IL87424A - Pharmaceutical compositions containing hypericin and/or pseudohypericin for treating retrovirus infections - Google Patents

Description

87424/2 t?i3 >o , 1 >o>->9 > >m-noD ΙΝ/Ί ) >O>-ID> >n o> on ηιηρΐ"» >-i>¾or> PHARMACEUTICAL COMPOSITIONS CONTAINING HYPERICIN AND/OR PSEUDOHYPERICIN, FOR TREATING RETROVIRUS INFECTIONS.

NEW YORK UNIVERSITY AND YEDA RESEARCH AND DEVELOPMENT CO.

C: 06998 I- IO O 87^24/2 PHARMACEUTICAL COMPOSITIONS CONTAINING HYPERICIN AND/OR PSEUDOHYPERICIN, FOR TREATING RETROVIRUS INFECTIONS.

The present invention relates to a pharmaceutical formulation for treating mammals suffering from an infection caused by a retrovirus, comprising a compound selected from hypericin, pseudohypericin and salts and mixtures thereof.

For many years the field of antiviral therapy has sought drugs that are capable of killing the invading pathogens without harming the host cell. The ability of viruses to physically invade a cell and to usurp the biochemical mechanisms of the cell in order to propagate their progeny, presents few unique biochemical features that can form the basis for selective inhibition of such viruses. Only a few compounds are known to possess selective antiviral activity. In particular, there are a wide variety of antiviral therapeutic agents, such as azidothymidine (AZT) , dideoxycytidine , acyclovir, ribavirin, and vibaridine, which owe their selective toxicity to the fact that they can inhibit viral functions more efficiently than they can inhibit cellular functions. In general, these agents are targeted against viral polymeraKes, phosp orylases, and nucleotide kinases. The use of these drugs iu limited due to their narrow spectrum of antiviral activity and their toxic side effects whan administered systemically to a host organism over long, periods of timei Interferonn ore antiviral polypeptides which tore currently in experimental therapoutlc use in humans'. However, their therapeutic value appears limited at the present time.

The production and purification of h man interferons require tedious procedures, the available quantities are limited and cytotoxic effects are also known to occur.

Recently it has become of great importance to find agents that are active against retroviruses, and in particular Human Immunodef ciency virus (HIV) which is responsible for Acquired Immune Deficiency Syndrome.

Therefore, the art is- constantly seeking new antiviral ge tii and in particular agents, which display high virus killing power with low cellular toxicity and are effective against retroviruses which have proven to be resistant to many conventional anti-viral agents.

. . . ■QDJECTS _0_F THE INVENTION ft is an o jec of the present invention to provide therapeuti agents having anti retrovi ral activity and low ce11 u.1 ar to:: i ci ty , Λ further object of the present invention is to provide pharmaceutical formulations for treating mammals suffering from retrovirus infections.

SUMMA Y. OF THE INVENTION It has now been unexpectedly discovered that certain aromatic poiycyclic dione compounds, exemplified by hypericin and pseudohypericin which are present in plants of the Family Hy e icum (St. Johnswort) and which have been isolated from the plant Hype cum, tr quetri.fol.ium, are effective against retroviruses including, for example, Friend Leukemia Virus (FV),' Radiation Leukemia Virus (RadLV) and human immunodef ciency virus (HIV, also known as HTLV III) .

T e presen invention provides pharmaceutical formulu- tions for treatiny mammals suffering from diseases caused by. retroviruses comprising an amount effective to inhibit such viruses of hypericin, pseudohypericin or mixtures thereof. The formulations ma also include physiologically-acceptable carriers and salts.

These and other aspects of the present invention will be apparent to thDse of ordinary skill in the art in light of the present description, accompanying claims and the appended drawings.

■ PRIEF" DESCRIPTION" F THE, DRAWINGS' " Figure .1 is a diagram showing the chemical structure of hypericin.

Figure 2 is a diagram showing the chemical structure of pseudohypericin.

It has now been unexpectedly discovered that certain aromatic polycyclic diones.. namely· hypericin and pseudohypericin are antiviral agents that are highly active against retroviruses. Th¾ anti-retroviral agents 'are compounds which have been isolated from the perennial, plant Hype i.cum, rj: guetri fol ίυτ .

As used herein, the term retrovirus refers to viruses containing an RNA genome and RNA-dependent DNA polymerase (reverse transcriptase) enzy ic activity. All retroviruses have common morphological, biochemical and physical properties that justify their inclusion into a single virus family. These parameters are summarized in Table A below.

