WO1990007952A1

WO1990007952A1 - Treatment methods and vaccines

Info

Publication number: WO1990007952A1
Application number: PCT/US1990/000275
Authority: WO
Inventors: Emil Bisaccia; Albert S. Klainer
Original assignee: Emil Bisaccia; Klainer Albert S
Priority date: 1989-01-10
Filing date: 1990-01-10
Publication date: 1990-07-26
Also published as: CA2007499C; AU638693B2; IL92996A; BR9007016A; JPH04502621A; FI105453B; CA2007499A1; AU4954590A; HUT62487A; FI913318A0; EP0453497A1; EP0453497A4

Abstract

Methods are provided for treating patients who are infected with a virus using a photoactive compound that, upon activation by exposure to electromagnetic radiation of a prescribed spectrum, such as ultraviolet light, inactivates and/or attenuates the virus and permits the so treated virus and/or virus infected cells to be presented to the immune system of the patient. Medicaments and an apparatus for carrying out said methods are also provided.

Description

TKEATMENT METHODS AND VACCINES

FIELD OF THE TWVFWTτr.M The present invention relates to t_he _fiel_d o_f immunology, particularly methods for inactivating and/or attenuating viruses and/or virus infecte_d cells, particularly virus infected CD₄ cells, using photopheresis and employing said inactivated and/or attenuated viruses and/or virus infected cells to engender an immune response. A particular aspect of the invention relates to the treatment of patients who are infected with a virus, particularly _an HI_V retrovirus, and who have an abnormally _low white ^bloo^d cell count, by using photopheresis in com^bination with the administration of a photoactive compound such as ⁸-methoxypsoralen. Compoun_ds for use in the manufacture of medicaments for the therapeutic treatment of such virus infections are also described. The invention also relates to vaccines against viruses, particularly HIV retroviruses , and methods for producing sai_d vaccines.

BACKGROUND OF THE INVENTION There are many debilitating infectious diseases which have been attributed to viral infections. There are also many different types of viruses including DNA viruses and RNA viruses.

Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first employ reverse transcription of the RNA of their genome into DNA ("transcription" conventionally describes the synthesis of RNA from DNA) . Once in the form of

DNA, the viral genome is incorporated into the host cell genome, allowing it to take full advantage of the host cell's transcription/translation machinery for the purpose of replication. Once incorporated into the host cell's DNA, the virus may persist for as long as the cell lives.

Certain retroviruses are known to cause a depression in an infected patient's white blood cell count. This sub-set of retroviruses which reduce white blood cell count are known as Human Immunodeficiency Viruses (HIV) .

Particular species of HIV retroviruses have been isolated from patients who suffer from Acquired Immune Deficiency Syndrome (AIDS) and have been given the designations HIV 1 and HIV 2, sometimes collectively referred to as "HTLV III", "LAV" or simply "HIV". These retroviruses will infect cells expressing the CD4 marker, such as human T- lymphocytes and monocytes. These cells are involved in the functioning of the immune system. This infection, in turn, results in the progressive loss of the CD4 T-cell population and disturbs the function of other CD₄ cells, such as monocytes, thereby reducing the patient's ability to combat other infections, and predisposing the patient to opportunistic infections which frequently prove fatal .

There are at least three clinical manifestations of AIDS infection. In the initial "carrier" state, the only indication of infection is either the presence of HIV antibodies in the blood¬ stream or the ability to culture the virus. The next stage is known as 'AIDS related complex' (ARC) and the physical symptoms associated therewith may include persistent general lymphadenopathy, general malaise, increased temperature and chronic infections. This condition usually progresses to the final, fatal AIDS condition, when the patient loses the ability to fight infection.

A particularly troublesome problem associated with combatting HIV retrovirus infections is that the RNA to DNA reverse transcription process is fraught with repeated mutation which makes it extremely . difficult for the body's immune system to recognize and attack infected cells along with the virus itself.

It is therefore, an object of this invention to provide a method of treating virus infections in patients having an abnormally low white blood cell count. It is a further object of the invention to provide a method of treating patients infected with an HIV retrovirus.

A still further object of the invention is to provide a vaccine against virus infections, particularly when the infection is caused by a retrovirus such as an HIV retrovirus.

Other objects of the invention will be apparent from the description of the invention which follows. BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention a method has been found for treating patients who are infected with a virus, particularly an HIV retrovirus, using a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum, such as ultraviolet light, for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient for the purpose of engendering an immune response to the viral infection. Psoralen compounds are particularly preferred for this purpose, especially the compound 8-methoxypsoralen — in which case UVA radiation is preferred for activating said compound.

An especially important feature of the present invention is that the treatment methods described herein are used in patients having an abnormally low number of circulating lymphocytes or white blood cells. In addition, it has been found by the inventors that the treatment methods according to the invention can be used to reconstitute the immune system's function in such patients. This is of tremendous importance in the treatment of ARC/AIDS patients who, in addition to having an HIV infection (which itself has heretofore been extremely difficult to treat) , also have a depressed immune system. Such patients generally cannot tolerate a reduction in the number of their lymphocytes or white blood cells without succumbing to opportunistic infections.

