Judy, and J. Newman, arrangement for effecting photoirradiation of tumor Photoactivation of Herpes simplex virus by Photofrin II and cancerous tissues in human or animal body. The and Light in A Flow Cell System, 1987 ASM Annual patent discloses the method of pulsing the electromag Meeting, Atlanta, Ga., 1-6 Mar. 1987). netic energy to activate, in situ, administered hemato Extracorporeal treatments of certain noninfectious porphyrin derivative contained within the tissue in the 15 cancers have been known for more than a decade. (H. body so that the surrounding flesh is not unduly af. Hyden, L. E. Gelin, S. Larsson, and A. Saarne, A New fected. 4. Specific Chemotherapy: A Pilot Study With An Extra This reference, however, neither teaches nor suggests corporeal Chamber. Rev. of Surgery (Philadephia), 31: the external purification of tissues such as blood, blood 305-320, 1974; H. Wolf, E. Langvad, and H. Hyden, plasma, serun, semen, skin or cornea utilizing a mixture 20 The Clinical Course In Patients With Renal Carcinoma of porphyrins to eradicate infectious pathogens and to Subjected To Extracorporeal Immunoadsorption, Brit provide purified and sterilized tissues such as blood, ish J. Urology, 53: 89-94, 1981). Recently, it was re blood plasma, serum, semen, skin or cornea which can ported that the activity of a noninfectious cancer, but be infused or introduced into the recipient's body. nevertheless potentially deadly, cutaneous T-cell lym The Stanco, et al. patent also fails to demonstrate the 25 phoma, could be controlled by extra corporeal photo tolerance of either blood, blood plasma, serum, or other chemotherapy. In the therapy, after patients were orally tissues outside the body toward the drug. Furthermore, given 8-methoxypsoralen, blood was removed from the no evidence was provided to demonstrate that either patient and the lymphoctye-enriched blood fraction blood, blood plasma, serum, or other tissues outside the was exposed to ultraviolet A. Subsequently, the dam body remains undamaged after the irradiation of such 30 aged lymphocyte-enriched blood fraction was returned blood, blood plasma, or serum, or other tissues contain to the body of the patient. An immune reaction to the ing the described drug. Similarly, neither the effective infused damaged cells then restricted the activity of the range of concentrations of the described drug nor the abnormal cancer cells in the patient's body. (R. Edel effective range of radiation is disclosed for its use out son, et al., Treatment of Cutaneous T-Cell Lymphoma side an animal or human body. 35 by Extracorporeal Photochemotherapy, New England Studies using fluorescence and laser techniques have J. Medicine, 316:297-303, 1987). suggested that hematoporphyin derivative (Hpd) would The purpose of the extracorporeal photochemoth bind to parasites Plasmodium berghei, P. vivax and P. erapy as reported by Edelson, et al., was not to damage falciparum. Moreover, whole animal studies utilizing a as many cells as possible outside the patient's body. mixture of Hpd and Chloroquine, an antimalarial drug, Rather, only a small fraction of the cells was damaged have reflected the reduction of the parasitemia in mice which was then reintroduced into the patient's body to infected with Chloroquine resistant P. berghei (F. serve as a "vaccine' for triggering an immune reaction Sogandares-Bernal, J. L. Matthews and M. M. Judy, in the body. HPD-Induced Reversal of Chloroquine Resistance to Despite the progress in photochemotherapy, no Malaria, a lecture presented at International Sympo 45 work, however, has been reported concerning the util sium on Malaria, held on Jun. 1-5, 1986, at Instituo ity of such method to eradicate viruses present either in Oswaldo Cruz, Rio de Janeiro, Brazil, also in press, human whole blood or in other body tissues outside the Men. Inst. Oswald Cruz). body. Similarly, no one has reported the use of photoin A few investigators have reported photoinactivating activation in a clinical setting to remove infectious bacterial viruses and animal viruses using heterocyclic 50 agents, such as viruses, bacteria, fungi and protozoa, dyes (Yamamoto, N., Nitrogen Fixation by A Facul from human whole blood used for transfusion. tatibe Bacillus, J. Bacteriology, 75: 403, 1958; Hiatt, C. Hepatitis-B virus causes a hepatitis infection which W., et al: Inactivation of Viruses by the Photodynamic may develop into cirrhosis in which the liver becomes a Action of Toluidine Blue, J. Inmunology, 84: 480-84, mass of fiber-like tissue. In hepatitis, liver function is 1960). The plaque formation capability of Herpes sim 55 impaired and, in some cases, the condition can be fatal. plex virus has also been reported to be hampered by the There are approximately 200,000 cases of reported hep treatment of virus in culture with a combination of a atitis infections per year in this country. In addition, hematoporphyrin derivative and visible light. (Lewin, there may be as many as a few million carriers of hepati A. A., et al., Photodynamic Inactivation of Herpes sim tis. plex Virus by Hematoporphyrin Derivative and Light, The human T-lymphotropic retrovirus (HTLV) is Proc. Soc. Exp. Biol. Med. 163; 81-90, 1980). the cause of acquired immune deficiency syndrome Similarly, it has been reported that, in culture, the (AIDS) which is invariably fatal. So far, the AIDS virus plaque formation by Herpes simplex virus type I, Cyto has infected more than a million people in this country megalovirus, or measles virus is reduced, by more than alone. About one-third to one-half of the infected indi 99%, by the combination effect of Hpd and Rhodamine 65 viduals will develop the disease. Worst yet, because the B dyelaser light, with an energy density of 20 J/cm2. On AIDS virus has a long and undeterminate period of the other hand, the echovirus type 21, which lacks an incubation, a person can unknowingly carry and spread envelope, is not affected under similar conditions (H. the deadly disease for years. The invariably fatal viral 5,030,200 5 6 disease AIDS can be transmitted by the exchange of Since there is so far no cure for AIDS, it is also desir body tissues or body fluids such as blood, blood prod able to have a safe method and apparatus to reduce the ucts, or semen. Indeed, hemophiliacs and other receiv viremia in AIDS patients to prolong the lives of such ing blood transfusions account for about 3% of the patients. reported AIDS cases in this country between 1981 and It is toward such goals that the present invention is late 1986. Artificial insemination, organ transplant, and directed. transplant of skin, cornea and other tissues can also transmit this fatal viral disease. SUMMARY OF THE INVENTION People infected with viruses may carry these agents According to the present invention an efficient and or their particles in their blood. Likewise, people at O economical method for treating body tissues to eradi tacked by infectious diseases often carry pathogenic cate infectious pathogenic biological contaminants, microorganisms or other contaminants in their blood. such as envelope-containing viruses, bacteria, malarial, Consequently, blood donated or sold to blood banks trypanosomes, and other parasites, which may be pres may be contaminated with virus or other biological and ent in said body tissues is provided wherein the contam pathogenic contaminants. 5 inants are eradicated prior to introduction of the treated Most blood samples are now being tested for the body tissues into the body of a patient. Broadly, the presence of certain virus. These fests usually involve the method comprises: determination of the presence of antibodies to various (a) introducing or admixing an effective, non-toxic viruses or the viral antigen itself, such as HBSAg. Al amount of a photoactive compound with the body though most of the tests employed to test blood samples tissue to produce a resulting body tissue, the photoac are generally quite accurate, they are not infallible. tive compound having a selectivity for binding to the Also, due to cost considerations, not all blood is tested infectious pathogenic biological contaminants present for the presence of pathogenic contaminants, including in the body tissue; viruses. Most importantly, because antibodies do not (b) maintaining the resulting body tissue in a suitable form immediately after exposure to the virus, a newly 25 container in which there is no net mass transfer; and infected person may unknowingly donate blood after (c) irradiating the resulting body tissue in the container becoming infected but before the antibody has a chance for an effective period of time with an effective level to manifest a positive test. It has also been documented of radiation such that the radiation penetrates the that some people infected with certain viruses simply do resulting body tissue and eradicates the photoactive not produce detectable antibodies against them. 30 compound-bound contaminants and produces a de A large number of diseases, some of which are either contaminated body tissue suitable for introduction fatal or of serious clinical importance, can be transmit into the body of a patient. ted by transfusion. Since pathogenic organisms are An object of the present invention is to provide an found in different fractions of whole blood, risks of 35 improved method for externally eradicating infectious post-transfusion diseases vary depending on the blood pathogenic biological contaminants from blood and product or component used. In general, the risk for any blood products. disease is directly proportional to the volume of blood Another object of the present invention is to provide transfused and to the numbers of infectious organisms an efficient and economical method to externally eradi contained therein. cate infectious pathogenic biological contaminants from It is clear that despite screening techniques, infections blood and blood products in a container so that the with viruses and other biological pathogenic contami blood and blood products are safe for introduction into nants still occur following blood transfusions. In the the body of a patient. setting of clinical medicine, the processing and handling Still another object of the present invention is to of body fluids, such as blood, imposes a threat of a 45 provide an efficient and economical method for exter number of possible infections to physicians and other nally eradicating infectious pathogenic biological con hospital workers, and patients. Currently, there is no taminants from blood or blood products so that the effective procedure for decontaminating the infected blood or blood products are safe for handling. body fluid, such as human whole blood or its formed Another object of the present invention is to provide elements. 