Title:  Method of treating humans with diseases characterized by high secretion of cytokines from macrophage cells

United States Patent:  6,407,068

Appl. No.:  421162

A method of treating an individual who is suffering from human immunodeficiency virus infection is disclosed. Methods of treating a human suffering from a disease characterized by high secretion of cytokines from macrophagecells and methods of reducing cytokine secretion are disclosed. The methods comprises administering an amount of ricin, abrin, modeccin, viscumin or volkensin effective to eliminate mononuclear phagocyte lineage cells.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "toxic lectins", "ribosomal inactivating proteins" (RIP), "RIPs", "double-chain ribosomal inactivating proteins" (DC-RIPs), and "DC-RIPs" are interchangeable and refer to Ricin, Abrin, Modeccin, Viscumin and Volkensin.

As used herein, "MPL cells" refers to cells of mononuclear phagocyte lineage. "Cells of the Mononuclear Phagocyte lineage" are called monocytes within blood vessels and become macrophages, Langerhan's cells, or Kupffer cells, depending on which tissue of the body is entered. Langerhan's cells, and possibly others, then occasionally migrate to the spleen to become Dendritic cells.

MPL cells that are persistently infected with HIV play a critical role in the development of AIDS. HIV infects MPL cells but replication is restricted and virus is produced at a severely reduced level, if at all. Viral infection of mononuclear phagocyte lineage cells which are resistant to cytopathic effects of the infection results in the establishment of chronic infection of the cells. Because viral replication is limited, infected MPL cells do not display viral proteins to the extent virally infected cells normally do. Accordingly, infected MPL cells harboring HIV are not identified and extricated by the immune system.

Interactions between infected MPL cells and T-cells results in virus dissemination. Fulfilling their normal role as antigen presenting cells, the macrophage present HIV to the T-cells. As the slowly increasing number of infected MPL cells continue to present HIV to helper T-cells, the helper T-cell population becomes infected in increasing numbers. As the result of this viral infection of T-cells and the subsequent viral replication therein, the number of helper T-cells decreases, thereby reducing the immune systems capacity to function. A weakened immune system allows for increased numbers of infected MPL cells which continue to present HIV to T-cells resulting in destruction of more T-cells. The immune system becomes more weakened as more T-cells are destroyed by HIV infection and more virus is produced. Eventually, the immune system is so compromised that the patient is susceptible to opportunistic infections which the patient cannot defend against. The patient dies from such infections.

The establishment of chronic infection of MPL cells renders therapies based solely upon inhibition of HIV in T-cells insufficient to fully combat HIV and delay the onset of AIDS for any appreciable time. MPL cells are reservoirs for HIV and it is suspected that infection of circulating CD4(+) T-cells is rare, occurring in only 1 of 12,000 cells. Targeting infected T-cells will not eradicate the virus. In the later stages of differentiation, MPL cells lose their CD4(+) marking, making "anti-CD4 therapy" ineffective against the true viral reservoir. Further, once the retrovirus has become a "pro-virus", reverse transcriptase becomes unnecessary and the currently available inhibitors are also ineffective. As a result, every HIV infection remains lethal. Bone marrow transplantation is likewise unable to prevent progressive HIV infection. Despite chemotherapy to completely eliminate the patient's bone marrow and circulating white blood cells followed by the reconstitution of a new immune system through the introduction of a donor's marrow, the virus still escapes destruction. The ablative chemotherapy does not eliminate tissue macrophages. Thus, despite some success in developing effective treatments, persistently infected MPL cells will remain and allow for the continued presence of the virus.

Although it is known that MPL cells represent a virus reservoir which provide for the chronic persistence of virus, there has been no teaching or suggestion of eliminating these cells. The focus of the problem posed by MPL cell virus reservoirs has been the effects that the progression of AIDS has on MPL cells in connection with the breakdown of natural immunity associated with HIV infection of MPL cells. Thus, it has been suggested that there is a need to address the abrogation of the normal effectiveness of MPL cells in natural immunity which results in the sensitivity an individual has to opportunistic infections. Rather than targeting MPL cells, the focus of attention has been to try to counteract the deleterious effects that the lack of functional MPL cells has on the HIV-infected individual caused. Moreover, another area of research has focussed on the need to control HIV infection and replication in macrophage cells but not the elimination of the cells themselves. A great deal of effort has been directed at the need for compounds which effectively prevent viral gene expression and viral replication while not being toxic to the cells. There exist no teachings or suggestions to target the virus reservoir for elimination. There are no drugs currently available which are designed to selectively eliminate MPL cells.