It has thus been found by the inventors that the treatment methods in accordance with the present invention are useful to control HIV and other retrovirus infections in patients having an abnormally low white blood cell level, without causing a harmful depression of the patient's immune system. Accordingly, the methods of the invention can be employed in the treatment of conditions such as ARC/AIDS without subjecting the treated patient to a risk of opportunistic infection. The treatment methods in accordance with the invention may also be used in such patients to improve the functioning of their immune systems. In addition, HIV retroviruses may also be responsible for causing diseases other than AIDS. The inventors believe that the inventive methods should be useful for treating such other diseases as well.

In accordance with the invention, a photoactive compound such as 8-methoxypsoralen is administered to the patient's blood, or some fraction thereof, in vitro or in vivo using conventional administration routes. A portion of the patient's blood is then treated (preferably, extracorporeally) using photopheresis, which comprises subjecting the blood to electromagnetic radiation in a wavelength suitable for activating the photoactive compound, such as ultraviolet light, preferably long wavelength ultraviolet light in the wavelength range of 320 to 400 nm, commonly called UVA light. The treated blood, or a fraction thereof, is returned to the patient (in the case of extracorporeal photopheresis) or remains in the patient (following in vivo photopheresis) .

Vaccines against viruses and methods of making same are also provided according to the invention. According to the invention, a photoactive compound (as described herein) is administered to the blood or some fraction thereof of a donor who is infected with a virus, such as an HIV retrovirus and/or who is suffering from AIDS or AIDS Related

Complex. At least a portion of the donor's blood is then treated using photopheresis, as described above. The treated blood or some fraction thereof (eg. , treated free isolated virus) may be used as a vaccine. In the case of treating an HIV infection it is preferred to use treated virus infected cells along with the treated virus in order to obtain the desired immune response.

Optionally, the treated blood is processed by conventional techniques to substantially remove the erythrocytes. The resulting processed fraction is then used as a vaccine which can be administered to a patient.

The invention also concerns medicaments for the therapeutic treatment of virus infections in patients having an abnormally low white blood cell count and for the therapeutic treatment of HIV infections, particullarly an HIV 1 infection or an HIV 2 infection, as well as for the therapeutic treatment of a patient who is an AIDS Carrier or who has AIDS or AIDS Related Complex. DESCRIPTION OF THE DRAWINGS Fig. 1 is a block diagram of a photopheresis apparatus which can be used to practice the inventive methods; Fig.2 is a graph of changes in CD₄ cells and

GP24 and GP120 antibody levels of patient No. 3 in Example 2;

Fig. 3 is a graph of changes in GP24 antibody levels of the five patients treated in Example 2; Fig. 4 is a graph of changes in GP120 antibody levels of the five patients treated in Example 2; and

Fig. 5 is a graph of changes in CD₄ helper cell percentages of the five patients treated in Example 2.

DETAILED DESCRIPTION OF THE INVENTION While it is not intended that the scope of the present invention be limited by any specific theory of operation, it is believed that viral infections, particularly those which are not controlled by the normal immunological response of a patient, can be treated using a photopheresis treatment according to the invention. It is believed by the inventors that the photopheresis treatment according to the invention not only treats the viral infection, but is believed by the inventors to (i) restore the ability of a treated patient's immune system (which has been weakened by the viral infection) to combat other infections, including non- viral infections, and (ii) restore the immune system's anamnestic response to previous infections. The photopheresis treatment method according to the invention is of particular value in the treatment of frequently mutating viral infections, such as retroviruses, for instance HIV retroviruses. In accordance with the photopheresis methods of the invention, treated infected cells as well as killed and/or attenuated virus, peptides, native sub-units of the virus itself (which are released upon cell break-up and/or shed into the blood) and/or pathogenic noninfectious viruses may be used.

Mutation of the viral antigen does not shield it from attenuation/inactivation during photopheresis and consequent generation of an immune response to the mutant forms of the viral antigen. Thus, the treatment methods according to the invention provide a dynamic autogenous vaccine against viral infections.

The inventive methods have been found by the inventors to be useful in the treatment of patients having a virus infection and who have an abnormally low white blood cell count and are particullarly useful in treating HIV retrovirus infections. The inventive methods are also particullarly useful for treating patients who are AIDS Carriers or who have AIDS or AIDS Related Complex.

According to the claimed methods, a photoactive compound is first administered to the blood of a patient who is infected with a virus. The photoactive compound may be administered in vivo (e.g. orally or intravenously) or may be administered in vitro to a portion of the patient's blood which has been removed from the patient by employing conventional blood withdrawal techniques. Alternatively, free virus is isolated from infected cells using conventional virus isolation methods which are known in the art. The photoactive compound can be administered to the infected cells prior to virus isolation or can be administered to the free isolated virus. In the case of treating HIV infection, however, it is presently preferred to use both treated virus and treated virus infected cells in the methods described hereinbelow.