50 an effective and economical method for external eradi It is, therefore, highly desirable to have a safe and cation of pathogenic enveloped viruses, other microor economical method and apparatus that will eradicate ganisms, or other pathogenic biological contaminants in pathogenic viruses, microorganisms, or parasites pres tissues intended for transplantation to humans. ent in human whole blood or blood products before Yet another object of the present invention is to pro such products are infused into a recipient, hence, infect 55 vide an effective and economical method for external ing the recipient with such disease producing agents. At eradication of pathogenic enveloped viruses, other mi the same time, properly decontaminated blood will also croorganisms, and parasites, or other pathogenic bio spare the daily threat of infections to hospital personnel logical contaminants in protein and other materials who must handle these body fluids. This need is even intended for intravenous administration to humans or more acute in a blood bank where donor blood and animals. blood products are stored and processed. Other objects, advantages and features of the present It is equally desirable to have a safe and economical invention will become clear from the following detailed method and apparatus that will eradicate pathogenic description when read in conjunction with the drawings viruses, microorganisms, or parasites present in human and appended claims. or animal tissues, such as skin, cornea, and semen, be 65 fore such tissues are introduced or transplanted into a BRIEF DESCRIPTION OF THE DRAWINGS recipient, hence, infecting the recipient with such dis FIG. 1 is a schematic representation of a cell assem eaSS. bly having a predetermined flow path in the form of a 5,030,200 7 8 flexible transparent tubing wherein fluids passing along ing a transfusion or transplantation of the body tissue is the flow path are exposed to radiation. substantially eliminated or at minimum the parasite or FIG. 2 is an enlarged cross-sectional view of the infectious agent burden of the tissue is considerably flexible transparent plastic tube of FIG. 1. reduced. FIG. 3 is a perspective view of the irradiation cell 5 The term "body tissue' as used herein is to be under being irradiated from the top of the cell. stood to include "body fluid', packed red blood cells, FIG. 3-a is a diagram showing a three-dimensional packed white blood cells, platelets, cryo precipitate rectangular coordinate system for the irradiation cell of from blood plasma, other plasma proteins, skin, cornea, FIG. 3. and other tissue from an animal or a human. FIG. 4 is a perspective view of the irradiation cell 10 The term “body fluid' as used herein is to be under being irradiated from both the top and the bottom of the stood to include whole blood, any formed elements of cell. the blood, blood plasma, serun, fluids containing said FIG. 4-a is a diagram showing a three-dimensional components, fluids from plasmapheresis, plasma fibrino rectangular coordinate system for the irradiation cell in gen, cryo-poor plasma, albumin, gamma globulins, se FIG. 4. 15 men, and other fluids introduced or intravenously in FIG. 5 is a perspective view of the irradiation cell jected into the body of a patient or an animal using being supported on the bottom by a reflecting mirror known administration techniques. The "body fluid' as surface while the cell is being irradiated from the top of used herein is to be understood to include body fluid the cell. prior to, or after, physical as well as chemical fraction FIG. 5-a is a diagram showing a three-dimensional 20 ation, separation or freezing. rectangular coordinate system for the irradiation cell in The term "external' as used herein is to denote out FIG. S. side the animal or human body. As is known in the art, FIG. 6 is a longitudinal sectional view of another the term "extracorporeal' means outside of, or unre embodiment of the flow cell. lated to, the body or any anatomical 'corpus'. FIG. 7 shows a perspective view of the irradiation 25 The words "no net mass transfer' as used herein are cell sandwiched between two transparent flat platens to denote the condition in which the body tissue that is while the assembly is being irradiated from two oppo maintained or stored in a container neither enters nor site directions, top and bottom, of the cell. leaves the container while being irradiated. In the case FIG. 8 shows the longitudinal sectional view of the of body fluids, the body fluids do not flow in and out of irradiation cell surrounded on the top and bottom with 30 the container during the process of illumination. The two arrays of tubular lamps which are depicted cross body tissues can be either kept static in the container or sectionally as small circles. Subjected to agitation or movement. Optically transpar FIG. 9 is a plot of normalized relative light intensity ent or "thin' body tissues can be irradiated in the con vs. the ratio of distance D with respect to distance h. tainer under static conditions. On the other hand, in the Distance D is the spacing between two adjacent lamps, 35 irradiation of optically "thick' or non-transparent body while distance h is the separation between the piane tissues, agitation may be required. Optically "thick' or containing the array of lamps and the illuminated sur non-transparent body tissues are those that absorb and face of the irradiation cell. Scatter light appreciably and will not allow light to FIG. 10 is a plot of normalized relative light intensity penetrate. Agitating the optically "thick' or non-trans along the length of the tubular lamp. The longitudinal parent body tissues in the container will promote the sectional view of the lamp is shown on top of the dia mixing, and hence the binding, of the body tissues with gran. the photoactive compound. Moreover, agitation will FIG, 11 is a cross-sectional view of a schematic dia enhance the probability of having every cell in, and gram of a closed container in the form of a flexible every part of, the body tissues exposed to incident radia transparent plastic bag which is partially filled by body 45 tion. fluids and kept static. The words "illuminated with light' and "irradiated FIG. 12 is a schematic diagram of the plastic bag of with radiation' are used interchangeably. FIG. 11 moving to the right. The method of the present invention provides for the FIG. 13 is a schematic diagram of the plastic bag of external decontamination of body tissues containing FIG. 11 moving to the left. 50 infectious pathogenic biological contaminants such that FIG. 14 is a perspective view of a schematic diagram the body tissues possess the desired therapeutic proper of a transparent closed container showing the possible ties and viability as normal, untreated body tissues. directions of reciprocating translational motions. Broadly, the method for treating body tissues to eradi FIG. 5 is a perspective view of a schematic diagram cate infectious pathogenic biological contaminants of a transparent closed container showing the possible 55 which may be present therein comprises: directions of reciprocatory rotational motions. (a) admixing an effective, non-toxic amount of a photo FIG. 16 is a cross-sectional view of a schematic dia active compound with the body tissue to produce a gram of a closed container in the form of a flexible resulting body tissue, the photoactive compound transparent bag which is partially filled with contami having an affinity for infectious pathogenic biological nated body fluids and has an air bubble. contaminants in the body tissue such that the photo active compound is selectively bound to such con DESCRIPTION OF THE PREFERRED taminants; EMBODIMENT (b) maintaining the resulting body tissue in a suitable The present invention provides a method for exter container in which there is no net mass transfer; and nally eradicating infectious pathogenic contaminants, 65 (c) irradiating the resulting body tissue in the container Such as enveloped viruses, microorganisms, parasites, with an effective level of radiation for a prescribed bacteria and the like, from body tissues that the likeli period of time so that the radiation penetrates hood of a person becoming infected as a result of receiv throughout the resulting body tissue in the irradiated 5,030,200 9 10 path and exposes the photoactive-compound-bound approximately 365, 505, 537, 575, and 615 nanome contaminants to the radiation so as to eradicate such ters, absorption peaks in the infrared spectrum at contaminants and produce a sterile body tissue. approximately 3.0, 3.4, 6.4, 7.1, 8.1, 9.4, 12 and 15 Photoactive compounds which are selective for in microns, absorption peaks in carbon-13 nuclear