AU645131B2 - Use of metalloporphyrins to potentiate aids therapy - Google Patents

Description

OPI DATE 02/03/92 AOJP DATE 09/04/92 APPLN. ID 84104 91 PCT NUMBER PCT/US91/05283 TREATY (PCT) INTERNA' (51) International Patent Classification 5 (11) International Publication Number: WO 92/02242 A61K 37/10, 31/70, 31/555 Al (43) International Publication Date: 20 February 1992 (20.02.92) (21) International Application Number: PCT/US91/05283 (74) Agents: BURKE, Henry, T. et al.; Wyatt, Gerber, Burke and Badie, 645 Madison Avenue, New York, NY 10022 (22) International Filing Date: 25 July 1991 (25.07.91) (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 560,191 31 July 1990 (31,07.90) US pean paten' CA, CH (European patent), DE (Euro- 735,090 24 July 1991 (24.07.91) US pean paten: )K (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (Euro- (71) Applicant: THE ROCKEFELLER UNIVERSITY [US/ pean patent), NL (European patent), SE (European pa- US]; 1230 York Avenue, New York, NY 10021 tent).

(72)Inventors: KAPPAS, Attallah 11(l York Avenue, #4L, New York, NY 10021 LEVERE, Richard, D. 5 Published Seymour Plice West, Armonk, NY 10504 ABRA- With international search report.

HAM, Na. r, G. 143 Charter Circle, Ossining, NY 10562 BUCHER, Doris, J. 129 East 92nd Street, New York, NY 10128 (US).

645131 (54) Title: USE OF METALLOPORPHYRINS TO POTENTIATE AIDS THERAPY (57) Abstract Methods and compositions for treating viral infections, especially retroviral infections such as AIDS comprising administration of a heme product in pharmaceutical compositions which may additionally contain an anti-retroviral drug such as AZT with or without erythropoietin.

WO 92/02242 PCT/US91/05283 -USE--FAL YRINS TO POTTE AIDS TRY RELATE b'PPLICATION This aplica on is a continuation in part application of commonly ed copending patent application srial nmnbpr n,7/NA -Qql.

BACKGROUND OF THE INVENTION This invention is concerned with methods and compositions for treating viral infections such as retroviral infections in mammals, especially humans.

The=,-troviruses are a broad group of RNA viruses w ich during their replication employ the reverse transcription enzyme (RT) to convert an RNA message to DNA. The retroviridae family of viruses includes Lentiviruses (visna, maedi, progressive pneumonia virus "slow viruses"), Spumaviruses (foamy viruses) and Oncornaviruses (types A, B, C, D, RNA tumor viruses). The retroviruses have been shown to infect murine, avian, feline, primate, and human species.

The human immunodeficiency virus (HIV-1) or human T-cell lymphotropic virus (HTLV-III) which causes Acquired Immune Deficiency Syndrome (AIDS), AIDS related complex (ARC) and AIDS related diseases is a retrovirus. Also, the feline leukemia virus (FeLV) of cats is a retrovirus.

Retroviruses of the HIV species such as HIV 1 and HIV 2 are cytopathic for helper/inducer T cells in vitro.

The HIV virus is the etiologic agent of the acquired immune deficiency syndrome (AIDS) and related diseases.

To date, thousands of cases of AIDS have been reported in WO 92/02242 PCT/US91/05283 2 the United States, and the incidence and prevalence of this disease continues to increase. During 1989 alone, 35,238 cases of AIDS were reported to the Centers for Disease Control with a total of more than 100,000 cases since 1981.

AIDS is almost always fatal within 1 to 2 years of the first clinical manifestations of illness. This disease was initially described and characterized in four high-risk groups (homosexual men, hemophiliacs, Haitians, and intravenous drug abusers); however, individuals belonging to no apparent high-risk groups have also developed the disease. AIDS is generally spread by intimate sexual contact or by the administration of infected blood products, and occasionally by the maternal-fetal route. Many patients who develop AIDS are asymptomatic when they transmit their disease to contacts because a 6-month to 5-year (or more) latency interval may exist between infection and clinical manifestations of illness.