The present anti-HIV therapies and strategies do not address critical mechanisms in the pathology of HIV infection. A more demanding and fundamental attack on the virus is necessary in order to effectively treat and/or cure a patient. In order to effectively combat HIV, important cells of the immune system must be eliminated i.e., destroyed. The viral reservoir must be removed in order to effectively oppose HIV dissemination and AIDS development. All efforts to eliminate the virus by natural means or otherwise are undermined by the persistent HIV infection of MPL cells. Accordingly, the MPL cells must be a target for elimination.

The invention relates to a method of treating HIV-infected humans by selectively eliminating the MPL cells. These virus reservoir cells play an essential role in the pathology of HIV and by specifically attacking them, it is possible to slow the progress of the infection and, especially when done in conjunction with other therapies, arrested the disease.

According to the invention, there are many different ways to delete MPL cells. Such methods include administration of double-chain ribosomal inactivating proteins such as Ricin, Abrin, Modeccin, Viscumin, and Volkensin. In addition, MPL cells may be eliminated by other means including, but not limited to, administration of silica or toxins enveloped within liposomes or by infusing long-acting adenosine analogs. DC-RIPs provide the most effective means to selectively eliminate the mononuclear phagocyte lineage reservoir. The double-chain ribosomal inactivating proteins such as Ricin, Abrin, Modeccin, Viscumin, and Volkensin are particularly useful as anti-HIV compounds because of their specific properties which render MPL cells more susceptible than other cell types to the toxic effects associated with these lectins.

MPL cells including Kupffer cells, Langerhans cells and pulmonary macrophages, are more sensitive to the effects of toxic lectins such as intact Ricin, presumably due to increased uptake through mannose receptors on these cells. However, Fc-receptor uptake and non-specific phagocytosis-pinocytosis also seem to be involved. Further, protein synthesis in MPL cells seems unusually sensitive to the effects of toxins such as Ricin.

Further, DC-RIP's are capable not only of providing mannose and galactose side-chains for MPL cells to bind, but also of binding certain other carbohydrates themselves. Because gp120, the HIV surface protein on infected T-cells, contains a number of these same carbohydrate moieties, it is probable the DC-RIP's have an independent affinity for infected T-cells beyond their effect on MPL cells. Internalized DC-RIP would cause T-cell death by apoptosis, as well as the abrupt termination of actively replicating virus. Patient blood samples may be cell sorted using fluorescent-labeled anti-gp120 antibody to demonstrate reduction with treatment, thereby indicating whether this additional effect actually takes place in infected humans.

MPL-selective toxins generally, and DC-RIPs in particular, can be used as effective anti-retroviral agents because when administered to infected persons at a proper dosage, these agents are selectively taken up by MPL cells which are thereby killed without causing toxic side effects to the patient. Intact double-chain ribosomal inactivating proteins such as Ricin, Abrin, Modeccin, Viscumin and Volkensin can be administered to a human infected with HIV to eliminate MPL cells and thereby reduce the availability of these cells to become persistently infected viral reservoirs.

Effective dosages of intact Ricin, Abrin, Modeccin, Viscumin and Volkensin sufficient to selectively eliminate MPL cells do so without side effects. Toxic lectins selectively bind to and are taken up by cells by a variety of mechanisms.

The HIV infected MPL cell reservoir that is exposed to Ricin, Abrin, Modeccin, Viscumin and Volkensin is removed through non-specific phagocytosis, mannose and galactose receptor-mediated phagocytosis, and Fc-receptor mediated phagocytosis. Moreover, there is an unusual sensitivity of protein synthesis in MPL cells for Ricin, Abrin, Modeccin, Viscumin and Volkensin. Accordingly, although these toxic lectins are highly toxic to all cells, MPL cells have a greater tendency to take in these lectins and, therefore, can be eliminated selectively by low doses of Ricin, Abrin, Modeccin, Viscumin and Volkensin which are otherwise non-toxic.

MPL cell elimination occurs through apoptosis; thus, virus particles are not allowed to escape the dying cell. Ingestion of the dying MPL cell by macrophages allows for virus to be killed in the lysosomes of the latter.

In cases of Fc-mediated phagocytosis, the developing antibody response of the patient against this foreign protein will not significantly reduce effectiveness. Antibody/toxin complexes can effectively eliminate MPL cells. This "acquired resistance" has been a major disadvantage of immunotoxins because the targeting antibody is neutralized and macrophages clear the complex.