mag fectious pathogenic biological contaminants, and which 5 netic resonance study at at least approximately 9.0, can be used in the eradication of such contaminants in 18.9, 24.7, 34.5, 62, 94.5, 130-145, 171.7 ppm relative accordance with the present invention, must satisfy the to a 37.5 ppm resonance peak of dimethyl sulfoxide following criteria: and additional absorption peaks in carbon-13 nuclear (1) normal body tissues remain undamaged in an envi- magnetic resonance study at approximately 27.9 ppm ronment outside of a human body when subjected to a 10 and 68.4 ppm relative to the resonance peak of tet combined action of irradiation and the photoactive ramethylsilane in deuterated chloroform solvent; and compound in levels effective to eradicate or destroy the at least 50 percent of the porphyrins in said mixture infectious pathogenic biological contaminants present being of said molecule having said molecular for in such body tissues; mula. (2) the photoactive compound is preferentially bound 15 The effective, non-toxic amount of the mixture of to the infectious pathogenic biological contaminants porphyrins admixed with the body tissue externally can either directly or indirectly by an antibody bridge or vary widely, depending to a large degree on the amount other linking molecule; and of infectious pathogenic biological contaminants pres (3) upon irradiation, the photoactive compound erad- ent or suspected to be present in the body tissue. How icates or destroys the infectious pathogenic biological 20 ever, to be effective the amount of the mixture of por contaminants to which the photoactive compound has phyrins utilized should be in excess of the amount of bound. such contaminants to insure that sufficient mixture of Photoactive compounds meeting the foregoing crite- porphyrins is present to bind all of the contaminants ria comprise porphyrins or hematoporphyrins. Such present in the body tissue. Generally, however, the compounds contain groups of pyrroles or substituted 25 amount of mixture of porphyrins admixed with the pyrroles combined in a specific pattern. The basic struc- body tissue to satisfy the above requirement is from tural grouping consists of phlorin or a group of four about 0.1 to about 50 micrograms of the mixture of pyrroles or combinations of pyrroles and substituted porphyrins per milliliter of body fluid. Desirably, the pyrroles combined into a larger ring structure. Two amount of the mixture of porphyrins used is from about such rings are covalently bonded to form a pair of units 30 2 to about 50 micrograms per milliliter of body fluid. each having four pyrrole groups or four groups at least To eradicate the infectious pathogenic biological some of which are pyrroles or substituted pyrroles. The contaminants which have been bound to the photoac resultant molecules have an absorption spectrum within tive compounds, the resulting mixture of body fluid and the range of wavelengths of from about 350 nm and the photoactive compound is subjected to radiation, about 1200 nm, and more desirably from about 350 nm 35 while maintaining the resulting mixture under flow to about 700 nm. conditions during exposure to the radiation. The preparation and purification of photoactive com- Any suitable source can be employed to irradiate the pounds comprising porphyrin and hematoporphyrins is photoactive-compound-bound contaminants provided disclosed in U.S. Pat. No. 4,649,151; and certain of such such source produces sufficient radiation to activate the photoactive compounds are commercially available 40 photoactive compound so as to eradicate or destroy from Johnson & Johnson as "Photofrin IITM" (Hpd) such contaminants bound to the compound and suffi and "Photofrin IITM" (a compound containing approx- cient energy to insure complete penetration of the radia imately 90% dihematoporphyrin ether, DHE). tion through the body fluid being treated, so that the The molecular structure of at least a portion of such treated body fluid is free of such contaminants. The photoactive compounds which render such compounds 45 operable source employed to irradiate the resulting useful in the practice of present invention is as follows: fluid has a wavelength of from about 350 nm to about 1000 nm and an energy density of from about 0.1 J/cm2 (Na)HO2C(CH2)2 CH3 H3C (CH2)2CO2H(Na)
H H Hs SH: H H (Na)HO2C(CH2)2 5-O- (CH)2CO2H(Na) H H
H3C CH3 H3C CH3 H H CH3 H--oh H--oh CH3 CH3 CH3 said molecules with said formula being fluorescent, photosensitizing, having the capability of selectively 65 binding to said pathogenic biological contaminants, to about 50J/cm2. Desirably, the irradiating source has forming a high molecular weight aggregate with a wavelength from about 600 nm to about 700 nm with absorption peaks in the visible spectrun in water at an energy density from about 1 to 20 J/cm2. Suitable 5,030,200 11 12 sources which satisfy such requirements are a Xenon is removed from the body of the patient and maintained 1000 W light fitted with infra-red blocking filters and in a container in which there is no net mass transfer. The 630-10 nm band pass filter or a laser light source emit blood is then agitated and irradiated with a light source ting radiation within (around 630 nm) the absorption having a wavelength of about 600 to 700 nm. The en spectrum of the photoactive compound and having the ergy density most often used to irradiate the blood in desired energy density. Another suitable source is a the flow cell is from about 1 J/cm2 to about 50 J/cm2. quartz halogen lamp. Alternatively, the patient's blood that is infected with As previously stated, the resulting mixture of body infectious biological contaminants is removed from the fluids and the photoactive compound is subjected to patient's body and then admixed with a sterile saline radiation while being maintained in a container. The 10 solution containing an effective amount of a mixture of agitation enhances the mixing of the photoactive com porphyrins to bind to all the infectious contaminants. pound with the body fluid to insure that the contami The saline solution containing the mixture of porphy nants in the body fluids are sufficiently bound to the rins is added at a rate that will maintain the concentra photoactive compound; and the agitation further en tion of the mixture of porphyrins in the resulting fluid to hances exposure of the chemically bound contaminants 5 be from about 0.1 to about 50 micrograms per milliliter to the desired level of radiation to insure eradication of of the infected blood. The preferred concentration, such contaminants. The desired agitation, mixing and however, is from about 2 to about 50 micrograms per exposure to irradiation of the resultant mixture of body milliliter of infected blood. The resulting mixture is then fluids can be achieved by moving the container in agitated and irradiated in the a container. The operable which the resultant mixture of body fluids are main 20 light source for irradiation has a wavelength of about tained and stored. The container can be moved in a 600 to 1000 nm, although the preferred wavelength reciprocatory translational motion such as laterally, range is from about 600 to 700 nm. The operable energy vertically, or circularly. Some containers can even be density that can be used to irradiate the resulting fluid in moved in a reciprocatory rotational motion along the the flow cell is from about 1 to 50 J/cm2, but the pre three coordinate axes of the container. Alternatively, an 25 ferred range is from about 1 to 20 J/cm2. agitating means comprising a movable inert agitating Due to diapedesis, a natural phenomenon in which object, such as a stirrer or a bar-shaped or ball-shaped some lymphocytes pass through the intact walls of agitator, can be used to promote and enhance the agita blood vessels and mix with other tissue fluids, not all tion. Preferably, the agitating object will be placed lymphocytes will be available, at one time, for the extra inside the container. The agitating means can be sonic, 30 corporeal treatment. Accordingly, the patient undergo mechanical, electrical, or magnetic. However, the level ing such extracorporeal treatment has to be subjected to of agitation of the body fluids being treated must be a series of extracorporeal treatments with an interval of maintained below a level where shearing of the body a few days ranging from 2 to 7 days. The duration of fluids or cells would occur. one extracorporeal treatment can range from about 3 to A suitable container can be an open container, or a 35 about 10 hours. closed container. It can be a bag, a tube, a box, a petri A pump is usually not required for the operation of dish, or others. Generally, the container is made up of extracorporeal treatment. At a systolic blood pressure inert transparent materials, such as plastics, other poly of 130 to 170 mm Hg, the blood can flow through a flow meric materials, glasses, or quartz. For an open con cell at a rate of about 80 to 140 ml/min through a chan tainer, it need not be made up of transparent materials. nel of typical cross-section of 0.7x 100 mm with a The internal surface of the container can either be length of about 150 to 200 cm. The flow cell for this smooth, roughened or rippled. The roughened or rip procedure can be constructed to contain a total of about pled surface can enhance the desired mixing. Further, to 120 ml of blood. insure that the body fluids in such a container are ex As discussed above, there is currently no cure for posed to a sufficient level of radiation to eradicate the 45 AIDS. Without an effective treatment, AIDS victims contaminants, the body fluids are exposed to a radiation live, on the average, about 2 years after diagnosis. It is density of about 5 J/cm2. only