In accordance with the present invention, the photoactive compound selected should preferably be one that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum, such as ultraviolet light, for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient. Psoralen compounds are particularly preferred for this purpose, especially the compound 8- methoxypsoralen — in which case UVA radiation is preferred for activating said compound.

Next, the portion of the patient's blood, or the free isolated virus, to which the photoactive compound has been administered is treated by subjecting the portion of the blood, or the free isolated virus, to photopheresis using said electromagnetic radiation — for example, ultraviolet light. The photopheresis step is preferably carried out in vitro using an extracorporeal photopheresis apparatus.

The photopheresis step in accordance with the present invention may also be carried out in vivo. A presently preferred extracorporeal photopheresis apparatus for use in the methods according to the invention is currently manufactured by Therakos, Inc., Westchester, Pennsylvania under the name TJVAR. A description of the Therakos UVAR photopheresis apparatus may be found in U.S. Patent No. 4,683,889, granted to R.L. Edelson on August 14, 1987, the contents of which are hereby incorporated by reference in their entirety.

As illustrated diagramatically in Fig. 1, the apparatus includes a pump 10 for removing blood from the patient via a donor needle placed in an appropriate vein of the patient; an irradiation chamber 20; a radiation source 30 in close proximity to the irradiation chamber and a centrifuge 40, preferably of the continuous type. The various parts of the apparatus, such as tubing collection bags for the blood and the like, which come in contact with -li¬

the patient's blood or some fraction thereof, are preferably replaceable so that they may be disposed of after each use to prevent the possibility of transmitting blood-borne infections from one patient to others who are subsequently treated with the apparatus.

The exposure of blood, or free isolated virus, to ultraviolet light in a photopheresis apparatus is within the ability of persons having ordinary skill in the art.

When the photopheresis step is carried out in vitro, at least a fraction of the treated blood, or the treated free isolated virus, is returned to the patient following the photopheresis treatment. Preferably, the treatment method described hereinabove is repeated at an interval of about once per week to about once every four weeks. Most preferably, in the treatment of HIV infection, the treatment methods described herein are administered on two successive days and repeated approximately once per month (ie, the patient preferably receives two treatments every month) .

In view of the disclosure contained herein, those persons who are skilled in the art will be able to adjust the treatment parameters — ie, dosage of the photoactive compound and electromagnetic radiation, periodicity of treatment (e.g., monthly, weekly, etc.) and the number of treatments administered in each period ( e.g., twice per month on two successive days) —depending on the condition of the patient and the patient's response to the treatment.

Preferred photoactive compounds for use in accordance with the present invention are compounds known as psoralens (or furocoumarins) which are described in U.S. Patent No. 4,321,919 the disclosure of which is incorporated herein by reference in their entirety.

The preferred photoactive compounds for use in accordance with the present invention include the following: psoralen;

8-methoxypsoralen;

4,5'8-trimethylpsoralen;

5-methoxypsoralen; 4-methylpsoralen;

4,4-dimethylpsoralen;

4-5'-dimethylpsoralen; and

4',8-methoxypsoralen

The most particularly preferred photoactive compound for use in accordance with the invention is 8-methoxypsoralen.

The determination of an effective dosage for in vitro virus inactivation of free isolated virus is within the ability of persons having ordinary skill in the art.

The photoactive compound, when administered to the patient's blood in vivo is preferably administered orally, but also can be administered intravenously and/or by other conventional administration routes.

The preferred dosage of the photoactive compound is in the range of about 0.3 to about 0.7 mg/kg of body weight although larger or smaller doses may be employed. When the photoactive compound is administered in vitro to only a portion of the patient's blood or fraction thereof, it is within the ability of those skilled in the art to calculate a dosage which is equivalent to said range based upon the volume of treated blood or fraction thereof. When administered orally, the photoactive compound should preferably be administered at least about one hour prior to the photopheresis treatment and no more than about three hours prior to the photopheresis treatment. The timing of administration may be adjusted up or down as needed depending on the bioavailability of the photoactive compound, its expected half-life, etc. If administered intravenously, the times would generally be shorter.

The photopheresis treatment in the treatment methods according to the invention is preferably carried out using long wavelength ultraviolet light (UVA) at a wavelength within the range of 320 to 400 nm. The exposure to ultraviolet light during the photopheresis treatment preferably has a duration of about three to four hours, although shorter or longer treatment periods may be used if desired.

Whatever the spectrum of electromagnetic, radiation, the exposure of virus infected cells and/or virus thereto, following administration of the photoactive compound, should be of sufficient intensity/duration to effectively inactivate and/or attenuate the virus. The selection of an appropriate wavelength for photopheresis as well as the exposure, depending upon the photoactive compound being employed and the conditions of treatment (e.g., in vivo exposure or in vitro exposure) , is within the ability of those skilled in the art in view of the present disclosure.