Historically, nucleosides have been among the best anti-retroviral drugs for treating DNA and RNA viral infections such as retroviral infections. For the treatment of AIDS, 3-azido-3'-deoxythymidine (AZT) has been the most successful of these types of therapeutic agents. However, because of the known deficiencies of AZT a large number of other drugs most of which are not nucleosides are currently under study both in vitro and in vivo. Several of them have reached the stage of clinical trials. These include: WO 92/02242 PCT/US91/05283 3 dextran sulfate AS 101 castanospermine Ampligen interferons

GM-CSF

ribavirin interleukin-2 doxorubicin foscarnet AL 721 suramin Ansumycin nefabutin HPA-29 The use of AZT and related nucleosides is described in detail in US. Patent 4,724,232, the disclosure of which is incorporated herein by reference.

Antiviral therapy for the treatment of AIDS is based on the assumption that continued retroviral replication is involved in both the pathogenesis and progression of the disease. Therefore, RT has been a major target for antiviral therapy in AIDS, and indeed, most of the agents now being investigated, including AZT, act on this enzyme.

The phosphorylated forms of these compounds inhibit HIV replication by acting as chain terminators. Reverse transcriptase of HIV is much more susceptible to the inhibitory effects of these phosphorylated aideoxynucleotides than mammalian DNA polymerases.

Administration of AZT has been shown to result in immunologic improvements and to confer a survival advantage in patients with AIDS.

The successful treatment of AIDS with AZT has, however, been limited due to its serious adverse effects including macrocytosis anemia, neutropenia and WO 92/02242 PCT/US91/05283 4 thrombocytopenia. AZT suppresses the proliferation of erythroid, granuclocyte, macrophage and primitive hematopoietic stem cells in a dose-related and timedependent fashion. AZT regimens are associated with significant bone marrow toxicity. In addition, long term treatment with AZT may create a selective pressure which affords replication advantage to viruses of drug-resistant phenotype. Such variants have been isolated from patients suffering from advanced HIV-1 associated disease, sometimes, as early as six months after initiation of treatment.

Heme or ferriprotoporphyrin IX is a red pigment comprised of four subunits called pyrroles; these subunits are chemically joined to form a single large tetrapyrrole (porphyrin) ring structure. A metal atom is chelated at the center of this porphyrin: in higher organisms this metal is iron and the porphyrin ring structure is called protoporphyrin IX. In physiological systems heme is bound to certain proteins; these hemoproteins bind oxygen at the site of the metal atom or they function as components of membrane bound electron transport systems. Cellular respiration, energy generation and chemical oxidations are dependent on these heme proteins. Hemin is the chloride salt of heme. Heme synthesis and heme degradation are critical to the maintenance of cellular heme homeostasis and hematopoietic differentiation.

Tsutsui and Mueller reported that the virionassociated RT activity of Rauscher murine leukemia virus was inhibited by hemin at a concentration of 100um. Hemin is the chloride of ferriprotoporphyrin IX (heme). The inhibition of RT by this large concentration of hemin was reversible and appeared to be directed against the enzyme rather than the template. On the other hand, hemin did not inhibit the activity of RT purified from avian myeloblastosis virus.

s Disclosure Of The Invention According to a first embodiment of this invention there is provided a method of treating a retroviral infection in mammals in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrine and mixtures thereof in an amount sufficient to effect such treatment.

According to a second embodiment of this invention there is provided a method of treating a retroviral infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic prophyrins and mixtures thereof in an amount sufficient to effect such treatment.

According to a third embodiment of this invention there is provided a method of treating a retroviral infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof together with an anti-HIV drug in amounts sufficient to effect such treatment.

According to a fourth embodiment of this invention there is provided a method of treating an HIV infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts, organometallic porphyrins and mixtures thereof together with AZT in amounts sufficient to effect such treatment.

According to a fifth embodiment of this invention there is provided a method of treating an HIV infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts, organometallic porphyrins and mixtures thereof together with AZT in amounts sufficient to effect such treatment and a sufficient amount of erythropoietin to reverse the adverse effects of AZT.