Double-chain ribosomal inactivating proteins selectively eliminate MPL cells. For example, the MPL cells of the liver, Kupffer cells, are 100 to 1000 times more sensitive to double-chain ribosomal inactivating proteins such as Ricin than are other cells such as hepatocytes. As discussed above, Zenilman (1989) has shown that a double-chain ribosomal inactivating protein, Ricin, injected intraperitoneally is capable of eliminating 33% of mouse Kupffer cells without morbidity after 24 hours. It is worth noting that the dose given in that experiment of 2.5 micrograms/kg is below the LD-50, that is, the dose at which 50% are expected to die, of 2.7 micrograms/kg, but death usually occurs 2-3 days later. Single-chain RIPs such as Ricin A-chain require 10,000 times higher doses (20 mg/kg given over 4 days) to achieve Kupffer cell elimination rates of 27% (See Zenilman et al., (1988) J. Surg. Res. 45:82-89).

Human studies have been performed to document the safety of Ricin. Fodstad, O. et al, (1984) Cancer Res. 44:862-865 which is incorporated herein by reference, disclose Phase I clinical studies using intact Ricin with cancer patients to determine the maximum tolerated doses. It is reported that doses up to 20 micrograms/square meter body surface are well below toxic levels.

HIV-infected MPL cells are more sensitive to the toxins than non-infected MPL cells. In general, viruses make cells 50-500 times more sensitive to toxins. Fernandez-Puentes, C. and L. Carrasco, (1980) Cell 20:769-775 disclose that viral infection increases cell membrane permeability. It is disclosed in U.S. Pat. No. 4,869,903, described above, that single-chain ribosomal inactivating proteins are 50-60 times more toxic in HIV infected lymphocytes compared to uninfected control lymphocytes.

Cellular protein synthesis in non-virally infected cells is inhibited by double-chain ribosomal inactivating proteins in concentrations of 3-10 nanograms/ml. With toxin efficacy increasing up to 500-fold during viral infection, double-chain RIP's kill cells in HIV(+) people at picogram/ml quantities.

Non-toxic, selective MPL cell elimination is possible at "reasonable" doses. The increased sensitivity of virally-infected cells dramatically improves the risk:benefit ratio, given the very steep dose-response curve for DC-RIP's.

MPL cell elimination from the body is transient; less than 20% of MPL cells can be eliminated by any single dose of a DC-RIP without causing non-specific damage. Therefore, the normal function of MPL cells in the body (red cell clearance, tumor surveillance, etc.) is not severely impaired. Unlike other lectins, Abrin and Ricin are not toxic to the central nervous system. Further, Abrin is not toxic to peripheral nerves, even when injected into them. Moreover, there is no bone marrow toxicity associated with the administration of intact Ricin or Abrin.

A typical treatment regimen will consist of administration at the highest dose possible, between about 1 and about 20 micrograms, depending on which of the five DC-RIP's is being used, without causing any undesired side effect, such as nausea, myalgia, or increased hepatic enzymes (SGOT, SGPT, Alkaline phosphatase, bilirubin). Treatment continues on a regular basis every 2-4 weeks until plasma HIV p24 antigen is no longer detected. Thereafter, the same double-chain ribosomal inactivating protein may be reinstituted, should p24 antigen reappear in the serum.

The development of atopic reaction or a substantial, specific IgE or IgG level against a given double-chain RIP, warrant switching to a second, immunogenically non-cross reactive double-chain RIP. It is known Ricin, Abrin and Modecccin are all immunogenically non-cross reactive. The development of small quantities of specific IgG should not interfere greatly with toxicity toward MPL cells, as Fc-receptor mediated phagocytosis should replace that mediated by carbohydrate; however, eventually the non-specific uptake by other cell lines possessing Fc receptors may lead to undesired toxicity (anemia, neutropenia, hematuria, etc.), and administrating a different DC-RIP is indicated.

The present invention provides a new weapon in the arsenal to treat HIV infection and can be used in concert with other therapies. Intact Ricin, Abrin, Modeccin, Viscumin or Volkensin can be used together with other anti-retroviral drugs, especially reverse transcriptase inhibitors such as Zidovudine or other available inhibitors of reverse transcriptase in standard doses. In general, the DC-RIP's can be used with any other anti-retroviral therapy. By practicing the present invention as a co-therapy with other treatments and medications, more than one mechanism by which the virus perpetuates itself in the patient can be targeted. Combatting the virus at multiple points of its infectious cycle hinders the progress of disease and the onset of AIDS is prevented or delayed.