When the photoactive compound is 8- methoxypsoralen, it is preferred in accordance with the invention to utilize an exposure to UVA radiation of about 2 Joules/meter^ based upon the surface area of the virus and virus infected cells undergoing treatment. When the photopheresis treatment according to the invention is carried out in vivo, careful attention should be paid to controlling the maximum radiant exposure so as to avoid unnecessary injury to the patient. Methods for calculating maximum radiant exposure to ultraviolet light are known in the art and, therefore, shall not be described herein.

In summary, the invention provides a novel treatment for patients who are infected by a virus and who have depressed immune systems as a result of such infection, as well as for patients who are infected with an HIV retrovirus or who are AIDS

Carriers or who have AIDS or AIDS Related Complex. Such patients cannot tolerate a treatment that would depress their immune systems.

The treatment methods according to the invention have been found by the inventors to be safe in this latter regard while also being effective in combatting HIV infection in humans. The . invention also provides methods for making vaccines. According to the invention, a donor who is infected with a virus, such as an HIV retrovirus, may be utilized to produce a vaccine against his infection as follows.

First, a photoactive compound as described hereinabove is administered to at least a portion of the donor's blood containing free virus and/or virus infected cells either prior to removal of the blood, either orally or intravenously, or after removal from the donor in which case it is administered in vitro. Optionally, a portion of the donor's blood could first be processed using known methods to substantially remove the erythrocytes and the photoactive compound is then administered to the resulting fraction.

In any case, the portion of blood (eg. , an enriched leukocyte fraction thereof) and/or free isolated virus to which the photoactive compound has been administered is subjected to a photopheresis treatment using electromagnetic radiation of a prescribed spectrum, e.g. ,ultraviolet light, preferably UVA, in the manner previously described. The treated blood, the treated portion thereof or the treated free isolated virus (as the case may be) is then administered back to the donor as an autogenous vaccine. It will be understood that in accordance with the present invention the treated virus can also be isolated from the treated blood or portion thereof following photopheresis treatment for use as a vaccine.

Additionally, in accordance with the present invention, the treated blood, which is itself a mixture of various blood components including peptides or polypeptides, eg., cytokines, lymphokines, monokines, etc., and/or the treated, portion of blood may be processed, as is within the ability of persons having ordinary skill in the art, to isolate a particular component or components which may be used in the treatment of the virus infection of the donor and/or may be used as a vaccine against the virus.

EXAMPLE 1 A male patient, 39 years of age, weighing approximately 70 kg and who had been diagnosed as having AIDS Related Complex was treated in accordance with the present invention as follows:

8-methoxypsoralen was administered orally to the patient during the afternoon of the first day of treatment at a dosage of 30 mg (i.e. about 0.4 mg/kg) . Approximately one hour after the 8- methoxypsoralen had been administered to the patient he was prepared for withdrawal of a portion of his blood for photopheresis treatment using said Therakos UVAR photopheresis machine. A centrifuge which is integral with the photopheresis machine was used to spin off substantially all of the erythrocytes from the withdrawn blood and these were subsequently returned to the patient. Next, approximately 300cc of plasma and 240cc of buffy coat (which includes the T- lymphocytes) were removed in six cycles (40cc of buffy coat per cycle) . The buffy coat and plasma were subjected to UVA light exposure beginning after 40cc of buffy coat had been collected in the first cycle. The irradiation was continued through all six cycles and then for an additional one and one-half hours for a total irradiation time of approximately four hours. The irradiated buffy coat and plasma were then returned to the patient. This process was repeated in the morning of the following day.

The blood parameters of the patient before receiving the photopheresis treatment according to the invention and five weeks after receiving the treatment are set forth in Table 1.

TABLE 1

Blood Parameters of ARC Patient Treated By The Photopheresis Method According To The Invention

85% 56-78%

22% 32-50%

62% 13-38%

T⁴/T⁸ RATIO 0.15 0.4 0.8-1.9

In addition, the patient, who had a negative mumps skin test (which was carried out in the usual manner known in the art) prior to receiving the treatment in accordance with the present invention as described hereinabove, developed a positive mumps skin test after six monthly treatments in accordance with the present invention— this positive mumps skin test being documented by skin biopsy demonstrating the characteristic histologic changes of delayed hypersensitivity. The above results demonstrate a reconstitution of normal immune function as a result of the treatment method according to the invention. This patient continued to receive the described treatment on two successive days on a monthly basis as part of a clinical study involving five patients, the results of which are discussed in Example 2 below in which this patient is identified as patient No. 1.

EXAMPLE 2

Five patients with ARC characterized by fatigue, lymphadenopathy, fever, absent skin-test reactivity, and decreased CD₄ cells volunteered and entered into a clinical study that was conducted in accordance with the provisions of the Institutional Review Board for the treatment of human subjects at Morristown Memorial Hospital, Morristown, New Jersey. Three homosexual males ages 27 to 39, one 42-year-old male reformed intravenous drug abuser, and one 27- year-old female reformed intravenous drug abuser comprised the study group. The range of time from the first known HIV antibody positivity until enrollment for the HIV positive patients was one to four years. Inclusion criteria included only patients not having undergone any previous therapy against HIV infection.