logical that if the viremia of an AIDS victim can As will become apparent, the desired exposure time be reduced, such a victim would have a better chance of can be readily controlled by the surface of body tissue staying alive longer, or at least the life in this victim can that is exposed, in combination with the agitation rate of 50 be improved. The extracorporeal photochemical treat the body tissue. However, care must be exercised to ment of infected blood from an AIDS patient can fulfill insure that irradiating of body tissue to eradicate or such a goal because the method described herein has destroy the contaminants does not heat the body tissue been shown to eradicate human immunodeficiency to a temperature sufficient to destroy the viability of the virus which causes AIDS. purified body tissue. Care must also be taken to insure 55 Where the body tissue is not in fluid form, said tissue that the agitation is not violent enough to cause damage is excised from the donor as a thin layer that is translu to cells. cent to light and is then suspended in a physiologically For the extracorporeal treatment of the blood of a acceptable saline solution containing an effective, non patient infected with infectious biological contaminants, toxic amount of the photoactive compound. The result a dosage from about 0.5 mg to about 40 mg of a mixture ing suspension is then subjected to radiation while main of porphyrins per kg of the body weight of the patient taining the resulting suspension under gentle agitation is commonly used. The mixture of porphyrins can be during exposure to the radiation. The gentle agitation administered to the patient either by oral or intravenous would enhance the mixing of the photoactive com route. The mixture of porphyrins can be given from pound and the tissues suspended in the solution to insure about one hour to about one week prior to the start of 65 that the infectious biological contaminants in the body irradiating the blood removed from the patient's body. tissues are sufficiently bound to the photoactive com Generally, the blood that contains the mixture of por pound; and the gentle agitation further enhances expo phyrins bound to the infectious biological contaminants sure of the chemically bound contaminants to the de 5,030,200 13 14 sired level of radiation to insure eradication of such Similarly, the irradiation cell for the irradiation of contaminants. The gentle agitation can be achieved by body tissues suspended in a physiologically acceptable placing the entire irradiation assembly in a laboratory saline solution has boundary surfaces of uniform thick shaker. Thus, the cell assembly can be gently agitated ness. The irradiation cell assembly is also illuminated while the body tissues suspended in sterile solution are with light of essentially uniform intensity and of appro being irradiated. priate wavelength to initiate photosensitization. With The operative amount of the mixture of porphyrins to such uniform irradiation, all areas of the body tissues be added to the suspension of body tissue in a physiolog receive essentially the same incident light fluence. ically acceptable saline solution is from about 0.1 to Hence, the results obtained can be accurately repro about 50 micrograms per milligram of the suspended 10 duced. body tissue. Desirably, however, the amount of the mixture of porphyrin added is from about 2 to 50 micro TABLE I grams per milligram of the suspended body tissue. The The following are enveloped viruses as divided into family and source employed to irradiate the resulting suspension common species or genus: has a wavelength of from about 350 nm to about 700 nm 15 Family Common Species or Genus Herpesviridae Human herpes simplex virus types and an energy density of from about 0.1 J/cm2 to about types I and II 20 J/cm2. Desirably, the irradiation source has a wave bovine mammilitis virus length from about 600 nm to about 700 nm with an herpes B virus of monkeys energy density from about 1 to about 20 J/cm2. Aseudorabies virus As previously set forth, the method of the present 20 equine rhinopneumontis virus varicella-zoster virus invention provides an economical and efficient means human cytomegaloviruses for externally eradicating infectious pathogenic biologi murine cytomegaloviruses, cal contaminants from body tissues so that the likeli Epstein-Barr virus hood of a person becoming infected as a result of a Baboon herpes virus 25 Chimpanzee herpesvirus transfusion or transplantation is substantially elimi Marek's disease herpes virus nated. The contaminants which can be effectively eradi Hinze virus cated from a body fluid using the present invention are Turkey herpes virus the enveloped viruses, other microorganisms, including Herpesvirus ateles Herpesvirus Saimiri other parasites and bacteria. The term "enveloped vi Infectious bovine rhinotracheitis virus rus' in all cases but one is understood to be a virus of 30 oviridae Aftrican swine fever virus which is encased within a modified host cell membrane, Frog virus group (Ranavirus) except for the Pox-virus which produce their own enve Iridovirus lope. Typical of such enveloped viruses are: Cytomega Chloriridovirus Poxviridae vaccinia virus lovirus, Herpes simplex virus, Pox virus, human im Smallpox virus munodeficiency virus, Epstein-Barr virus, and others as 35 cowpox virus set forth in Table I. Other viruses include Dengue virus monkeypox virus and yellow fever virus. buffalopox virus camelpox virus Microorganisms, parasites and bacteria which can be ectromelia of mice virus effectively eradicated by the method of the present rabbitpox virus invention include: Plasmodium vivax, P. malariae, P. Orf virus virus of milker's node falciparum, P. ovale, and Trypanosoma cruzi Bacillus avipoxvirus subtilis, Streptococcus faecalis, and Borrelia burgdorferi, sheep pox virus the bacteria that cause Lyme disease; Babesia microti; goat pox virus Leptospira; Borrelia; Haemobartonella; and Barcher lumpy skin disease (Neethling) virus lichia. 45 myxoma virus of hares fibroma viruses of rabbits In order to more fully describe the method for eradi fibroma viruses of squirrels cating infectious pathogenic biological contaminants swinepox virus from body fluids in accordance with the present inven Yaba monkey virus tion, reference is now made to FIG. 1 through FIG. 16 molluscun contagiosum virus Hepadnaviridae human hepatitis B virus (HBV) of the drawings wherein schematic representations of 50 woodchuck hepatitis virus cell assemblies and containers useful in the practice of ground squirrel hepatitis virus the invention are set forth. duck hepatitis virus In irradiating blood or blood products to achieve Orthomyxoviridae influenza virus, types A, B, and C Paramyxoviridae Newcastle disease virus of fowl photodynamical killing of infectious pathogens with the human parainfluenza viruses preferred embodiment, a closed container with uniform. 55 Sendai virus thickness of surfaces containing the blood or blood mumps virus products is illuminated with light of essentially uniform paramyxoviruses measles virus intensity and of appropriate wavelength to initiate pho rinderpest virus of cattle tosensitization. Alternatively, the blood or blood prod canine distemper virus ucts can be maintained in an open container and illumi 60 peste-des-petits-ruminants. virus nated with light of essentially uniform intensity and of of sheep and goats respiratory syncytial virus of man appropriate wavelength to initiate photosensitization. bovine respiratory syncytial virus With such uniform irradiation, all units of blood or pneumonia virus of nice blood products maintained and stored in the container Rhabdoviridae rabies virus receive essentially the same incident light fluence. In 65 vesicular stomatitis virus of: horses, cattle and swine this way, fluid flow and illumination conditions, demon chandipura virus strated experimentally to result in killing of the patho lyssavirus gen, can be accurately reproduced. duvenhage virus 5,030,200 15 16 TABLE I-continued TABLE I-continued The following are enveloped viruses as divided into family and The following are enveloped viruses as divided into family and common species or genus: common Species or genus: Family Common Species or Genus Family Common Species or Genus Lagos bat virus murine mammary tumor virus mokola virus feline complex leukemia virus Bunyaviridae bunyavirus (Bunyamwera, Bwamba, feline complex sarcoma virus California, Capim, Guama, woolly monkey sarcoma virus phlebovirus koongol, patois, simbu Gibbon leukemia virus and tete viruses) 10 mason-Pfizer virus sandfly fever virus hamster leukemia virus Rift Valley fever virus of sheep and rat leukemia virus ruminants bovine lymphoma virus Nairovirus human T cell leukemia viruses: Crimean-Congo hemorrhagic fever types l and 2 etc. viruses spumavirinae; syncytial and foamy Uukuvirus 15 viruses of humans, monkeys, cattle, Uukuniemi virus Cats Hantaan virus visna virus of sheep Korean hemorrhagic fever virus Maedi virus Filoviridae ebalo virus progressive pneumonia viruses of Marburg virus sheep Nodaviridae Nodamura virus 20 "human immunodeficiency viruses Togaviridae Alphaviruses (includes HTLV III/LAV) HIV, aura virus HTLV IV, LAV-2, STLV-III G? Chikungunya virus Arenaviridae Junin virus eastern equine encephalitis virus lassa virus getah virus machupo virus Inayaro virus 25 pichinde virus middleburg virus lymphocytic choriomeningitis virus mucamba virus assa fever virus indumu virus pichinde virus O Nyong-nyong virus arenavirus pixuna virus Other virus-like agents kuru virus ross river virus 30 viroids-prions Creutzfeldt-Jakob disease virus Semliki forest virus scrapie virus sindbis virus transmissible mink encephalopathy una virus Aleutian disease of mink