TABLE A GENERAL PHYSICAL PROPERTIES OF KNOWN RETROVIRUSES Nucleic acid linear positive-sense single-stranded RNA (60S-70S) composed of identical subunits (30S-35S); 5' structure (m7G5ppp5NmpNp) ; poly denylated 3' end; repeated sequences at 3' and 5' ends? tRNA base-paired to genome complex Protein about 60% by weight; gag, internal structural proteins; pol , reverse transcriptase; env, envelope proteins Lipid about 35% by weight; derived from cell membrane Carbohydrate about 4% by weight; associated with envelope proteins Physiochemical density 1.16-1.18 g/ml in sucrose, 1.16-1.21 properties g/nil in cesium chloride; sensitive to lipid solvents, detergents, and heat inactivation (56*C, 30 min) ; highly resistant to UV- and X-irradiation Morphology spherical enveloped virions (80-120-nm diameter), variable surface projections (8-nm .... . diameter), icosahedral capsid containing a ribonucleoprotein complex with a core shell (nucleoid) All retroviruses have similar overall chemical compositions. In general, they comprise about 60-70% protein, 30-40% lipid, 2-4% carbohydrate, and about 1% RNA. The envelope of retroviral particles is derived from the cell-surface membrane, and most, if not all, of the lipids in viral particles are located in the unit-membrane envelope of the virion. Non limiting examples of retroviruses include Friend Leukemia Virus (FV) , Radiation Leukemia Virus (RadLV) and Human Immunodeficiency Virus (HIV) . 1 It has now been surprisingly discovered that the aromatic polycyclic dione compounds of the present invention inhibit the replication of Friend Leukemia virus and Radiation Leukemia virus, both in vivo and in vitro in mice. Even more surprisingly, this inhibition occurs without any significant toxicity to the recipient mammals, as shown in the examples below, wherein liver function assays, such as the levels of the enzymes lactate dehydrogenase (LDH) and serum glutamyl aminotransferase (SGOT) and those of bilirubin, which were increased due to FV infection (and are associated with the pathogenic effects of this virus) were returned to near normal values after administration of the antiviral compounds of the present invention.

The Merck Index (10th Ed. p. 710) discloses that hypericin is an antidepressant and that 'ί-small quantities appear to have a tonic and tranquil iz ing effect on the human organism." In addition, hypericin has been said to produce photosensitivity upon ingestion; Oxford, Raistick Biochem J . 34 , 790 (1940) .

Hypericin and pseudohypericin both display antiviral activity when administered to mice after retroviral infection. Hypericin was effective in inhibiting RadLV infection and pseudohypericin showed inhibitory activity against FV infection in mice as shown in Example II below. Moreover, a 50:50 mixture of hypericin and pseudohypericin led to an almost complete inhibition of the reverse transcriptase enzyme activity of RADLV-infected mouse cells in culture, as shown in Example 2 below. The same level of inhibition can be achieved by administering either hypericin or pseudohypericin alone.

The aromatic polycyclic diones appear to be capable of crossing the blood-brain barrier, as they have been previously administered as tranquil izing agents to humans. This is significant because, in the case of HIV, infection of the brain and central nervous system has been observed in infected individuals. It is also known that HIV can infect cells of the brain and central nervous system. Indeed, it has been recently found that gpl20, a virally encoded polypeptide, is directly cytotoxic to cells of neuronal origin. Therefore, treatment of mammals suffering from diseases caused by these viral agents is a most important application of the present invention.

Hypericin and pseudohypericin, the aromatic polycyclic diones of the present invention can be used in treating mammals suffering from infections caused by retroviruses. These antiviral dione compounds are preferably obtained by extracting them from plants of the species Hypericum as detailed in Example 1 below, or alternatively may be chemically synthesized using the methods of Brockmann, M. et al, U.S. Patent No. 2,707,704, issued May 3, 1955 and of Brockmann, H. et al Tetrahedron Letters 2.2.: 1991-199 , 1974, both incorporated herein by reference. Due to the wide distribution and availability of the St. Johnswort plant throughout the world and the relatively convenient and inexpensive procedure for the extraction and purification of the compounds of the present formulation (as detailed in Example I below) , the extraction procedure is preferred when small amounts (i.e. grams) are desired. However, for the production of large scale amounts (kilograms and greater) chemical synthesis is preferred.

Hypericum is a genus -from the family Guttifereae . It has been related also to the families Hypericaceae and Clusiaceae . The genus is geographically fairly widely distributed. It is known for its content of ether-containing oils, and for the occurrence in small glands of red fluorescent pigments, .represented among others by the aromatic polycyclic structural compounds hypericin and pseudohypericin (Figure 1) . Plants of the genus Hypericum have been reported to grow in very wide areas in Middle and Eastern Europe, Asia, North and South Africa. Additionally, there are reports of its occurrence in certain parts of the American continent, Australia and New Zealand. The plant is indexed in The Audubon Society Field Guide to North American Wild Flowers, Eastern Region, pg. 558, Chanticleer Press, Inc., NY, the disclosure of which is incorporated by reference.