Each patient received a monthly regimen of the treatment substantially as described in Example 1 repeated on two succesive days. The dosage of 8- methoxypsoralen employed varied between 0.4 and 0.6 mg/kg and was administered orally.

Four of the patients received a total of twelve treatments each over the six month course of the study, that is, on two successive days at monthly intervals for six months. The fifth patient voluntarily discontinued his treatment after the fifth month and only received ten treatments. On the basis of the physical examination, monthly antibody and antigen studies, and CD₄ percentage, all five patients had either a stabilization of their disease or a positive response to the treatment. None of the patients experienced damage to their immune systems during the course of the treatment.

By strictly clinical criteria, all five patients demonstrated improvement with respect to lymph node size. Energy index and general state of "well being" improved in four. At the start of treatment, the increase in energy level lasted for approximately one week post-treatment. By the third month of treatment, a more consistent rise in energy level persisted.

Most notably, the first patient treated (the patient in Example 1, above) , a homosexual male who before treatment was able to walk up a flight of stairs only with difficulty, is now capable of jogging three and one-half miles per day and of lifting weights.

The one patient who did not seem to have a persistent rise in energy level was the female reformed intravenous drug abuser with three small children. Although her fatigue had been chronic prior to her entry into the study, no persistent change in energy level was observed after therapy. Additional clinical observations include increased appetite and weight stability in four of the patients, the exception being the female who lost 3.3 kg over the six month treatment period. Ly phadenopathy disappeared in all five patients by the third month and has remained absent. The four male patients also regained delayed cutaneous hypersensitivity after six months of therapy. During the six-month period of treatment all patients were able to manage common community-acquired upper respiratory tract infections without difficulty.

Other than normal variations, hemoglobin and hematocrit, reticulocyte count, and platelet count remained stable. There were minor variations in the white blood count, and circulating total lymphocytes remained stable in the four male patients; however, the single female patient showed a decrease in white blood count (4,000 prior to treatment to 2,000 after six months of therapy) and a parallel decrease in lymphocytes. In all cases, ESR, urinalysis, and SMAC-20 remained unchanged with the exception of minor rises in serum SGOT and SGPT. EBV titers remained constant in all but one patient in whom the IgG capsid antigen decreased from a titer of 1:1280 to a titer of 1:160 paralleling an increse in HIV antibody titers. CMV titers remained unchanged in all patients. Those patients who were P24 antigen negative remained negative; those who were positive remained positive, and no changes in titer occured by the end of the six-month period. Viral cultures of blood for HIV turned negative in one patient (Patient No. 3) but remained positive in the others after the reported therapy.

The course of the patient whose HIV culture turned negative (Patient No. 3) is outlined in Fig. 2. The Gp 24 antibody levels of all five patients over the course of treatment are plotted in Fig. 3. The Gp 120 antibody levels of all five patients over the course of treatment are plotted in Fig. 4. Finally, the CD₄ helper cell percentages of all five patients over the course of treatment are plotted in Fig. 5. A summary of the clinical characteristics and laboratory results is provided in Table 2 below. TABLE 2

Summary Of Clinical Characteristics

And Laboratory Results

Patient No. 1

Age: 38 Sex: Male

Predisposing Factor: Homosexual

Known Duration Of HIV Antibody Positivity: 3 years PRE-TREATMENT POST-TRE EN

Delayed Skin Test neg. pos.

Hypersensitivity

Virus Culture (blood) pos. pos.

Patient No. 2 Age: 30 Sex: Male Predisposing Factor: Homosexual

Known Duration Of HIV Antibody Positivity: 2 years

GP120 Antibody Level 0.03 0.14 (absorbance)

Delayed Skin Test neg. pos.

Hypersensitivity

Virus Culture (blood) pos. pos.

Patient No. 3

Age: 27

Sex: Male Predisposing Factor: Homosexual

Known Duration Of HIV Antibody Positivity: 4 years

PRE-TREATMENT POST-TREATMENT

Weight(kg) 63.6 64.7

CD Cells(%) 34* 33** GP24 Antibody Level 0.72 1.69

(absorbance)

GP120 Antibody Level 1.04 2.07

(absorbance)

Delayed Skin Test neg. pos. Hypersensitivity

Virus Culture (blood) pos. neg.

* Data for this patient's pre-treatment CD₄ Cells(%) is not available. The value shown in the table was determined for blood drawn one month after the patient received his first treatment.