"Synthetic hemes" in this specification include a wide variety of organometallic porphyrins in which the chelated atom is a metal other than iron such as tin, chromium, of 4 cobalt, zinc or manganese or analogous compounds in which the porphyrin ring structure is modified as in protoporphyrins or mesoporphyrins. Typical synthetic hemes which might be mentioned by way of example are tin protoporphyrin (SnPP), tin mesoporphyrin (SnMP), tin diiododeuteroporphyrin (SnI 2 DP) and the corresponding zinc, chromium, manganese and cobalt compounds all of which are known or can be prepared by known procedures. All are useful in the practice of this invention. Tin protoporphyrin and tin mesoporphyrin are preferred because they are readily available and especially active.

Other heme derivatives useful in the practice of the invention include acid addition salts of heme, particularly amino acid salts of heme including L-amino acids such as arginine. heme arginate is especially preferred for use in the invention, although other non-toxic acid addition salts of inorganic and organic acids especially the naturally occurring L-amino acids may also be employed.

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(4 r j IN:\LIBUU00167:KEH Sof 4 WO 92/02242 PCT/US91/05283 6 Because of the aforesaid disadvantages of AZT and AZT type nucleosides in the treatment of retroviral infections, much time, effort and money has been expended to find replacement drugs, or at least drugs which will enhance the antiviral effects of the known therapeutic agents so as to permit lower doses, to reduce toxic sequellae or permit use of the therapeutic agents for longer periods.

BRIEF SUMMARY OF THE INVENTION It has now been discovered that heme, synthetic hemes and heme acid addition salts either alone or coadministered with anti-HIV drugs are useful for the treatment of AIDS. For convenience, heme, heme acid addition salts and synthetic hemes will sometimes hereinafter be referred to as "heme products." Heme products will enhance the activity of antiviral compounds especially antiretroviral compounds such as AZT. The invention is particularly useful with therapeutic agents of the nucleoside type such as AZT or dideoxyinosine (DDI). The invention also includes within its scope pharmaceutical compositions containing one or more heme products, an anti-HIV infection drug such as AZT together with erythropoietin (EPO). It has been observed that the combination of heme products and EPO is effective to reverse the adverse effects of the anti-HIV drug.

WO 92/02242 PCT/US91/05283 7 The invention therefore comprises methods and compositions for the treatment of retroviral infections of mammals especially humans in need of such treatment. The invention is especially applicable to controlling AIDS infections of humans.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1, 2, 3 4 and 5 illustrate by means of graphs the results of studies described herein utilizing hemin, AZT, and combinations of hemin and AZT, and combinations of these agents with EPO.

DETAILED DESCRIPTION OF THE INVENTION As used in this description, the term "coadministration" means that the therapeutic agents are administered simultaneously or sequentially at time intervals sufficiently close so that the administration of one agent has a beneficial effect on the action of the other. Normally, they will be administered parenterally in one dosage unit although the invention is not so limited. In fact, the one component of the therapeutic mixture, e.g. the nucleoside can be administered ora ly and the heme product parenterally.

For convenience, the invention will be described principally as it relates to heme, AZT and EPO, but as is clear from the foregoing, the invention is not limited to these materials.

WO 92/02242 PCT/US91/05283 8 A number of tests were conducted to determine the antiviral efficacy of heme alone or with various concentrations of AZT utilizing an AZT sensitive strain of HIV, an AZT resistant strain of HIV, both isolated from human patients, and AZT sensitive strain HTLV-III B obtained from ERC Bioservices Corporation.

Other tests were conducted to establish the ability of the combination of heme products and EPO to reverse the adverse effects of anti-retroviral drugs such as AZT.

Cells and viruses In this study of heme products coadministered with AZT a total of three strains of HIV-I were utilized. Two strains were isolated from blood obtained from AIDS patients. One isolate was obtained from a patient who had been on AZT therapy for four months; this isolate is defined as AZT-resistant (at luM AZT), in Table 1. The other isolate was derived from a patient who had never received AZT and is referred to as AZT-sensitive (at luM AZT) in Table 1. The third HIV strain (HTLV-IIIB) was obtained from ERC Bioservices Corporation (Rockville, MD) through the NIAID AEDS Research and Reference Reagent Program.

HTLV-IIIB 4) was replicated in the H9 cell line 6) (ERC Bioservices Corp.) The AZT-resistant patient-derived HIV was also adapted to the H9 cell line but the AZT-sensitive HIV (Table 1) could not be adapted WO 92/02242 PCT/US91/05283 9 to these cells. The two HIV strains were passaged in H9 cells in IMDM (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and subcultured at 3-4 days.