Toxic lectins which can be used in the method of the present invention include Ricin, Abrin, Modeccin, Viscumin and Volkensin. A preferred toxic lectin used in the method of the present invention is Ricin.

A preferred embodiment of the present invention is a method of treating a human suffering from HIV which comprises administering an effective amount of Ricin. A preferred dosage range is between about 0.0001 and about 30 micrograms. A more preferred dosage range is about 1 to about 20 micrograms.

Intact Ricin is very easy and inexpensive to produce. Large quantities can be produced by well know methods from readily available starting materials. Simmons B. M. and J. H. Russell, (1985) Analytical Biochemistry 146:206-210, which is incorporated herein by reference, describe a single affinity column step method for the purification of Ricin toxin from castor beans.

Abrin can be extracted from Abrus precatorius seeds. Olsnes, S. and A. Phil, (1973) Eur. J. Biochem. 35:179-185, incorporated herein by reference, disclose a method of extraction and purification of Abrin from semen jegwiriti.

Modeccin can be extracted from the root of Adenia digitata. Olsnes A. et al., (1978) J. Biol. Chem. 253(14):5069-5073, incorporated herein by reference, disclose a method of extraction and purification of Modeccin from Adenia digitata.

Viscumin can be extracted from Viscum album L. (mistletoe). Ziska, P. et al., (1978) Experientia 34:123-124 incorporated herein by reference, disclose a method of extraction and purification of Viscumin from Viscum album L.

Volkensin can be extracted from Adenia volkensii. Barbieri, L. et al., (1984) FEBS 171(2):277-279 incorporated herein by reference, disclose a method of extraction and purification of Volkensin from Adenia volkensii.

Fodstad, O. et al., discussed above and incorporated herein by reference, disclose a phase I study of Ricin on cancer patients. The purification, formulation and administration of Ricin is described. Tolerated dose levels are disclosed.

To practice one embodiment of the invention, intact Ricin can be purified following the teachings of Simmons and Russell or Fodstad et al. Ricin can be formulated as described by Fostad et al. and administered to patients infected with HIV. Non-toxic doses effective for the selective eliminating macrophages can be determined by routine methods.

In addition to the destruction of MPL cells in order to eliminate them as viral reservoirs, the present invention provides further benefits. Virally infected MPL cells secrete cytokines and amplify T-cell:macrophage interaction, causing more rapid dissemination of the infection. Elimination of activated macrophages will reduce cytokine (Il-1, TNF, Il-6) levels, lower infected macrophage/T-cell interactions, and slow progression of the illness.

It is contemplated that according to another aspect of the invention, the selective elimination of MPL cells can be undertaken to reduce cytokine levels that occur during certain illnesses and disorders. Any disease characterized by high secretion of macrophage cytokines including Interleukin-1 or tumor necrosis factor, for example, can be treated by selective elimination of MPL cells, especially the administration of DC-RIPs. Such diseases include alcoholic cirrhosis, rheumatoid arthritis, systemic vasculitis, multiple sclerosis, guillain-barre, uveitis, inflammatory bowel disease, diabetes mellitus, atherosclerosis, systemic lupus erythematosus, Sjogrens syndrome, malaria or leishmaniasis infection or others. Furthermore, while there are no other known retroviral infections in humans besides HIV type 1 and 2, other diseases such as, for example, systemic lupus erythematosus, Sjogrens syndrome or other collagen vascular disease may represent retroviral infections. If chronic retroviral infection of MPL cells plays a role in these disease, the invention can be used to selectively eliminate the MPL cells. There are certain parasitic infections, such as Leishmaniasis, which localize to MPL cells. Accordingly, such infections can be treated by selective elimination of MPL cells, especially by administration of DC-RIPs. Finally, MPL cells which are not killed by doses of chemotherapy and which are responsible for acute or chronic graft-versus-host disease during bone marrow or other organ transplantation, may be eliminated and will vastly increase the potential of this therapy.

Claim 1 of 26 Claims

What is claimed is:

1. A method of treating an individual that has been identified as suffering from a disease comprising high secretion of cytokines from macrophage cells comprising:

administering to said individual parenterally by subcutaneous, intramuscular or intravenous injection of intact, unconjugated double chain ribosomal inactivating protein in an amount therapeutically effective in reducing the number of mononuclear phagocyte lineage cells in said individual, wherein said reduction in mononuclear phagocyte lineage cells ameliorates the disease by effecting a reduction in cytokines secreted by macrophage cells.

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