**

This patient received only ten treatments over a five month period at which point he voluntarily opted to discontinue receiving treatment. Three months after voluntarily discontinuing treatment this patient's CD4 Cells(%) had risen to 40. However, five months after discontinuing treatment the CD₄

Cells(%) fell to 24. Patient No. 4 Age: 42 Sex: Male

Predisposing Factor: Reformed IVDA Known Duration Of HIV Antibody Positivity: 2 years

PRE-TREATMENT -

Delayed Skin Test neg. pos.

Hypersensitivity Virus Culture (blood) pos. pos.

Patient No. 5

Age: 27 Sex: Female

Predisposing Factor: Reformed IVDA

Known Duration Of HIV Antibody Positivity: 2 years

PRE-TREATMENT POST-TREATMENT

Weight(kg) 61.0 57.7 CD₄ Cells(%) 12 13

GP24 Antibody Level 1.41 2.89

(absorbance)

GP120 Antibody Level 0.46 1.95

(absorbance) Delayed Skin Test neg. neg.

Hypersensitivity

Virus Culture (blood) pos. pos.

EXAMPLE 3

Upon completion of the six month course of therapy described in Example 2, wherein patient Nos. 1,2,4 and 5 received treatment on two successive days at monthly intervals, the course of therapy was modified with each patient receiving only one treatment per month instead of two. It was found by the inventors that each patient's CD4 Cell(%) fell off markedly. The original course of therapy, wherein the patients received treatment on two successive days once per month was reinstituted with the result that in patients 1 and 2 the CD₄ Cell(%) rose. Data for CD₄ Cell(%) in patients 4 and 5 is not yet available.. The individual results were as follows: Patient No. 1

At the conclusion of the original six month course of therapy at two treatments per month, this patient's CD₄ Cell(%) was 34. After receiving only one treatment per month for five months, the CD₄ Cell(%) fell to 18. The original course of therapy was reinstituted and after two months the CD Cell(%) rose to 39. Patient No. 2

At the conclusion of the original six month course of therapy at two treatments per month, this patient's CD₄ Cell(%) was 31. After receiving only one treatment per month for four months, the CD₄

Cell(%) fell to 10. The original course of therapy was reinstituted and after two months the CD₄ Cell(%) rose to 27. Patient No. 4 At the conclusion of the original six month course of therapy at two treatments per month, this patient's CD₄ Cell(%) was 33. After receiving only one treatment per month for four months, the CD Cell(%) fell to 7. The original course of therapy has been reinstituted but data on changes in the CD₄ Cell(%) is not yet available. Patient No. 5

At the conclusion of the original six month course of therapy at two treatments per month, this patient's CD Cell(%) was 13. After receiving only one treatment per month for four months, the CD₄ Cell(%) fell to 8. The original course of therapy has been reinstituted but data on changes in the CD₄ Cell(%) is not yet available. Medicaments made using the photoactive compounds herein may be formulated using standard techniques which are already known in the art and, therefore, shall not be described in detail herein. By way of illustration, the photoactive compounds may be formulated using conventional excipients in the form of tablets, capsules and the like which would be suitable for oral administration. Alternatively the photoactive compounds described herein may, if desired, be formulated for parenteral administration by intravenous or intramuscullar routes. The medicaments can also be formulated as injectable solutions or suspensions for in vitro administration to a blood fraction which has been removed from an infected donor.

It should be understood that while the foregoing description has been provided to illustrate the present inventions, it is not intended to limit the scope of the inventions as various modifications to the inventions described herein may be made by persons having ordinary skill in the art without departing from the spirit and scope thereof as defined in the following claims.

Claims

We Claim:

1. A method for treating a patient having a virus infection and an abnormally low number of lymphocytes or white blood cells, said method comprising the steps of: a. administering to the patient's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient; and b. treating at least a portion of the patient's blood to which the photoactive compound has been administered, said treatment comprising subjecting the portion of blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electromagnetic radiation being administered in amounts which are pharmaceutically effective for controlling the viral infection.

2. The method of claim 1, wherein the administration of the photoactive compound is performed in vitro.

3. The method of claim 1, wherein the administration of the photoactive compound is performed in vivo.

4. The method of claim 2, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of blood to the patient following step (b) .

5. The method of claim 3, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of the blood to the patient following step (b) .

6. The method of claim 1, wherein the electro-magnetic radiation is ultraviolet light and the photoactive compound is a psoralen compound.

7. The method of claim 6, wherein the psoralen compound is selected from the group consisting of, psoralen, 8-methoxypsoralen, 4,5',8- trimethylpsoralen, 5-methoxypsoralen, 4-methylpsoralen, 4,4-dimethylpsoralen, 4,5'- dimethylpsoralen, and 4',8-dimethylpsoralen. 8. The method of claim 7, wherein the psoralen compound is selected from the group consisting of 8-methoxy¬ psoralen, psoralen and 4,5',

8-trimethylpsoralen.