Infection of H9 with HIV H9 cells (0.5 x 106/ml) were infected with the tstrains of HIV (HTLV-IIIB and AZT-resistant patient isolate). Graded concentrations of hemin (Sigma, St.

Louis) (0-15.OuM) were added to the two groups of cultures. The cultures were maintained in RPMI 1640 media supplemented with 10% FBS at 37 0 C with replacement of media containing hemin twice during 7 days. An aliquot of was collected with an equal volume of FBS at day 7 and frozen at -70 C until assayed.

p24 assay Samples were assayed in duplicate for the HIV antigen, p24, with the Abbott HIV antigen detection system according to the Abbott protocol Control (200ul) or diluted sample (180ul) was added with 20 ul Triton X-100 to each well, followed by a polystyrene bead coated with human antibody to HTLV-IIIB. The assay plates were held at room temperature for 16-20 hours. After washing the beads with water, rabbit antibody to HTLV-III (200ul) was added to every well and the assay plates incubated at for 4 hours. After washing the beads, 200ul of goat anti-rabbit IgG conjugated with horseradish peroxidase was added to each well and the assay plates incubated at for two hours. After a final wash, the beads were WO 92/02242 PCT/US91/05283 10 transferred from assay plates to tubes and 300ul freshly prepared OPD (o-phenylenediamine.2HC1) substrate solution with 0.02% hydrogen peroxide added to each assay tube containing one bead. The assay tubes were incubated at room temperature in the dark for 30 minutes. One ml of IN sulfuric acid was added to each tube to stop the reaction.

The absorbance of controls and treated cultures was determined spectrophotometrically at 492nm.

Cultiviation of HIV in Peripheral Blood Lymphocytes (PBL) Human PBLs (0.5x106/ml) from healthy donors were infected in vitro with HIV isolated from two AIDS patients (Table AZT (Burroughs Wellcome) was added to the cultures at a final concentration of luM, while hemin was added at a final concentration of luM or 10uM in a total volume of 2ml. The cultures were maintained at 37 0 C with a replacement of 50% of the media containing AZT, hemin or a combination of AZT and hemin twice within 7 days.

Aliquots were collected with an equal volume of FBS on day 7 and frozen at -70 0 C until assayed.

Cell Viability Cell viability was assessed by the trypan blue exclusion method. In all experiments reported in this disclosure, cell viability was greater than 72%.

In the experiments shown in Table 1, AZT (luM) completely inhibited HIV replication in the drug-sensitive strain (Table 1, patient 1) on day 7 in in vitro cultures WO 92/02242 PCT/US91/05283 11 of PBL. Hemin did not alter the antiviral action of AZT when administered concurrently with the drug. Further, hemin alone (10uM) had a substantial inhibitory effect on viral replication. For the AZT-resistant HIV-isolate (Table 1, patient hemin alone or AZT, in the concentrations studied had no antiviral activity.

However, when a combination of AZT (luM) and hemin (luM or was used, HIV replication was inhibited completely.

To extend these observations, additional experiments with hemin and AZT were carried out to study HIV replication in the H9 cell line. In these experiments the AZT-resistant HIV from patient 2 was adapted to H9 cells.

HTLV-IIIB was also replicated in the H9 cell line and served as the AZT sensitive strain. It did not prove possible to adapt the AZT-sensitive HIV isolate from patient 1 to the H9 cells. H9 cells were infected with the two strains of HIV and graded amounts (0-luM) of AZT were added to the two groups of cultures. The cultures were maintained at 37 C in RPMI 1640 supplemented with FBS with replacement of 50% of media containing hemin (1 or 10uM) twice within 7 days. An aliquot of 0.3ml was collected and an equal volume of FBS added at day 7 and frozer. at -70 0 C until assayed. For the HTLV-IIIB AZTsensitive HIV, the 50% inhibition concentration (IC 50 of AZT was 0.008uM (Fig. Hemin alone at a concentration of 10uM, almost completely blocked replication (Fig. 1).

The addition of hemin with AZT reduced the IC50 of the drug markedly (Fig. because of the efficacy of hemin alone at the 10uM dose, augmentation of the virucidal effect of AZT could not be determined.