The aromatic polycyclic diones of the present invention can be utilized for the treatment of mammals suffering from diseases caused by retroviruses such a's Acquired Immune Deficiency Syndrome (AIDS) . Due to their potency and lack of cellular toxicity, the aromatic polycyclic diones of the invention will be particularly useful as specific antiviral therapeutic agents for these disorders. Currently there are no broad spectrum agents available to treat these retroviral .

The present invention further includes salts of hypericin or pseudohypericin which retain their anti-viral activity. Salts in which the base is of the alkaline or amine type are particularly comprehended within the scope of the present invention.

When treating mammals suffering from infections caused by retroviruses according to the present invention, the determination of the most effective compound (or mixtures thereof) for treatment of the particular retrovirus responsible for the infection can be ascertained by routine experimentation using suitable in vitro systems, such as that described in Example IV below for human immunodeficiency virus or in Example II for FV and Radiation Leukemia Virus (RadLV) in experimental animals .

When employed to treat AIDS, vire ia (i.e. the presence of virus in the blood stream) or sepsis (viral contamination of bodily fluids) caused by retroviruses, the aromatic polycyclic dione compounds of the present invention may be administered orally, topically or preferably parenterally, and most preferably by intravenous administration of dosages broadly ranging between about 200 micrograms and about 12,000 micrograms per kilogram (kg) of body weight per treatment and preferably between about 400 and about 4,000 micrograms per kg body weight per treatment. The duration and number of doses or treatments required to control a patient's disease will vary from individual to individual, depending upon the severity and stage of the illness and the patients condition. A typical treatment may comprise intravenous administration of from about 1000 to about 3000 micrograms per kg of body weight of one or more of the compounds of the present invention. The total dose required for each treatment may be administered in divided doses or in a single dosage. The antiviral treatment may be administered one or two times a week, or more, as determined by the patients condition and the stage of the patients disease.

The aromatic polycyclic dione compounds of the present invention can be incorporated in conventional, solid and liquid pharmaceutical formulations (e.g. tablets, capsules, caplets, injectable and orally adroinisterable solutions) for use in treating mammals that are afflicted with enveloped virus infections. The pharmaceutical formulations of the invention comprise an effective antiviral amount of the aromatic polycyclic diones of the present invention (as disclosed above) , or mixtures thereof as individual components as at least one of the active ingredients. The quantity of effective dose supplied by each capsule, tablet or injection is relatively unimportant since the total dosage can be reached by administration of one or a plurality of capsules, tablets, injections or a combination thereof. In addition, such formulation may comprise inert constituents including phar- maceutically-acceptable carriers, diluents,- fillers, salts and other materials well,-known in the art depending upon the dosage form utilized. The capsules · employed may be comprised of any pharmaceutically acceptable material, such as gelatin or cellulose derivatives. The tablets may be formulated in accordance with conventional procedure employing solid carriers well known in the art. Examples of solid carriers include starch, sugar, bentonite and other commonly used carriers. For example, a preferred parenteral dosage form may comprise a sterile isotonic saline solution, containing between about 0.2 (200 micrograms) milligrams per kilogram of body weight and about 12 milligrams (12,000 micrograms) per kilogram of body weight of one or more of the antiret oviral compounds, including mixtures thereof. Propylene glycol, benzyl alcohol, ethanol or other biologically acceptable organic solvents may be used as diluents, carriers or solvents in the preparing solid and liquid pharmaceutical formulations containing the anti-retroviral compounds .

Hypericin and pseudohypericin may be administered separately, or combined (mixed) in a single dosage form, or coadministered at the same time. Selection of the particular agent to be employed will be determined by ascertaining the antiviral compound or mixture that works best for a particular patient.

The compounds in the formulations of" the present invention may e ideally-suited for co-administration with other agents such ,as immune system cells, factors such as T-cells or interleuKin-2 , cytotoxic agents, lymphokines suc as interferons or the like, that are known to have some effectiveness against retroviruses.

The present invention is described below in specific working examples which are intended to illustrate the invention without limiting the scope thereof.

EXAMPLE I; EXTRACTION OF HYPERICIN AND PSEUDOHYPERICIN FROM ST. JOHNS ORT .

Hypericin, (C30H1608, molecular weight 504.43) referred to herein as Hy, and pseudohypericin (C30H16°9 referred to herein as Ps, molecular weight 520.43) were obtained as detailed below.