9. The method of claim 8, wherein the psoralen compound is 8-methoxypsoralen.

10. The method of claim 9, wherein the 8- methoxypsoralen is administered in a dosage of about 0.3 to about 0.7 mg/kg.

11. The method of claim 10, wherein the 8- methoxypsoralen is administered orally.

12. The method of claim 10, wherein the 8- methoxypsoralen is administered intravenously.

13. The method of claim 9 , wherein steps (a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

14. The method of claim 10, wherein steps (a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

15. A method for producing a vaccine against an HIV retrovirus infection in an infected donor, said method comprising the steps of: a. administering to at least a portion of the donor's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient for the purpose of engendering an immune response to the viral infection; and b. then treating the portion of the donor's blood to which the photoactive compound has been administered, said treatment comprising subjecting at least a fraction of said portion of blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electro-magnetic radiation being administered in amounts which are pharmaceutically effective for attenuating and/or inactivating at least some of the HIV retrovirus which is present in the treated fraction.

16. The method of claim 15, wherein the fraction of blood is processed to substantially remove erythrocytes.

17. The method of claim 16, wherein the fraction of blood is processed to substantially remove erythrocytes prior to performing step (b) .

18. The method of claim 16, wherein the fraction of blood is processed to substantially remove erythrocytes after performing step (b) .

19. The method of claim 15, wherein the fraction which is treated in step (b) consists essentially of free isolated HIV retrovirus.

20. The method of claim 15, further comprising processing the treated fraction of blood following step (b) to isolate treated HIV retrovirus for use as a vaccine against an HIV retrovirus infection.

21. A method for treating an infected patient who is either an AIDS carrier or who has AIDS or AIDS Related Complex, said method comprising the steps of: a. administering 8-methoxypsoralen to at least a portion of the patient's blood; b. then subjecting at least a portion of the patient's blood to which the 8-methoxypsoralen has been administered to extracorporeal photopheresis using UVA light; and c. infusing at least a fraction of the portion of blood treated in steps (a) and (b) back into the patient, said 8-methoxypsoralen and said UVA light being administered in amounts which are pharmaceutically effective for inhibiting the progression of the patient's infection.

22. The method of claim 21, wherein the dosage of 8-methoxypsoralen which is used in step (a) is between about 0.3 to about 0.7 mg/kg administered orally to the patient no more than about 3 hours prior to photopheresis.

23. A method for treating a patient who is an AIDS carrier or who has AIDS or AIDS Related Complex and who has infected leukocytes, said method comprising the steps of: a. administering to the patient's infected leukocytes a photoactive compound which is capable of binding to nucleic acids in the leukocytes upon activation of the photoactive compound by exposure to ultraviolet light; and b. then treating at least a portion of the patient's leukocytes to which the photoactive compound has been administered by subjecting the. portion of leukocytes to photopheresis using ultraviolet light, said photoactive compound and said ultraviolet light being administered in amounts which are pharmaceutically effective for inhibiting the progression of the patient's infection.

24. The method of claim 23, wherein the photoactive compound is 8-methoxypsoralen.

25. A method for increasing the number of a patient's non-infected T-cells in a patient having an abnormally low number of T-cells due to a viral infection, such as an HIV infection, said method comprising the steps of: a. administering 8- methoxypsoralen to at least a portion of the patient's blood in a dosage range between about 0.3 to about 0.7 mg/kg; and b. then exposing at least a fraction of the portion of he patient's blood to UVA light, said 8- methoxypsoralen and said UVA light being administered in amounts which are pharmaceutically effective for increasing the number of the patient's non-infected T-cells.

26. A method for treating an infected patient who is either an AIDS carrier or who has AIDS or AIDS Related Complex, said method comprising the steps of: a. administering to the patient's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient; and b. treating at least a portion of the patient's blood to which the photoactive compound has been administered, said treatment comprising subjecting the portion of blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electromagnetic radiation being administered in amounts which are pharmaceutically effective for controlling the viral infection.

27. The method of claim 26, wherein the administration of the photoactive compound is performed in vitro.

28. The method of claim 26, wherein the administration of the photoactive compound is performed in vivo.

29. The method of claim 27, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of blood to the patient following step (b) ,

30. The method of claim 28, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of the blood to the patient following step (b) .

31. The method of claim 26, wherein the electro-magnetic radiation is ultraviolet light and the photoactive compound is a psoralen compound.

32. The method of claim 31, wherein the psoralen compound is selected from the group consisting of, psoralen, 8-methoxypsoralen, 4,5'.,8- trimethylpsoralen, 5-methoxypsoralen,

4-methylpsoralen, 4,4-dimethylpsoralen, 4,5'- dimethylpsoralen, and 4',8-dimethylpsoralen.

33. The method of claim 32, wherein the psoralen compound is selected from the group consisting of 8-methoxy- psoralen, psoralen and 4,5',8-trimethylpsoralen.

34. The method of claim 33, wherein the psoralen compound is 8-methoxypsoralen.

35. The method of claim 34, wherein the 8- methoxypsoralen is administered in a dosage of about

0.3 to about 0.7 mg/kg.