WO 92/02242 PCT/US91/05283 12 In the AZT-resistant HIV strain from patient 2, the of AZT alone in the H9 cells exceeded luM. Hemin alone at luM had no effect on virus replication; at a concentration of 10uM, substantial inhibition 40%) of virus growth was observed (Fig. 2).

The dose-response effect of hemin alone on the replication of HIV in H9 cells of both the AZT-resistant and the AZT-sensitive strains of HIV was also determined.

Viral replication of the drug sensitive strain was reduced by 50% at a hemin concentration of <0.05uM (Fig. for the drug-resistant strain the comparable inhibitory concentration of hemin was about These data indicate that hemin (10uM) alone was able to inhibit replication of an AZT-sensitive isolate in cultured PBL and that in combination with AZT, hemin in concentrations of luM or 10uM greatly enhanced AZT efficacy against a drug resistant HIV strain in such culture. Against both AZT-sensitive and AZT-resistant viral strains grown in H9 cells, hemin alone displayed virucidal properties and also augmented the antiviral actions of AZT against the AZT-sensitive viral isolates.

The doses of hemin required for these actions were markedly smaller than the dose of hemin required to inhibit virus-associated RT activity of Rauscher murine leukemia virus and are significantly less than those used in other mammalian cell systems where they have proved to be non-cytotoxic WO 92/02242 PCT/US91/05283 13 To determine the effects of a combination of heme and EPO on the adverse effects of AZT, normal human bone marrow hematopoietic colonies were grown in methyl cellulose cultures to establish the effects on erythroid colony growth according to the procedure of Iscove et al with 1.0 u/ml to 6u/ml EPO added (Epo Toyobo, Osaka Japan) for CFU-E and BFU-E. CFU-E growth was scored at 7 days and BFU-E was scored at 14 days. The cultures contained 0.1 um/L AZT and growth was observed with and without added hemin. The results are shown in Fig. As seen in Fig. 5, EPO alone at all concentrations tested was not able to overcome AZT's cytotoxic effect.

In all cultures without hemin, a similar degree of inhibition of CFU-E growth was obtained at all concentrations of Epo, and a greater degree of inhibition of BFU-E growth was obtained at all Epo concentrations greater than 1U/ml. On the other hand, hemin was able to protc CFU-E and BFU-E colony growth against the cyto-....ic effect of AZT. Addition of hemin (10 umol/L) to cultures suppressed by AZT (0.1 umol/L) increased CFU-E colony count by up to 279% and BFU-E count by 282%. The maximal stimulatory effect of hemln was in the presence of Epo 1 U/ml for CFU-E and 2 U/ml for BFU-E colony formation.

The preferred compositions of this invent on are designed for parenteral administration. The antiviral agent or agents will be in isotonic, aqueous, buffered solution, typically, made isotonic by the addition of sodium chloride, glucose or other standard solute. The WO 92/02242 PCT/US91/05283 14 compositions may also contain propylene glycol, sesame oil or other inert excipient. As suggested above, AZT and similar agents may be administered separately by the oral route.

The therapeutic compositions of the invention will contain a heme product and may additionally contain one or more of the compounds useful for the treatment of retroviral infections, especially HIV infections attributable to HIV 1 or HIV 2. The heme products and antiretroviral agents will be present in amounts effective usefully to treat a mammal such as a human afflicted with such infections. Anti-HIV nucleosides such as AZT and DDI are preferred. As shown above, the compositions may also contain EPO.

Useful dosage ranges for the active agents of the invention will vary with the selected agent, the age, weight, general physical condition of the patient, and other factors readily evaluated by the attending physician or veterinarian.

With the heme products of the invention, a typically useful dosage range whether or not another anti-HIV agent such as AZT is present will be from about 0.5 to 50 mg/kg body weight. The dosage range for the anti-HIV agent is of the same order of magnitude usually employed with these drugs although, because of the beneficial contribution of the heme products to the compositions, the concentration of the anti-HIV agent in a particular composition may be less than recommended when the agent is used alone.

WO 92/02242 PCT/US91/05283 15 To provide for treatments at such dosage levels, various dosage units will be provided. Typical dosage units may contain from 0.25 to 50 mg/kg of heme product whether or not used alone. The concentration of anti-HIV drug will be of the same order of magnitude as when used alone.