The herb of the whole St. Johnswort plant was harvested at its flowering time, (July throuyh October in the Eastern hemisphere) , dried at 55*C, cut and milled, and then extracted with acetone (about 5-10 liters β5£αο¾¾.· 0ne kilogram of the -material was plac d in ..a .Soxhlet /(Kimax, available from Fisher Scientific, New Brunswick, NJ) and extracted until the extracting solvent was colorless (about · five to ten hours). The solution containing the aromatic polycyclic diones had a red fluorescent color with absorption and fluorescence spectra as described in Scheibe, Schentaq. Ber . 75; 2019, 1942, BrocJanann, M. , Natureweiss 38: 47, 1951, both incorporated herein by reference.

The solvent (containing the aromatic polycyclic diones) was evaporated under reduced pressure to complete dryness of the residue, yielding 95 grams. This residue was then further fractionated on a chromatographic column, packed with silica gel 60 (0.06-0. 0mm, Malinckrodt, American Scientific Products, McGaw Park, IL) . A dry chromatographic procedure was utilized whereby 25 grams of the above obtained residue was dissolved in about 500 ml acetone, added to an* equal amount of silica gel 60, and evaporated on a jRotavapor (Buchi, American Scientific Products) with swirling until the mixture was homogeneous and dry. The mixture was then placed on top of a column containing one kilogram of silica gel 60 and eluted with chloroform until the solvent reached the bottom of the co.lumn. This was followed by washing the column with a solvent mixture compris-ing chloroform-acetone-methanol , 75:15:10 (Vol/Vol/Vol). When the red color became. 1/5 of the original intensity, the concentration of chloroform was reduced and the column eluted with a solvent mixture comprising chloroform-acetone-methanol in the ratio of 55:15:10 respectively (Vol/Vol/Vol) . Fractions of 250ml were collected. Each fraction was monitored on thin layer chromatoplates (VWR, San Francisco, CA}' and the Rf value of the two main red fluorescent spots under ultraviolet light at 254nm was determined. The developing solvent mixture was chloroform-acetone-methanol , 55:15:10 as above. The chromatog-raphy was completed in about two days.

Further purification and separation of the two main components was obtained by two rounds of flash chromatography using a silica gel 60 (mesh 0.04-0.06) under pressure as described in Gas Chromatography. Princip es , Techniques and Appl ications . A.B. Littlewood, ed . Academic Press. New York 1970, incorporated herein by reference.

Two main components were identified: hypericin (Hy) , R 0.45, in a yield of 0.19 grams; and pseudohypericin (Ps) , Rf 0.35, in a yield of 0.73 grams. The NMR spectrum analysis of the two components were the same as those previously reported (Brockmann, H, et al, Tetrahedron Letters 1:37, 1975, incorporated by reference) .

The compounds were stored at 4°C in absolute ethanol, in the dark, until use.

EXAMPLE II: ANTIVIRAL ACTIVITY IN MAMMALS The effects of the compounds of the present invention on the infection of mammals with the retroviruses Friend Leukemia Virus (FV) and Radiation Leukemia Virus (RadLV) was examined as follows.

( ) Friend Leukemia Virus Infection Friend Leukemia virus (FV) an aggressive retrovirus induces an acute erythroleukemia m sensitive strains of mice such as BALB/c and NIH SWISS mice (as described in Friend, C. J- Exp. Med. 105 : 307-324, 1957; Friend, C. et al Nat. Cancer Inst . Mon.ogr. 22.: 505-522, 1966; Friend, C. et al Proc . Natl . Acad . Sci . U.S.A. 68.: 378-383, 1971, all incorporated by reference herein) . The malignant transformation is the result of the combined activities of the spleen focus forming virus (SFFV) and the ecotropic murine Friend Leukemia helper virus (F-MuLv) . The acute erythroleukemia is characterized by hepatosplenoinegaly (a marked increase in the size of the spleen and liver) and a severe anemia.

Friend Leukemia virus was prepared by homogenizing the enlarged spleen of a mouse previously infected with FV, 10 days after intravenous virus injection. The spleen was homogenized in phosphate buffered saline in a volume equal to 10 times the weight of the isolated spleen.

The effects of Hy and Ps on the increase in spleen size (splenomegaly) of BALB/c mice (Jackson Labs, Bar Harbor, ME) was examined. In these experiments, the virus (10^ focus forming units - FFU) was inoculated intravenously, and the indicated doses of the antiviral compounds of this invention were administered to the BALB/c mice intraperitoneally 24 hours later. The animals were then sacrificed ten days later and their spleens weighed. The results are summarized in Table 1 below.

TABLE 1 The effect of administering compound Ps (diluted in phosphate buffered saline with 1% ethanol) on the splenomegaly in BALB/c mice following inoculation with Friend erythroleukemia virus.