Venezuelan equine encephalitis virus NOTE: western equine encephalitis virus under Retroviridae Whataroa virus 35 HTLV II, hunan T-lymphotropic virus type Ill rubella virus LaV. Lymphadenopathy virus mucosal disease virus HIV. human immunodeficiency virus STLV-IIG simina T-lymphotropic virus type III border disease virus HTLV IV, human T-lymphotropic virus type IV hog cholera virus HTLV III and LAV are now usually referred to as HIV Flaviviridae flavivirus Brazilian encephalitis virus Bussuguara virus 40 Red blood cells (rbc) containing hemoglobin absorb dengue virus and scatter light appreciably. In normal human whole iiheus virus Israel turkey meningoencephalitis blood, the concentration of rbc is approximately 46% virus by volume. Thus, in normal human whole blood, the Japanese Bencephalitis virus attenuation of light intensity I "Zee Zero', Z0, pene kunjin virus 45 trating from an illuminated surface as described by Kyasanur forest disease virus Beer's law is given after penetration to depth Zby langat virus louping ill virus modoc virus Murray valley encephalitis virus ntaya virus 50 where I0 is the light intensity at the illuminated surface omsk hemorrhagic fever virus and the constants A and S are the absorption constant powassan virus St. Louis encephalitis virus and scattering constant, respectively. For blood, these spondwnei virus are, approximately, A=5cm and S= 10 cm at 630 tick-borne encephalitis nm light wavelength. Using these numbers, it can be Uganda S virus 55 seen that the Beer's Law light intensity decreases to US bat salivary gland virus wesselsbron virus about 0.47, 0.22, and 0.10 of Ioat depths of 0.05, 0.1, and west nile fever virus 0.15 cm, respectively. Thus, with depth, whole blood or yellow fever virus thick suspensions of rbc becomes optically thick. One zika virus way to overcome the thickness is to treat an optically european tick-borne encephalitis 60 thin layer of blood having very large lateral surface far eastern tick-borne encephalitis virus extent under static fluid conditions. Alternatively, the Russian tick-borne encephalitis same result can be achieved by exposing an appreciable Retroviridae type Concovirus group volume of thin layer of blood to the light, each small type B oncovirus group volume element of blood is brought to the surface hav type D retrovirus group 65 avian complex leukemia virus ing an optically thin depth for light exposure. This alter Rous sarconna virus native way can be brought about through fluid mixing murine complex leukemia virus with no net fluid volume displacement from the con mouse sarcoma virus tainer, no net mass transfer. It is necessary for each 5,030,200 17 18 small volume to be exposed to a sufficient light fluence be treated photochemically, the cell 30 would function (J/cm) for photosensitized inactivation of infectious as a flow cell to direct the flow of body fluids. If, on the agents to occur. This requirement necessitates a mini other hand, it is the thin layer of body tissue to be sub mum residence time within the near surface fluid region jected to photochemical treatment, the cell would serve by repetitive and adequate mixing motion. Such mixing 5 to hold the suspension of body tissue in a physiologi notion can be attained in a number of different ways. cally suitable saline solution. Generally, blood or blood components are retained Still referring to FIG. 3, the uniform width of the inside a sterile bag without having the sterility of an irradiation cell 30 is denoted by W, while the uniform enclosed system broken at anytime subsequent to col length of the cell is L. In this scheme, the incident light lection from the donor. Then the bag is subjected to 10 Io and the falloff of light intensity I with depth along Z movement to create the mixing motion. Alternatively, axis is everywhere uniform on any plane of constant an agitating object inside the bag can be used to create depth from the irradiated surfaces. Additionally, flow the mixing motion. rate within such a layer with much larger cross-sec FIG. 1 shows a schematic diagram of a flow cell used tional area than the previously used tubing is considera in the original experiments. Whole blood or other body 15 bly enhanced. fluid was infused via a syringe driven by an infusion FIG. 3-a shows the decrease of the intensity of unidi pump. The blood or body fluid was constrained to flow rectional irradiation as the irradiating light travels along through a flexible transparent plastic tube, 11, which the depth, d, of the irradiation cell 30, as depicted in was looped along the planar surface of a 2 in. X2 in. area FIG. 3. The geometry of the irradiation is graphically of transparent glass slide, 12, which served to support 20 the tube in a plane perpendicular to the irradiating light illustrated as a three-dimensional rectangular coordi beam. The tube was attached to the glass slide 12 by nate system (X, Y, Z). The two horizontal axes are X epoxy glue shown as 14. Tube diameter was 0.05 cm and Y, while the vertical axis is Z. The depth, d, of the and the length of the tube illuminated was 40 cm. Irradi irradiation cell 30 is defined by the Z axis; the width, W, ation was essentially uniform with irradiance Io of 25 of the irradiation cell 30 is defined by X axis; and the 1.04X 10-2 w/cm2 at 630-5 nm wavelength. Flow rate length, L, of the irradiation cell 30 is defined by Y axis. of body fluid was 9.8 x 10-4 cm3/min. The flow time The origin F represents point 31 at the upper left hand for each unit of volume element of body fluid within the corner of the irradiation cell 30. illuminated region was about 8 minutes. Irradiation of Referring still to FIG. 3-a, the Z component, I, of the 2 in.X2 in. planar area occupied by the looped flow 30 the irradiating light Io, as represented by line 32, de tube for the 8 min. flow time resulted in a fluence of creases in intensity as the light travels through the Ec=5 J/cm2 delivered to the planar area of the looped thickness d of the cell 30. The decrease in intensity is a tube. function of the distance of the path travelled by the FIG. 2 shows the enlarged cross-sectional view of the irradiation, as is shown by line 32 which approaches flow tube, 21, sitting on a planar support with the inci 35 zero as the distance travelled by light along the thick dent light, Io, coming in along the direction as shown by ness d increases. aOWS. FIG. 4 shows the irradiation cell 30 being irradiated The corresponding average fluence
TABLE IV RESULTS: Human Blood From Patients. With AIDS Donor BL BL BDD BD Date 2/1/87 2/11/87 4/14/87 4/4/87 Tine O925 O925 O900 0900 hrs. Light O yes O yes Photofrin O 2.5 O 2.5 ug/mL. Sodium 152 155 160.7 63.0 mM Potassiurn 3.8 3.8 3.32 3.43 mM Calcium 0.10 0.007 0.06 0.06 nM Glucose 75 75 66 67 mg/dL 66 67 mg/dL Osmotic Frag. Begin: 44 44 46 44 % 50%: 44 42 41 41 % complete: 34 34 34 34 % Sugar Wat. Iys 0.07 0.030 0.000 0.000 ODG 540 nm Fibrinogen: 378 315 227 183 mg/dL. PT: 1.6 13.0 13.0 14.5 secs. APTT 29.5 35.4 47.9 50.1 SeCS. Factor Def. Scn. Factor II neg. neg. neg. neg. Factor V neg. neg. neg. neg. Factor VII neg. neg. neg. neg. Factor X neg. neg. neg. neg. Factor VIII neg. neg, neg. neg. IX neg. neg. neg. neg. XI neg. neg. neg. neg. XII neg. neg. neg. neg.
WBC 2.5 2.2 2.3 2.3 thsnds. RBC 2.9 2.45 5.33 5.86 millins. HGB 9.0 7.7 15.6 16.8 g/dL. HCT 27.1 22.9 47.3 52.4 % MCV 92.9 93.3 88.7 89.3 f. MCH 30.8 31.5 29.2 28.7 pg. MCHC 33.5 33.7 33.0 32. g/dL. RDW 9.2 9. l,6 1.9 % Platelets 120 122 47 45 thsnds. Differential Total polys 43 30 25 28 7. Segs 35 54 20 2 c bands 8 12 5 7 C. lymphs 39 54 36 31 % 5,030,200 31 32 TABLE IV-continued RESULTS: Human Blood Fron Patients. With AIDS Donor BL BL BDD BD Date 2A1 1/87 2/11/87 4/4/87 4/14/87 Tine O925 O925 O900 O900 hrs. Light O yes O yes Photofrin O 2.5 O 2.5 ug/mL. OCS 16 12 9 9 9. eS 2 4. 30 30 % baso 2 4. 30 30 9% Direct Coombs neg (poly) 1 -- wk neg. --A- specific polysp. 1 -- wkIgG i + IgG C3 neg. C3 neg. sal. con - neg. Serum Elect Alburnin: 46.34 42.93 48.77 52.61 % alpha 1-Glob 3.17 2.73 3.07 3.14 % alpha 2-Glob 2.42 12.0 9.10 7.5 % beta - glob 4.22 2.46 3.90 3.6 % gamma - glob 23.84 20.86 25.6 23.58 % O2 dissoc. NR NR 28 25 mm Hg at p50 Comments Hemolysis of sample (LIPPBL) noted after approx. 2.5 ml. had been run through flow cell. Noted apparent high sed-rate of patient. The abbreviations used in Tables II to IV are as foi- 25 lows: -continued ABBREVIAED UNITS FULL NAME pg picogram ABBREVIA- p partial pressure TION FULL NAME Photofrin Photofrin II TM, from Johnson and Johnson, 3O containing approximately 90% of It can be seen from these Tables that photo-irradia dihematoporphyrin ether, DHE tion in the presence of Photofrin IITM did not have a Osmotic Frag Osmotic Fragility deleterious effect on blood. It is important in evaluating Sugar Wat, lys. Sugar Water lysis tests pT Prothrombin Time the data to appreciate the scaling down of normal labo APTT Activated Partial Thromboplastin Time 35 ratory procedures that occurred in these examples. The Factor Def. Scm. Factor Deficiency Screen flow rate attained during these experiments was about CBC Complete Blood Count 23 microliters per minute. Handling small aliquots on WBC White Blood Cell RBC Red Blood Cell blood in a darkened room and avoiding them drying out HGB Hemoglobin presented logistical problems. In some control samples HCT Hematocrit 40 appreciable hemolysis occurred, this was found to arise MCV Mean Corpuscular Volume from kinking of the cuvet tubing following repeated use MCH Mean Corpuscular Hemoglobin Concentration Total polys Total polymorphonucleocytes in the high intensity light. This kinking caused pressure Segs segmented neutrophils to build up in the sample, resulting in lysis. Whenever lymphs. lymphocytes this problem arose