For AZT, the dosage level when administered with a hemin product of the invention will be from about 5 to 250 mg/kg body weight. Such levels may be conveniently achieved by providing parenteral dosage units containing from about 1 to 80 mg/kg of AZT.

If the composition of the invention contains EPO, the dosage range will normally be from 50 to 300 mg/kg body weight. Typically dosage units will contain from about 10 to 100 units/kg of EPO.

Heme arginate is preferred over heme for use in this invention because the acid addition salt is more water soluble than heme or hemin which is the form of heme usually utilized. Additionally, it can be administered intravenously without the complications associated with intravenous use of heme. Additionally, it is more stable than heme. Both products are normally from animal sources and must be purified by tedious and expensive procedures to reduce or eliminate undesirable contaminants, and to be certain the product is free of virus or other infectious organisms.

WO, 92/02242 PCT/US91/05283 16 The synthetic hemes are especially preferred for the practice of this invention not only because they are soluble at useful concentrations in the buffered, isotonic, aqueous compositions of the invention, and may be administered by any parenteral route including intravenously, but also for several other reasons. They may be synthesized chemically, are not subject to viral or other contamination if handled with ordinary care and, in contrast to the natural products, may be produced in lyophylized forms which are stable for years.

The compositions of this invention will be prepared by the usual procedures employed for such purposes utilizing standard readily available components.

Typically, the pH of the buffered compositions will be from about 7 to 8, preferably 7.4 to WO 92/02242 WO 9202242PCr/US91/05283 17 TABLE 1 HIV Antigen, P24, pg/mi Inhibition) Treatment AZT-sensitive AZT-resistant AZT-sensitive HIV Isolates AZT 0 11800 0 101000 (0.0) AZT lum 42 (100.0) 90000 (11.0) Hemin lum 104000 (12.0) 99500 Hemin 10um 35000 101500 (0.0) AZT lum Hemin lum 42 (100.0)* 1400 (99.0)* AZT linn Hemin 10um 29 (100.0)* 145 (100.0)* *p values 0.01 0.001 WO 92/02242 PCT/US91/05283 18

REFERENCES

1. Tsutsui, and G.C. Mueller. 1987. Hemin inhibits virion-associated reverse transcriptase of murire leukemia virus. Biochem. and Biophys. Res. Comm., 149:628.

2. Ho, T.R. Rota, R.T. Schooley, J.C. Kaplan, J.D.

Allan, J.E. Groopman, L. Resnick, D. Felsenstein, C.A. Andrews, and M.S. Hirsch. 1985. Isolation of HTLV-III from cerebrospinal fluid and neural tissues of patients with neurologic syndromes related to the acquired immunodeficiency syndrome. N. Engl. Jr.

Med., 313:1493.

3. Popovic, M.G. Sanrgadharan, E. Read, and R.C.

Gallo. 1984. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science, 224:497.

4. Gallo, S. Salahuddin, M. Popovic, G.M. Shearer, M. Kaplan, B.F. Haynes, T.J. Palken, R.Redfield, J.

Oleske, B. Sofai, G. White, P. Foster, and P.D.

Markham. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science, 224:500.

WO 92/0)2242 PCT/US91/05283 19 Popovic, E. Read-Connole, and R.C. Gallo. 1989.

T4 positive human neoplastic cell lines susceptible to and permissive for HTLV-III. Lancet, ii:1472.

6. Mann, S.J. O'Brien, D.A. Gilbert, Y. Reid, M.

Popovic, E. Read-Connole, R.C. Gallo, and A.F.

Gazdar. 1989. Origin of the HIV-susceptible human CD4 cell line H9. AIDS Res. and Human Retroviruses, 5:253.

7. Spivak, D.C. Barnes, E. Fuchs, and T.C. Quinn.

1989. Serum immunoreactive erythropoietin in HIVinfected patients. JAMA, 261:3104.

8. Yarchoan, R.W. Klecker, K.J. Weinhild, K.H.

Lyerly, E. Gelman, R.M. Blum, G.M. Shearer, H.

Mitsuya, J.M. Collins, C.E. Myers, P.D. Markham, D.T.

Durack, S.N. Lehrman, D.W. Barry, M.A. Fischl, R.C.