Control Mice FV inoculated Mice (PBS) (106 FFU) 2094 Spleen weight (gms) 1.0272 Spleen weight (gms) 1834 Spleen weight (g s) 0.9596 Spleen weight (gms) 1790 Spleen weight (gms) 1.2432 Spleen weight (gms) 1669 Spleen weight (gms) _ 1.1174 Spleen weight (gms) +0.0178 x=l.0865+0.1226 Net change from control=0.9019 (cont'd) Friend Virus Friend Virus (106 FFU) (106 FFU)+ + PS 80 meg/mouse 2 injections PS 80 meg/mouse 0.2831 Spl-een weight (gms) 0.2457 Spleen weight (gms) 0.2761 Spleen weight (gms) 0.3400 Spleen weight (gms) 0.2215 Spleen weight (gms) 0.2938 Spleen weight (gms) _ 0.1810 Spleen weight (gms) _ 0.1956 Spleen weight (gms) x=0.2404+0.0482 X=0.2687+0.0621 Net change from control=0.0558 Net change from control=0.08 1 % Inhib=93.82 % Inhib=90.70 Control Mice Friend Mice (PBS) (2xl05 FFU) 2094 Spleen weight '('ijjms) 0.8911 Spleen weight (gms) 1834 Spleen weight ( ms^ 0.9211 Spleen weight (gms) 1790 Spleen weigJrt (gms) 0.8004 Spleen weight (gms) 1669 Spleen weight (gms) _ 0.8662 Spleen weight (gms) X=0.1846+0.0178 .x=0.8697+0.0513 Net change from control=0.6851 Friend Virus (2x10= FFU) Friend Virus (2x10= FFU) + PS 80 meg/mouse 2 inject PS 80 meg/mouse 0.3457 Spleen weight (gms) iC4924 Spleen weight (gms) 0.2784 Spleen weight (gms) i0.2469 Spleen weight (gms) 0.2208 Spleen weight (gms) 0.2722 Spleen weight (gms) _ 0.1791 Spleen weight (gms) 0.2438 Spleen weight (gms) x=0.2560+0.0723 138+0.1197 Net change from contxol=0.©714 Net change from control=0.1292 % Inhib=89.58 % nhib=81.15 The data in Table 1 show the inhibition of splenomegaly, with median inhibition of 53-8%, following a single injection of 80 micrograms per jnoikse of Ps. A median inhibition of 89.6% in spleen enlargement was observed when 80 micrograms per mouse of "Fs. was administered in a single injection to mice that had previously been inoculated with 0.5ml of the virus preparation (corresponding to 2xl05 FFU of virus) . When two daily consecutive injections of Ps, each comprising 80 micrograms per mouse of the compound were administered, the median inhibition of splenomegaly was 90.7% with a viral preparation containing 106 FFU and 81.7% with a viral preparation containing 2xl05 FFU (Table 1) .

The above results show a marked decrease in the malignant transformational capacity of the Friend Leukemia Virus (as measured by decreased splenomegaly) following the intraperitoneally administration of Ps 24 hours after infection. (2) Co-administration with Friend Leukemia Virus A different experimental design was also tested involving the simultaneous intravenous co-administration of Ps with the FV complex. In this case, the viral preparation was mixed with Ps at various concentrations and the mixture was injected into the mouse tail vein in a final volume of 0.5ml. The mice were sacrificed ten days later, the spleens weighed and the level of inhibition of splenomegaly subsequently determined. The results are summarized in Table 2.

TABLE 2 The effect of intravenous co-administration of pseudo-hypericin (diluted in PBS with 1% EtOH) with FV, on viral-induced splenomegaly.

Spleen Weights (grams) Controls Exptl Expt2 Expt3 PBS PBS + l¾EtOH FV FV+PS 5mcq FV+PS 20 meg FV+PS 50mcg 0.1304 0.1862 1.1499 0.3425 0.1655 0.1830 0.1490 0.1567 1.0657 0.3766 0.1426 0.1674 0.1362 0.1386 0.9597 0.4005 0.1433 0.1422 0.1515 1.1347 0. 255 0.1966 0.1365 x=0.1417 X=0.1605 X=l.0774 X=0.3862 X=0.1614 X=0.1572 ±0.0101 +0.0240 +0.0866 +0.0353 +0.0253 +0.0217 % inhibition as compared to the group receiving Ps in PBS + 1% EtOH = 75.44% 100% 100% As shown in Table 2 above, 100% inhibition of splenomegaly was found when Ps was administered with the viral complex at concentrations of 20 micrograms per mouse and 50 micrograms per mouse (average mouse weight approximately 150 grams). A mean inhibition of 75.44% was found when 5 B7424/3 micrograms per mouse was co-administered with the virus.