the cuvet causing the problem was OCS. monocytes 45 discarded. Viability of the irradiated samples containing eosin eosinophils baso basophils Photofrin IITM was based on the following parame Serum Elec Serum Electrophoresis ters: Potassium and glucose were well within the limits Gob Globulin established for blood banked whole blood; RDW which IgG Immunoglobulin G is the standard deviation of red cell size was unchanged C3 Complement fragment 3 50 O2 dissoc. Oxygen dissociation or lower than in the controls; mean corpuscular hemo sal. con Saline concentration globin concentration (MCH) showed no elevation; os neg. Negative notic fragility was unaffected; sugar water lysis tests NR No result showed no lysis following irradiation, but the same sed-rate sedimentation-rate results are usually found in banked blood and is there 55 fore not a contraindication for transfusing the blood. The following abbreviated units are also used in Ta Serum electrophoresis could only be quantified as a bles II to IV. percentage and not in absolute terms. In two of the samples from patients with AIDS, pro longation of the APTT test occurred. One patient, how ABBREWATED UNITS FULL NAME ever, had already had a prolonged APTT. In both these hrs hours ug/ml microgram per milliliter patients, the factor deficient screen test indicate that this mM microgram per milliliter prolongation did not arise from selective destruction of ng/dl milligram per deciliter any of the coagulation factors but might have arisen O.D., S4O Optical Density at 540 nanometer from changes in the optical qualities of the plasma SecS seconds 65 Thous. Thousand per microliter which could affect the optical clot detection devices. Mil. Millions per microliter In summary, photoirradiation of human blood outside g/d gran per deciliter the human body in the presence of a photoactive com 1. femtoliter pound such as Photofrin TM did not affect the viability 5,030,200 33 34 of the human blood as determined by currently ac amounts of Photofrin IITM resulted in a similar reduc cepted blood bank criteria. tion in viral infectivity. Blood samples containing the Effect of Photofrin IITM and Light on Human Whole three different concentrations of Photofrin II TM Blood Containing Herpes simplex Virus Type I showed no evidence of cellular toxicity when assayed in the vero cells as described above. Herpes simplex virus type I (HSV-I), the MacIntyre strain, was purchased from the American Type Culture TABLE V Collection (ATCC) and propagated in Vero cells PHOTOINACTIVATION OF BLOOD SPIKED WITH (ATCC) to a concentration of 106-107 plaque forming HERPES SIMPLEX TYPE-1 (HSV-1) units (PFU) per milliliter (ml). This solution was used as O CARRIED OUT INA FLOW CELL SYSTEM stock virus. Twenty milliliters of blood were collected Concentration PFU/In PFU/ml % Reduction. In from a healthy volunteer in acid citrate dextrose and the of PIAml in Dark in Light PFU-Light to Dark 2.5 pig 1 x 10 1.2 x 10 99.88 blood was divided into 0.9 ml aliquots. A volume of 0.1 10 ug 9 x 10 1.25 x 10 99.86 ml of stock virus was added to eight separate aliquots of 20 ug 9 x 103 0.5 x 10 99.94 blood. 5 PFU: Plaque forming unit; Photofrin TM (From Johnson and Johnson. A photo P: Photofi I v. active dye that contains approximately 90% of pure HSV-1: Diluted 1:10 in blood and irradiated yielded 1 x 10'PFU/ml, Virus diluted dihematoporphyrin ether (DHE)), was added to dupli in blood and not irradiated yielded 5 x OPFU/ml. cate tubes of the virus-blood mixture and a set of tubes containing blood only. The concentrations of Photofrin Effect of Photofrin IITM and Light on Human IITM employed were 2.5, 10 and 20 g/ml. Samples Immunodeficiency Virus (HIV) representing each concentration of Photofrin IITM in the blood virus mixture were irradiated while moving The human immunodeficiency virus (HIV) has also through the flow cell by light at a wavelength of ap been irradiated in the flow cell. Approximately 4X 10 proximately 635 nm with an energy density of about 5 25 infectious units (IU)/ml of cell-free HIV was prepared J/cm2. Approximately 30-60 minutes elapsed between in tissue culture. The stock virus was then diluted 1:2 in the addition of each concentration of Photofrin IITM tissue culture medium and six 1 ml aliquots of HIV were and exposure of light in the flow cell. During the hold prepared. Irradiation studies were carried out in a simi ing period, the samples were maintained at 4 C. Except lar manner as described above for the photoinactivation during the time of irradiation, all manipulations were 30 of HSV-I in blood. All samples were, however, main carried out with minimal exposure to extraneous light. tained at room temperature throughout the experiment. During a 15-30 minute period of time, approximately One of the above aliquots of HIV was passed through 0.7 ml of the irradiated sample was collected. A sample the flow cell in the dark. A second aliquot of HIV was of virus mixed with blood but not containing Photofrin irradiated in the flow cell at 5 J/cm2 for approximately IITM was also irradiated under the same conditions. 35 During the holding period and the period of irradiation 30 minutes which yielded approximately 0.7 ml of irra the duplicate samples of virus and blood containing diated fluid. Three other samples, each containing dif. different concentrations of Photofrin IITM were held ferent concentrations of Photofrin IITM, namely, 2.5, in the dark. In addition, samples of human blood (with 10 and 20 g/ml, were irradiated. All six samples were out virus) containing different concentrations of Photo then placed in cell culture at five different dilutions: frin IITM and a sample of blood spiked with virus only Neat (no dilution) 10-1, 10-2, 10-3, and 10-4. Super were also maintained in the dark. natants from each culture were collected at prescribed Each sample including the stock virus were assayed intervals and assayed for HIV activity by the reverse on vero cells for PFU/ml of HSV-I. The assay con transcriptase assay, according to the procedure re sisted of growing vero cells in minimal essential medium 45 ported by Chanh, et al. (Eur. Mol. Biol. Organization.J., with Hanks balanced salt solution supplemented with 5:3065-3071, 1986). 10% fetal bovine serum, L-glutamine and antibiotics. It can be seen from Table VI that an appropriate Herpes buffer (2%) was added for growth in open amount of Photofrin IITM in combination with light at plates (twelve well microplates from Costar). Ten fold a wavelength of 625-635 nm having an energy density dilutions of each sample were prepared and 0.1 ml of the 50 of 5 J/cm2 achieved a near complete kill of human appropriate dilution for each individual sample was immunodeficiency virus (HIV), the culprit for AIDS, in adsorbed at 37 C. for 1.5 hours on a cell monolayer the flow cell. from which the growth medium had been removed. After the adsorption period, the cell monolayer was TABLE VI washed and an overlay consisting of equal volumes of 55 PHOTOINACTIVATION OF A CULTURE OF HUMAN 2X strength L-15 medium and 2% methylcellulose was IMMUNODEFICIENCY VIRUS IN A FOW added. Following an appropriate incubation time at 37 CELL SYSTEM C. (about 4 days), the overlay medium was removed. Concentration Infectious Units Per Millilieterb % Kill Monolayers were fixed with methanol and stained with of PIAll In Dark In Light (5 J/cm2) In Light O 2 x 10 2 x 10 O giemsa to elaborate the presence of plaques. The 2.5 ug a- C C plaques were counted using a dissecting microscope at a 10 g d OO magnification of 20X. 20 ug d OO The results of the experiment can be observed in Pl: Photofrin ty Table V. It is clear that Photofrin IITM in a concentra The infectious units of the human immunodeficiency virus were unaffected 65 whether or not the culture of virus was channelled through the flow cell systern. tion as low as 2.5 g/ml, in combination with light at a As determined by reversed transcriptase activity, wavelength of 625-635 nm having an energy density of C: Percent kill could not be determined. 5 J/cm2, achieved a near total (larger than 99.88%) kill No detectable activity of the enzyme reverse transcriptase. of HSV-I in human whole blood. The use of larger 5,030,200 35 36 These studies suggest that the major target for photo body prior to introduction of the decontaminated body dynamic damage of Photofrin IITM in viruses is enve fluids into the body of a patient, said method compris lope related. ing: The method of the present invention provides an admixing an effective, non-toxic amount of a photo effective and efficient means for eradicating infectious 5 active compound with the body fluid to produce a pathogenic contaminants from body fluids outside the resulting body fluid, the photoactive compound body of an animal or a human. having an affinity to be selectively bound to the Unlike the metabolic clearance of a photoactive drug contaminants; in normal tissues observed in a live animal or human as maintaining the resulting body fluid in a suitable con described in the U.S. Pat. No. 4,649,151 to Dougherty 10 tainer in which there is no net mass transfer; and et al., animal or human blood outside the body is not irradiating the resulting body fluid in the container subject to this physiological or metabolic action by with an effective level of essentially uniform inten various body organs. Consequently, there is no mecha sity of radiation in the region of the visible spec nism for detoxification or "cleaning' of the human trum, with a wavelength range of from about 400 whole blood outside a body. 15 nm to about 1000 nm, for an effective period of Suprisingly, despite the absence of the physiological time such that the radiation penetrates the resulting or metabolic "cleansing' mechanism, human whole fluid and exposes the photoactive-compound blood outside the human body shows a remarkable bound contaminants to the radiation so as to eradi tolerance towards a photoactive compound such as cate such contaminants while maintaining the via Photofrin IITM. By all accounts, the human whole 20 bility of said body fluids to produce viable decon blood remains unchanged in the presence of certain taminated body fluids. concentrations of this photoactive compound. Equally 2. The method as recited in claim 1 further compris surprising, external irradiation of normal human whole ing the step of selecting a body fluid from the group blood containing a photoactive compound with a speci consisting of whole blood, blood plasma, serum, and fied wavelength of absorption causes no detectable 25 fluids fron plasmapheresis. damage to the whole blood when used at selected con 3. The method as recited in claim 1 further compris centrations and intensity. Although outside the human ing the step of selecting a body fluid comprising of body, after such treatment, the human whole blood is See. still unchanged by all currently acceptable Standards. 