36. The method of claim 35, wherein the 8- methoxypsoralen is administered orally.

37. The method of claim 35, wherein the 8- methoxypsoralen is administered intravenously.

38. The method of claim 34, wherein steps (a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

39. The method of claim 35, wherein steps (a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

40. A method for treating a patient who has an HIV retrovirus infection, said method comprising the steps of: a. administering to the patient's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g. , DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient; and b. treating at least a portion of the patient's blood to which the photoactive compound has been administered, said treatment comprising subjecting the portion of blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electromagnetic radiation being administered in amounts which are pharmaceutically effective for controlling the HIV retrovirus infection.

41. The method of claim 40, wherein the administration of the photoactive compound is performed in vitro.

42. The method of claim 40, wherein the administration of the photoactive compound is performed in vivo.

43. The method of claim 41, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of blood to the patient following step (b) ,

44. The method of claim 42, wherein the photopheresis is conducted extracorporeally and wherein said method further comprises the step of returning the treated portion of the blood to the patient following step (b) .

45. The method of claim 40, wherein the electro-magnetic radiation is ultraviolet light and the photoactive compound is a psoralen compound.

46. The method of claim 45, wherein the psoralen compound is selected from the group consisting of, psoralen, 8-methoxypsoralen, 4,5',8- trimethylpsoralen, 5-methoxypsoralen, 4-methylpsoralen, 4,4-dimethylpsoralen, 4,5'- dimethylpsoralen, and 4',8-dimethylpsoralen.

47. The method of claim 46, wherein the psoralen compound is selected from the group consisting of 8-methoxy- psoralen, psoralen and 4,5',8-trimethylpsoralen.

48. The method of claim 47, wherein the psoralen compound is 8-methoxypsoralen.

49. The method of claim 48, wherein the 8- methoxypsoralen is administered in a dosage of about 0.3 to about 0.7 mg/kg.

50. The method of claim 49, wherein the 8- methoxypsoralen is administered orally.

51. The method of claim 49, wherein the 8- methoxypsoralen is administered intravenously.

52. The method of claim 48, wherein steps (a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

53. The method of claim 49, wherein steps

(a) and (b) are performed on two succesive days, said steps (a) and (b) being performed on the first day and repeated on the following day, at an interval of between about one to four weeks between each such two day treatment.

54. A method for treating a patient who has an HIV retrovirus infection, said method comprising the steps of: a. administering 8-methoxypsoralen to at least a portion of the patient's blood in an amount which upon photoactivation is sufficient to bind to substantially all free and/or cell associated HIV retrovirus as well as to infected cells in said portion; b. then subjecting at least a portion of the patient's blood to which the 8-methoxypsoralen has been administered to extracorporeal photopheresis using UVA light in an amount which is sufficient to inhibit the progression of the patient's HIV infection; and c. infusing at least a fraction of the portion of blood treated in steps (a) and (b) back into the patient.

55. The method of claim 54, wherein the dosage of 8-methoxypsoralen which is used in step (a) is between about 0.3 to about 0.7 mg/kg administered orally to the patient no more than about 3 hours prior to photopheresis.

56. The use of a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus, characterized in that the photoactive compound is used for the manufacture of a medicament for the therapeutic treatment of a virus infection in an infected patient who has an abnormally low number of lymphocytes or white blood cells.

57. The use according to claim 56, characterized in that the photoactive compound is a psoralen compound.

58. The use according to claim 57, characterized in that the psoralen compound is 8- methoxy psoralen.

59. The use of a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus, characterized in that the photoactive compound is used for the manufacture of a medicament for the therapeutic treatment of an HIV virus infection.

60. The use according to claim 59, characterized in that the photoactive compound is a psoralen compound.

61. The use according to claim 60, characterized in that the psoralen compound is 8- methoxy psoralen.

62. The use of a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e.g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus, characterized in that the photoactive compound is used for the manufacture of a medicament for the therapeutic treatment of a patient who is an AIDS Carrier or who has AIDS or AIDS Related Complex.

63. The use according to claim 62, characterized in that the photoactive compound is a psoralen compound.

64. The use according to claim 63, characterized in that the psoralen compound is 8- methoxy psoralen.

65. The use of a photopheresis apparatus comprising a pump (10) , an irradiation chamber (20) , and a radiation source (30) , characterized in that the photopheresis apparatus is used for the therapeutic treatment of a virus infection in an infected patient who has an abnormally low lymphocyte or white blood cell count.

66. The use of a photopheresis apparatus comprising a pump (10) , an irradiation chamber (20) , and a radiation source (30) , characterized in that the photopheresis apparatus is used for the therapeutic treatment of an HIV virus infection.

67. The use of a photopheresis apparatus comprising a pump (10) , an irradiation chamber (20) , a radiation source (30) , and a centrifuge (40) , characterized in that the photopheresis apparatus is used for the therapeutic treatment of a patient who is an AIDS Carrier or who has AIDS or AIDS Related Complex.