These results show the effectiveness of the anti-retroviral compounds in the present formulations, in that as little as 5 micrograms per mouse was effective in inhibiting viral transformation by this aggressive RNA tumor virus. (3) Effect of ¾ on liver function of FV infected animals As a further demonstration of the antiviral activity of Ps, the effect of FV infection, in the presence and absence of Ps, on liver functions was monitored by analyzing the serum of infected mice for liver-associated proteins.

BALB/c mice were inoculated with FV at a concentration of 2 x 10^ FFU. Each group of animals contained 4 mice, and analyses were done in pooled aliquots.

TABLE 2 EFFECT OF FRIEND VIRUS WITH/WITHOUT PS (IN PBS *l% EtOH) ON LIVER FUNCTIONS Total CHOLEST PROTEIN ALBUMIN BU AP LDH SGOT SGPT GOT mg/dl gm/dl gm/dl mg/dl IU/1 IU/1 IU/1 IU/1 miu/ml PBS 122 5-7 3-3 0.2 226 1075 132 10 3 IVFV 118 5.5 3.4 0.4 200 6710 4 0 110 4 IVFVIPPS 109 5.7 3.4 G.l 226 3303 213 99 ND IPFV 111 -4 3-3 0.2 223 . 3330 24 100 3 IPFVIVPS 137 5.5 3.2 0.3 200 2305 244 135 4 PBS ■ phosphate buffered saline IVFV ■ intravenous administration of Friend Virus IVFVIPPS * intravenous administration of Friend Virus, intraperitoneal injection of Ps compound IPFV = intraperitoneal administration of Friend Virus IPFVIVPS - intraperitoneal administration of Friend Virus, intravenous injection of Ps compound In Table 3, BU = Total Bilirubin, AP = Alkaline phosphatase, LDH = Lactate dehydrogenase, SGOT = Serum glutamyl aminotransferase, SGPT = Serum glutamyl peptidyltransferase , and GGT = Gamma-glutamyl-transpeptidase .

As shown in Table 3, intravenous inoculation with FV led to an increase in LDH, SGOT and SGPT enzyme activity and to an increased total serum bilirubin accumulation in infected mice. Such increases are indicators of liver function damage, well-known in the art. Administration of Ps, both intravenous-ly and intraperitoneally, led to a reduction in these virally-induced increases. ( 4 ) Antiviral Effect on Radiation Leukemia Virus Infection (RadLV) The intrathymic inoculation of murine radiation leukemia virus in susceptible strains of mice results in the malignant transformation of the thymocytes and the development of overt leukemia (as described in Meruelo, D. et al J. Exp . Med . 147 ; 470-487, 1978, incorporated herein by reference). This transformation has been shown to be associated with the major histocompatibility complex (MHC) which determines resistance or susceptibility to the leukemogenic effects of the virus. Among the early events which follow the viral infection in some mouse strains is a dramatic increase in the cell surface expression of class I H-2 antigens. This increase is then followed by a reduction in the expression of these antigens after the malignant transformation and subsequent development of leukemia. The changes in H-2 antigen expression have been utilized to monitor viral infectivity and to determine the effects of Hy and Ps on the level of infectivity of this virus.

Murine radiation leukemia virus (RadLV) was inoculated intrathymically into B10.T(6R) mice as described in Meruelo et al (supra) . Groups of six mice were treated 24 hours later with 30 micrograms per mouse of compound Hy administered intraperitoneally in isotonic saline solution. The mice were sacrificed ten days later and the expression of Class I H-2 antigens determined by analysis with a fluorescence activated 87424/2 cell sorter {Ortho Cytofluorograph Model 50H, Ortho Diagnostic Systems. Westwood, MA) (FACS) , using X-56 anti-H-2D^ mouse monospecific antibodies (as described in Meruelo, et al supra; similar antibodies are available as ATCC No. HB75. American Type Culture Collection, Rockville, MD) . The results are shown in Table 4 below.

TABLE 4 EFFECT OF HY AND PS ON THE INCREASE IN H-2 ANTIGEN EXPRESSION IN THYMOCYTES AFTER INTRATHYMIC INOCULATION OF MOUSE RADIATION LEUKEMIA VIRUS IN B10.T(6R) MICE.