4. The method as recited in claim 1 further compris Although the infectious biological contaminants, such 30 ing the step of selecting a photoactive compound com as parasites, bacteria or viruses encapsulated by a pro prising a mixture of porphyrins, at least a portion of the tein envelope, are totally different from human malig molecules of said porphyrin mixture having the molecu nant tumor cells, a photoactive compound such as lar formula: (Na)HOC(CH2)2 CH3 H3C (CH2)COH (Na)
H H CH3 CH3 H (Na)HO2C(CH2)2 C-C-C (CH3)2COH (Na) H
HC CH3 HC CH3 H H CH3 H-C-OH H-C-OH CH3 th. CH3 Photofrin IITM has been found to have a selective affinity toward these infectious pathogenic contami nants contained in human whole blood outside the said molecules with said formula being fluorescent, human body. Moreover, such a compound can be pho photosensitizing, having the capability of selec toactivated outside the human body to cause the demise tively binding to said pathogenic biological con of these infectious biological pathogenic contaminants 55 taminants, forming a high molecular weight aggre to which the compound has preferentially attached. gate with absorption peaks in the visible spectrum It is clear that the present invention is well adapted to in water at approximately 365, 505, 537, 575, and carry out the objects and attain the ends and advantages 615 nanometers, absorption peaks in the infrared mentioned herein as well as those inherent in the inven spectrum at approximately 3.0, 3.4, 6.4, 7.1, 8.1, 9.4, tion. While presently preferred embodiments of the 60 12 and 15 microns, absorption peaks, in carbon-13 invention have been described for purposes of this dis nuclear magnetic resonance study at at least ap closure, numerous changes may be made which will proximately 9.0, 18.9, 24.7, 34.5, 62, 94.5, 130-145, readily suggest themselves to those skilled in the art and 171.7 ppm relative to a 37.5 ppm resonance peak of which are accomplished within the spirit of the inven dimethyl sulfoxide and additional absorption peaks tion disclosed and as defined in the appended claims. 65 in carbon-13 nuclear magnetic resonance study at We clain: approximately 27.9 ppm and 68.4 ppm relative to 1. A method for eradicating infectious pathogenic the resonance peak of tetramethylsilane in deuter biological contaminants from body fluid outside the ated chloroform solvent; and 5,030,200 37 38 at least 50 percent of the porphyrins in said mixture pathogenic biological contaminants comprising a mala being of said molecules having said molecular for rial parasite. mula. 17. The method as recited in claim 1 further compris 5. The method as recited in claim 4 wherein the ing the step of selecting body fluid which contains amount of said mixture of porphyrins admixed with said pathogenic biological contaminants comprising a try fluid is from about 0.1 to about 50 micrograms per milli panosomal parasite. liter of body fluid. 18. The method as recited in claim 1 further compris 6. The method as recited in claim 5 wherein the ing the step of agitating the resulting body fluid in the amount of said mixture of porphyrins admixed with said container. fluid is from about 2 to 50 micrograms per milliliter of 10 19. The method as recited in claim 18 wherein the body fluid. step of agitating is carried out by moving the container. 7. The method as recited in claim 1 wherein the level 20. The method as recited in claim 18 wherein the of radiation is produced by a light source having a step of agitating is carried out by the aid of an agitating wavelength of from about 400 to about 1000 nm and an e.S. energy density of from about 0.1 to about 50 J/cm2. 5 21. The method as recited in claim 20 wherein said 8. The method as recited in claim 7 wherein the level agitating means comprises an agitating object. of radiation is produced by a light source having a 22. The method as recited in claim 1 wherein said wavelength of from about 600 to about 700 nm and an container is kept static. energy density from about 1 to about 20 J/cm2. 23. A method for externally purifying a blood prod 9. The method as recited in claim 1 further compris 20 uct to eradicate pathogenic biological contaminants ing the step of selecting body fluid which contains selected from the group consisting of envelope-contain pathogenic biological contaminants comprising an en ing viruses, bacteria, malarial, and trypanosomal para velope-containing virus. sites prior to introduction of the decontaminated blood 10. The method as recited in claim 9 further compris products intravenously into a patient, said method com ing the step of selecting the envelope-containing virus 25 prising: from the group consisting of Herpesviridae, Poxviridae, admixing an effective amount of a photoactive com Iridoviridae, Hepadnaviridae, Orthomyxoviridae, and pound with the blood products to bind the contam Paramyxoviridae. inant with the photoactive compound, said photo 11. The method as recited in claim 9 further compris active compound comprising a mixture of porphy ing the step of selecting the envelope-containing virus 30 rins, at least a portion of the molecules of said por from the group consisting of Rhabdoviridae, Bunyaviri phyrin mixture having the molecular formula:
H3C (CH2)COH (Na) SH: SH: H H 5-O- H H
CH3 HC CH
H--oh CH CH3 said molecules with said formula being fluorescent, dae, Filoviridae, Nodaviridae, and Togaviridae. 50 photosensitizing, having the capability of selec 12. The method as recited in claim 9 further compris tively binding to said pathogenic biological con ing the step of selecting the envelope-containing virus taminants, forming a high molecular weight aggre from the group consisting of Flaviviridae, Retroviridae, gate with absorption peaks in the visible spectrum and Arenaviridae. in water at approximately 365, 505, 537, 575, and 13. The method as recited in claim 12 further con 55 615 nanometers, absorption peaks in the infrared prising the step of selecting the Retroviridae comprising spectrum at approximately 3.0, 3.4, 6.4, 7.1, 8.1, 9.4, a human immunodeficiency virus. 12 and 15 microns, absorption peaks in carbon-13 14. The method as recited in claim 1 further compris nuclear magnetic resonance study at at least ap ing the step of selecting body fluid which contains proximately 9.0, 18.9, 24.7, 34.5, 62, 94.5, 130-145, pathogenic biological contaminants comprising a bac 171.7 ppm relative to a 37.5 ppm resonance peak of teria selected from the group consisting of Streptococcus dimethylsulfoxide and additional absorption peaks faecalis and Bacillus subtilis. in carbon-13 nuclear magnetic resonance study at 15. The method as recited in claim 1 further compris approximately 27.9 ppm and 68.4 ppm relative to ing the step of selecting body fluid which contains the resonance peak of tetramethylsilane in deuter pathogenic biological contaminants comprising a Bor 65 ated chloroform solvent; and relia burgdorferi bacteria. at least 50 percent of the porphyrins in said mixture 16. The method as recited in claim 1 further compris being of said molecules having said molecular for ing the step of selecting body fluid which contains mula; 5,030,200 39 40 maintaining the blood product containing the por bacteria selected from the group consisting of Strepto phyrins-bound contaminants in a suitable con coccus faecalis and Bacillus subtilis. tainer, exposable to a radiation source capable of 34. The method as recited in claim 23 further com irradiating the blood product and in which there is prising the step of selecting body fluid which contains no net mass transfer; and pathogenic biological contaminants comprising a Bor irradiating the blood product in the container with an relia burgdorferi bacteria. essentially uniform intensity of radiation source in 35. The method as recited in claim 23 further con the region of visible spectrum, with a wavelength O prising the step of selecting blood product which con of from about 400 nm to about 1000 nm for an tains pathogenic biological contaminants comprising a effective period of time to permit the radiation to malarial parasite. penetrate through the blood product in the con 36. The method as recited in claim 23 further com tainer and eradicate the contaminants while main 5 prising the step of selecting blood product which con taining the viability of components in said blood tains pathogenic biological contaminants comprising a product. trypanosomal parasite. 24. The method as recited in claim 23 wherein the 37. The method as recited in claim 23 further com amount of said mixture of porphyrins admixed with said 20 prising the step of agitating the resulting body fluid in blood product is from about 0.1 to about 50 micrograms the container. per milliliter of blood product. 