Gallo, D.P. Bolognesi, and S. Broder. 1986.

Administration of 3'-azido'3'-deoxythymidine, and inhibitor of HTLV-III/LAV replication to patients with AIDS or AIDS-related complex. Lancet, 1:575.

9. Brown, J.D. Lutton, J.C. Nelson, N.G. Abraham, and R.D. Levere. 1987. Microenvironmental cytokines and expression of erythroid heme metabolic enzymes.

Blood Cells, 13:123.

Iscove NN, Sieber F, Winterhalter KH. Erythroid colony formation in cultures of mouse and human bone marrow analysis of the requirement of erythropoietin WO 92/02242 PCT/US9 1/05283 20 by gel filtration and affinity chromatography on agarose concanavalin. An. J. Cell Physilo) 1974 ;83:309-316.

Claims (15)

1. A method of treating a retroviral infection in mammals in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof in an amount sufficient to effect such treatment.

2. A method of treating a retroviral infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts, organometallic porphyrins and mixtures thereof i an amount sufficient to effect such treatment.

3. A method of treating a retroviral infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts e e organometallic porphyrins and mixtures thereof together with an anti-HIV drug in amounts sufficient to effect such treatment.

4. A method of treating an HIV infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts, organometallic porphyrins and mixtures thereof together with AZT in amounts sufficient to effect such treatment. A method of treating an HIV infection in humans in need of such treatment which comprises parenteral administration of a buffered, isotonic, aqueous solution s containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts, organometallic porphyrins and mixtures thereof together with AZT in amounts sufficient to effect such treatment and a sufficient amount of erythropoietin to reverse the adverse effects of AZT.

6. The method as claimed in any one of claims 1 to 5, wherein the acid addition io salt is an arginate or a hydrochloride.

7. The method as claimed in claim 6, wherein the acid addition salt of heme is heme arginate.

8. The method as claimed in any one of claims 1 to 5, wherein the organometallic porphyrin is tin protoporphyrin.

9. The method as claimed in any one of claims 1 to 5, wherein the organometallic porphyrin is tin mesoporphyrin. The method as claimed in any one of claims 1 to 5, wherein the pharmaceutical agent is heme.

11. A pharmaceutical composition useful for treating a retroviral infection in mammals comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid IN:\UBUU]00157:KEH 22 of 4 WO 92/02242 PCT/US91/05283 23 addition salts of heme, organometallic porphyrins and mixtures thereof together with an anti-retroviral drug in an amount sufficient to effect such treatment. I\1a-. A pharmaceutical composition useful for treating a retroviral infection in humans comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof together with an anti-retroviral drug in an amount sufficient to effect such treatment. 13 A pharmaceutical composition useful for treating an HIV infection in humans comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof together with an anti-HIV drug in amounts sufficient to effect such treatment. I f. A pharmaceutical composition useful for treating an HIV infection in humans comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof together with AZT in amounts sufficient to effect such treatment. i34. A pharmaceutical composition useful for treating an HIV infection in humans comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent I selected from the group consisting of heme, acid addition salts of heme, organometallic porphyrins and mixtures thereof together with an anti-HIV drug and erythropoietin in amounts sufficient to effect such treatment.

16. A pharmaceutical composition useful for treating an HIV infection in humans s comprising a buffered, isotonic, aqueous solution containing a pharmaceutical agent selected from the group consisting of heme, amino acid addition salts of heme, organometallic porphyrins and mixtures thereof together with AZT and erythropoietin in amounts sufficient to effect such treatment.

17. The composition as claimed in any one of claims 11 to 16, o1 addition salt is an arginate or a hydrochloride.

18. The composition as claimed in claim 17, wherein the amino of heme is heme arginate.

19. The composition as claimed in any one of claims 11 to organometallic porphyrin is tin protoporphyrin.

20. The composition as claimed in any one of claims 11 to organometallic porphyrin is tin mesoporphyrin.

21. The composition as claimed in any one of claims 11 to wherein the acid acid addition salt 16, wherein the 16, wherein the 16, wherein the 16, wherein the pharmaceutical agent is heme. Dated 28 October, 1993 The Rockefeller University Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON QS s. 1;i IN:\buu)00167:KEH 24 of 4