Base line Virus induced 30mcg/mouse Hy 30mcg/mouse PS (untreated mice) (inoculated mice) (inoculated with RadLV and Hy) % of mean % of mean % of mean % of mean cells fluorescent cells fluorescent cells fluorescent cells fluorescent stained intensity stained intensity stained intensity stained intensity with in with in with in with in antibody fluorescent antibody fluorescent antibody fluorescent antibody fluorescent units units units units 9 .I (606.8) 97.3 (752.9) 76.7 (625.5) 97.4 (713.3) 76.0 ( 1 .7) 91.7 (599.0) 77-2 (596.9) 94.3 (639) 45.1 (440.3) 92.5 (599.6) 96.3 (723.6) 76.Ο (518.7) 94.0 (703.1) 84.8 (663.5) 89-5 (596.7) 96.3 (729.2) 54.0 (513.6) 87.4 (558.8) 97-3 (719.9) 97.8 (657-3) x = 78.02 x = 94.85 x = 81.13 {treated 30mcg - base line } (1) % inhibition = 100- { } x 100 = 82% {virus induced - base line } As can be seen from the data in Table 4, Hy exerted a significant inhibition in the increase in H-2 expression in four of the six mice tested (Table 4), indicating a significant reduction in the effective capacity of the virus following the intraperitoneal administration of compound Hy. This occurred as late as 24 hours after the initial viral inoculation. No effects on H-2 expression induced by RadLV were found with compound Ps. 8742 1 EXAMPLE III: EFFECTS OF PS AND HY ON RNA-DEPENDENT DNA POLYMERASE (REVERSE TRANSCRIPTASE) Retroviruses (RNA tumor viruses) carry within the virion the reverse transcriptase enzyme which, upon infection 16a 087424/2 17 of the cell with the virus, recruits the cellular synthetic machinery and transcribes a complementary DMA (cDNA) copy of the virion R11A using the viral RHA genome as a template. The determination of levels of reverse transcriptase (RT) activity in the growth medium of cells infected with retroviruses is a well-known method with which to evaluate the release of infectious virus particles by cells and has been used to measure the antiviral activity of the components of the present invention. The effect of a mixture of both Hy and Ps at a concentration of 10 micrograms per ml (of each compound) on the RadLV RT activity was examined.

The mixture of the two compounds was administered 2, 4 and 16. hours post-infection and the supernatants were harvested and assayed for RT activity according to the method of Stephenson et al, Virology 4_8 : 749-756, 1972, and Weissbach et al, J. Virol . 10: 321-327, 1972, both references incorporated herein by reference .

T nr.r.ny ,'ns performed ns follows: a ) Preparation of Virus RadLV-infected lymphoblastoid cells, line B10.T(6R) were pelleted by centri fugation at 4"C, 3500 rpm for 15 minutes. The top 2/3 of the supernatant (10ml) was removed for assay. The supernatant was then centrifuged at 40,000 rpm for 1 hour at 4"C in 12ml ultracentrifuge tubes, using a fixed-angle ultracentrifuge rotor (Ti70 rotor, Beckman Instruments, Fullerton, CA) · The supernatant was carefully decanted and the tu ¾s dried. The pellet was resuspended in 200 microliters of a buffer containing 0.02M Tris/HCl, pH 7.8, 0.1 M NaCl, 0.001M dithiothreitol , and 0.2% Triton X-100. The mixture of the buffer and the virus-containing pellet was vortexed and incubated on ice for 30 minutes before use.

The reverse transcriptase assay was performed in a volume of 100 microliters containing components as shown in Table 5. 87424/3 TABLE 5 Reagent Stock μΐ of Stock final concentration per assay per assay (100 μΐ) solution A: 0.5M Tris/HCl pH 7.8 (37°) 50 mM G.6M C1 10 μΐ 60 mM solution B: 2.0 mM Mn acetate 10 μΐ 0.2 mM solution C: 40 mM Dithiothreitol (DTT) 5 μΐ 2 mM Triton X-100 (10#) 1 μΐ 0.1% poly(rA).(aT)12 (10 A260 U/ml)1 Μ μΐ 0.4 A26Q/ml (20 meg/ml) dTTP (2 x lO*4 H) ~~ 10 μΐ 2 x 10"5 M ^H-dTTP (500 uCi/ml)2 10 μΐ 5 yCi 2500 Ci/ml 50 μΐ obtained from Pharmacia Fine Chemicals, Piscataway, NJ obtained from New England Nuclear, Boston, MA 50 microliters of the reaction mixture was mixed with 0 microliters of the above obtained supernatant and incubated for 1 hour at 30°C, The reaction was stopped by the addition of 0.1 ml of 0.06 M sodium pyrophosphate, vortexed and incubated on ice. 2 ml of 10% trichloroacetic acid (TCA) containing 0.3 M sodium pyrophosphate wasiiadded, .'the mixture vortexed and incubated on ice -for 10—20 minutes. TCA-precipitable radioactiv ty was determined after -filtration of samples using glass fiber filters < 2. cm Whatman GFC, Whatman, Cliftons NJ) « These results are shown in Figure 2.