38. The method as recited in claim 37 wherein the 25. The method as recited in claim 24 wherein the step of agitating is carried out by moving the container. 39. The method as recited in claim 37 wherein the amount of said mixture of porphyrins admixed with said step of agitating is carried out by the aid of an agitating blood product is from about 2 to about 50 micrograms calS. per milliliter of blood product. 40. The method as recited in claim 39 wherein said 26. The method as recited in claim 23 wherein the agitating means comprises an agitating object. 30 level of radiation is produced by a light source having a 41. The method as recited in claim 23 wherein said wavelength of from about 400 to about 700 nm and an container is kept static. energy density of from about 0.1 to about 50 J/cm2. 42. A method for extracorporeal treatment of the 27. The method as recited in claim 26 wherein the blood of a patient infected with infectious pathogenic level of radiation is produced by a light source having a 35 biological contaminants said method comprising: wavelength of from about 600 to about 700 nm and an removing blood from the body of a patient infected energy density of from about 1 to about 20 J/cm2. with infectious pathogenic biological contami 28. The method as recited in claim 23 further com nants; prising the step of selecting blood product which con 40 adding to said blood an effective, non-toxic amount of tains pathogenic biological contaminants comprising an photoactive compound having an affinity to be envelope-containing virus. selectively bound to the infectious contaminants; 29. The method as recited in claim 28 further con maintaining said treated blood in a suitable container prising the step of selecting the envelope-containing 45 in which there is not net mass transfer; virus from the group consisting of Herpesviridae, Pox irradiating said contaminated blood admixed with viridae, Iridoviridae, Hepadnaviridae, Orthomyx photo-active compound in the container with an oviridae, and Paramyxoviridae. effective level of essentially uniforn intensity of 30. The method as recited in claim 28 further com 50 radiation in the region of visible spectrum, with a prising the step of selecting the envelope-containing wavelength range of from about 400 nm to about virus from the group consisting of Rhabdoviridae, 1000 nm, for an effective period of time such that Bunyaviridae, Filoviridae, Nodaviridae, and Togaviri the radiation penetrates the blood and exposes the dae. 55 photoactive-compound-bound infectious contami nants to the radiation so as to eradicate such infec 31. The method as recited in claim 28 further com tious contaminants while maintaining the viability prising the step of selecting the envelope-containing of components in said blood to produce a viable virus from the group consisting of Flaviviridae, Re and decontaminated blood; and troviridae, and Arenaviridae. returning said viable and decontaminated blood to 32. The method as recited in claim 31 further com the patient's body. prising the step of selecting the Retroviridae comprising 43. The method as recited in claim 42 further com a human immunodeficiency virus. prising the step of selecting a photoactive compound 33. The method as recited in claim 23 further com 65 comprising a mixture of porphyrins, at least a portion of prising the step of selecting blood product which con the molecules of said porphyrin mixture having the tains pathogenic biological contaminants comprising a molecular formula: 5,030,200 41 42
(CH2)COH(Na)
(CH2)2COH (Na)
CH3
CH3 H--oh H--oh CH3 CH3 CH3 said molecules with said formula being fluorescent, photosensitizing, having the capability of selec pathogenic biological contaminants comprising an en tively binding to said pathogenic biological con 20 velope-containing virus. taminants, forming a high molecular weight aggre 52. The method as recited in claim 51 further com gate with absorption peaks in the visible spectrum prising the step of selecting the envelope-containing in water at approximately 365, 505, 537, 575, and virus from the group consisting of Herpesviridae, Pox 615 nanometers, absorption peaks in the infrared viridae, Iridoviridae, Hepadnaviridae, Orthomyx spectrum at approximately 3.0, 3.4, 6.4, 7.1, 8.1, 9.4, 25 oviridae, and Paramyxoviridae. 12 and 15 microns, absorption peaks in carbon-13 53. The method as recited in claim 51 further com nuclear magnetic resonance study at at least ap prising the step of selecting the envelope-containing proximately 9.0, 18.9, 24.7, 34.5, 62, 94.5, 130-145, virus from the group consisting of Rhabdoviridae, 171.7 ppm relative to a 37.5 ppm resonance peak of Bunyaviridae, Filoviridae, Nodaviridae, and Togaviri dimethyl sulfoxide and additional absorption peaks 30 dae. in carbon-13 nuclear magnetic resonance study at 54. The method as recited in claim 51 further com approximately 27.9 ppm and 68.4 ppm relative to prising the step of selecting the envelope-containing the resonance peak of tetramethylsilane in deuter virus from the group consisting of Flaviviridae, Re ated chloroform solvent; and troviridae, and Arenaviridae. at least 50 percent of the porphyrins in said mixture 35 55. The method as recited in claim 54 further com being of said molecules having said molecular for prising the step of selecting the Retroviridae comprising mula. a human immunodeficiency virus. 44. The method as recited in claim 43 further com 56. The method as recited in claim 42 further com prising the step of administering said mixture of porphy prising the step of selecting a blood which contains rins to the patient prior to the removal of the blood 40 pathogenic biological contaminants comprising a bac from the patient's body for irradiation. teria selected from the group consisting of Streptococcus 45. The method as recited in claim 44 further con faecalis and Bacillus subtilis. prising the step of administering said mixture of porphy 57. The method as recited in claim 42 further con rins to the patient between about thirty minutes and prising the step of selecting body fluid which contains about one week prior to removal of the patient's blood 45 pathogenic biological contaminants comprising a Bor for irradiation. relia burgdorferi bacteria. 46. The method as recited in claim 44 further com 58. The method as recited in claim 42 further com prising the step of administering said mixture of porphy prising the step of selecting the pathogenic biological rins at a dosage from about 0.5 mg to about 40 mg per contaminants comprising a malarial parasite. kg of body weight of the patient. 50 59. The method as recited in claim 42 further com 47. The method as recited in claim 42 further com prising the step of selecting a blood which contains prising the step of admixing said mixture of porphyrins pathogenic biological contaminants comprising a try with said blood in an amount from about 0.1 to about 50 panosomal parasite. micrograms per milliliter of said blood. 60. The method as recited in claim 42 further com 48. The method as recited in claim 47 further com 55 prising the step of agitating the resulting body fluid in prising the step of admixing said mixture of porphyrins the container. with said blood in an amount from about 2 to about 50 61. The method as recited in claim 60 wherein the micrograms per milliliter of said blood. step of agitating is carried out by moving the container. 49. The method as recited in claim 42 wherein the 62. The method as recited in claim 60 wherein the level of radiation is produced by a light source having a 60 step of agitating is carried out by the aid of an agitating wavelength of from about 600 to about 1000 nm and an leans. energy density of from about 1 to about 50 J/cm2. 63. The method as recited in claim 62 wherein said 50. The method as recited in claim 49 wherein the agitating means comprises an agitating object. level of radiation is produced by a light source having a 64. The method as recited in claim 42 wherein said wavelength of from about 600 to about 700 nm and an 65 container is kept static. energy density of from about 1 to about 20 J/cm2. 65. A method for extracorporeal treatment of the 51. The method as recited in claim 42 further com blood of a patient infected with infectious pathogenic prising the step of selecting a blood which contains biological contaminants said method comprising: 5,030,200 43 44 administering to the patient infected with infectious range of from about 400 nm to about 1000 nm, for pathogenic foreign biological contaminants an ef an effective period of time such that the radiation fective, non-toxic amount of photoactive com penetrates the blood and exposes the photoactive pound having an affinity to be selectively bound to compound-bound infectious contaminants to the the infectious contaminants; radiation so as to eradicate such infectious contami waiting a period of time for the photoactive com nants while maintaining the viability of compo pound to be taken up by the blood; nents in said blood to produce a viable and decon removing the blood from the body of the patient; taminated blood; and maintaining the blood in a suitable container in which returning the viable and decontaminated blood to the there is no net mass transfer; O patient's body. irradiating the blood admixed with photoactive com 66. The method as recited in claim 65 wherein the pound in the container with an effective level of waiting period of time is from about one hour to about essentially uniform intensity of radiation in the one week. region of visible spectrum, with a wavelength k sk xx xt xk 5
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