The data in Figure 2 show that incubation of virally-infected cells with the 50: 0 mixture of Hy and Ps led to at least a 50% decrease in the detectable RT activity at 2 and 4 hours post-infection and further inhibited this enzyme approximately 15% when assayed 16 hours postinfection. 18 87^24/1 EXAMPLE IV; INHIBITION OF HIV BY THE ANTI-RETROVIRAL COMPOUNDS OF THE PRESENT FORMULATIONS The activity of Ps, Hy, and mixtures thereof against human immunodeficiency virus (HIV) may be investigated in the following manner. HIV-infected, 0KT4+ lymphoblastoid cells, such as clone H9 (described by Popovic, M., et al, Science, 224 : 497-500 (1984 ) , incorporated by reference) is maintained in RPMI-1640 medium (GIBCO, Grand Island, New York) containing 20$ fetal calf serum (Flow Laboratories, Inglewood, CA) . Triplicate cultures of cells, seeded at a concentration of about 4 x io5 cells per ml, are exposed to polybrene (2 micrograms per 18a ml, Sigma Chemical Co., St. Louis, MO), infected with 2xl08 HTLV ΪΙΙ particles per 4xl05 H9 cells, and cultured in the presence or absence of Ps, Hy and mixtures thereof as in Example 2 above.

The antiviral activity of the compounds of the present invention is determined by monitoring the reverse transcriptase activity and the expression of HIV proteins p24 and pl7, as described in Sarin, P.S. et al, J. Nat. Cancer Inst. 78 ; 663- 665, 1987, incorporated herein by reference.

EXAMPLE V: Expression of HTLV III gacr proteins p24 and pl7.

H9 cells (2xl05) , either uninfected or HTLV III infected, are continuously exposed to various concentrations of Ps, Hy and mixtures thereof at concentrations between 5 and 100 micrograms per ml for 4 days. . The percentage of cells expressing p24 and pl7 gag proteins of HTLV III is determined by indirect immunofluorescence. icroscopy with the use of mouse monoclonal antibodies to HTLV-III pl7 and p24 (available in numerous commercial sources such as those in HIV serum antigen detection kits from Abbott Labs, North Chicago, IL, and from Du Pont,...Wilmington, DE) . ..The positive cells are visualized by treatment with fluorescein-labeled goat anti-mouse IgG (Cappell Laboratories, Cochranville, PA) . The experiments are performed in duplicate (at least three times) .

EXAMPLE VI; Determination of reverse transcriptase activity Uninfected H9 or H9 cells infected with HTLV-III (500 virus particles/cell) are exposed to various concentrations of Ps, Hy and mixtures thereof as above. At day 4, supernatants of the cultures are collected and virus particles are precipitated with polyethylene glycol and obtained by centrifugation as described above in Example 2 for FV. The virus pellet is suspended in 300 microliters of buffer containing 50 mM Tris-HCl (pH 7.5), 5 iuM dithiothreitol , 250 mM KCl, and 0.25% Triton X-100. Reverse transcriptase activity in these samples is analyzed in a 50-jjl reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5mM dithiothreitol, 100 mM KCl, 0.01% Triton X-100, 10 μΐ dTlsrAn as template primer, 10 mM MgCl2, 15 »M [3H]dTTP (New England Nuclear, Boston, MA), and 10 microliters of disrupted virus suspension. After incubation for 1 hour at 37 "C and subsequent addition of 50 micrograms of yeast tRNA (Sigma Chemical, St. Louis, MO), the incorporation into cold trichloroacetic acid-insoluble fraction is assayed as in Example 2 above. Assays are performed in duplicate and repeated three times.

Claims (9)

1. A pharmaceutical formulation for treating mammals suffering from an infection caused by a retrovirus, comprising an effective amount for treating retrovirus infection in a mammal of a compound selected from the group consisting of hypericin, pseudohypericin and salts and mixtures thereof.

2. The pharmaceutical formulation of claim 1, wherein said compound comprises hypericin.

3. The pharmaceutical formulation of claim 1, wherein said compound comprises pseudohypericin.

4. The pharmaceutical formulation of claim 1, which comprises a mixture of hypericin and pseudohypericin.

5. The pharmaceutical formulation of any of the preceding claims, comprising a parenteral dosage form.

6. The pharmaceutical formulation of claim 5 which comprises an injectable solution.

7. · The pharmaceutical formulation of any of claims 1 to , comprising a solid oral dosage form selected from the group consisting of a tablet and a capsule.

8. The dosage form of claim 7 comprising a pharmaceutically acceptable carrier or diluent. 21

9. The pharmaceutical formulation of any of the preceding claims, wherein said effective amount comprises between about 20G milligrams and about 12,000 milligrams of said compound. For the Applicants, Sanford T. Colb & Co. C: Ο6998 I-IO9O 22