JPH02504283A - Antiviral compositions and methods for treating retroviral infections comprising aromatic polycyclic dione compounds and nucleoside analogs - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Antiviral composition containing an aromatic polycyclic dione compound and a nucleoside analog compound Methods for treating viral and retroviral infections Conventional technology passed away This invention provides that an aromatic polycyclic dione compound in an amount exhibiting an antiviral effect is Contained with midine, dideoxycytidine or related nucleoside analogs Pharmaceutical drugs used for the treatment of retrovirus infections in mammals and this pharmaceutical product Regarding how to use the agent.

The pathogenic range of retroviruses includes slow progressive encephalitis lentivirus, various types of leukemia, oncogenic transformation that causes disease, carcinoma, and sarcoma and the recently identified H. IV (Human Immunodeficiency Virus) y Virus) infection. Retroviral infections of mammals (particularly HI There are only a limited number of therapeutic agents that have therapeutic power against V), and they only affect the viral replication cycle. Disturb a certain period of time.

This means that the host's immune system can effectively fight against various types of retroviruses. The inability to act is due to the genetic Wa of infectious retroviruses. Related. For example, viruses can be transmitted directly through cell-to-cell contact. possible, has high recombinant activity, and is long-lasting through integration into the host genome. It can remain hidden within cells for long periods of time. Also, HIV can be infected causes immune response suppression by reducing the function of CD+47 cells in individuals who have It is known that

Furthermore, recently, two related viruses have been linked to AIDS (acquired immune system infection). Acquired Immune Deficiency Syndrome HTV-1 and HIV-2 It was named. At the nucleotide level, the genome of HrV-1 and that of HIV-2 are Although their genomes only show about 50% homology, the two viruses have genetic differences. The structure of these two viruses is the same, and the mechanism by which these two viruses attack and destroy human cells is are considered to be almost the same. Therefore, in the following description, these two Luz is called HIV.

The most effective antiviral treatments to date are nucleoside analogs. 2°, 3°-dideoxycytidine and 3°-azido-3°-deoxythymidine (AZT). These two substances are phosphorylated within the cell and then By being incorporated into the viral DNA by the viral reverse transcriptase, Causes termination of complementary DNA (cDNA) before it has been completely synthesized. vinegar. However, the use of these drugs is limited due to their high toxicity to the host. Ru.

AZT is currently being used to treat HIV-induced immunodeficiency syndrome (AIDS). It is. AZT increases immune function and in some patients, system dysfunction is at least partially ameliorated, and other AIDS-related conditions are also ameliorated. It is good.

However, suppression of bone marrow function occurs in proportion to the dose of AZT, leading to anemia. It has been found that this can cause symptoms such as cancer and leukopenia. This means that AID This shows the limitations of stasis for the treatment of S. Nevertheless, the current Currently, AZT is the only drug effective against AIDS. Also, AIDS patients dideoxycytidine was used for treatment, but this drug was more toxic than AZT. It has been proven that

For this reason, there are several treatments available for the treatment of retrovirus infections, especially HIV infections. It is necessary to develop a pharmaceutical drug using a leoside-like compound, and in this pharmaceutical drug, The toxicity of nucleoside analogues may be undetectable in patients suffering from the disease. It must be possible to reduce the content of the compound. and/or have an increased antiviral effect. Ru.

The object of the present invention is to provide antiviral drugs for the treatment of retroviral infections in mammals. It is an object of the present invention to provide sexual compositions and methods for treating retroviral infections.

Another object of the present invention is to minimize the toxic effects of nucleoside analogs. Contains nucleoside analogues such as AZT in a highly controlled form, and Antiviral composition effective against retrovirus infection in mammals and the same This provides a treatment method using

Hatsuto 9! effect The present inventors have discovered that nucleoside compounds such as AZT and polycyclic dione compounds more effective due to the interaction of hyvericin and pseudo≦vericin. It was discovered by chance that it exhibited a strong antiretroviral suppressive effect. Therefore, this Combinations of antiviral drugs such as or may have a more beneficial effect on patients suffering from other retroviral infections. It is something to give. Until now, there are no drugs other than AZT that are effective in treating AIDS patients. The above discovery is a very important discovery because it has never been discovered before.

The present invention provides an antiviral drug containing an amount of a nucleoside analog compound that exhibits antiviral effects and hyvericin. , pseudohybericin, salts and mixtures thereof. containing a physiologically acceptable salt and a diluent, as well as a physiologically acceptable salt and a diluent. Retrovirus infection of mammals, characterized by giving it to retrovirus-infected animals. It provides a method for treatment.

Furthermore, the present invention provides an antiviral compound containing an amount of a nucleoside analog compound that exhibits an antiviral effect; selected from the group consisting of ricin, pseudohybericin, salts and mixtures thereof; and a physiologically acceptable salt and a diluent. Compositions for treating retroviral infections in mammals are provided.

The above-mentioned features of the present invention include the description of the present application including the claims and attached drawings. This will become clear by comparing it with conventional technology.

■Plate two blood star l theory alliance Figure 1 shows Friend leukemia virus treated with various concentrations of pseudohypericin. It is a graph showing the survival rate of infected mice.

Figure 2 shows Friend leukemia virus-infected mice administered with various concentrations of hibericin. 2 is a graph showing the survival rate of fish.

FIG. 3 shows various concentrations of AZT and various concentrations of hyvericin or pseudohypercin. Graph showing the survival rate of Friend leukemia virus infected mice administered with Vericin It is.

FIG. 4 shows the effects of hybericin and pseudohyvericin administered to the rats at various concentrations. 1 is a graph showing the survival rate of mice infected with leukemia virus.

of - of lightning The present inventors have discovered that nucleoside-like compounds represented by AZT and aromatic polycyclic di- Because the on-compounds hibericin and pseudohypericin interact with each other. , nucleoside-like compounds that have no effect when used alone. The concentration of ocide-like compounds, i.e., within the concentration range that is not toxic to mammals. effective anti-inflammatory drugs with nucleoside analogues (e.g. drugs like AZT). We discovered that it is possible to perform retroviral therapy. This discovery is A AIDS patients who have difficulty tolerating therapy using ZT, and those with AZT toxicity Some AIDS patients may have to discontinue AZT therapy due to It is very important. In addition, two antiviral therapeutic drugs are being used to treat AIDS. can be used to treat patients infected with retroviruses such as It will be possible to administer antiretroviral therapy.

Nucleoside analogs and polycyclic dione compounds each have different mechanisms of retroactivity. inhibits the replication of virus. As mentioned above, AZT and related nucleotides Compounds similar to cDNA cause termination of viral cDNA during unfinished synthesis. vinegar.

However, in the polycyclic dione compound of the present invention, the virus It may also inhibit replication, e.g., the release of virus particles from infected cells. inhibitory effect on the assembly of viral particle components within infected cells. Inhibitory effects or direct virus particle inactivation are possible.

In addition, a dose of the above compound that inhibits virus replication in an in vitro system is also provided. Even with cyclic dione compounds, cultured mouse cells infected with Friend leukemia virus There was no change in the expression of total viral mRNA and urinary antigens in the cell system (results). Results not listed).

Furthermore, only the purified reverse transcriptase was incubated with the polycyclic dione compound. The activity of reverse transcriptase was not affected even when the sample was heated (results not shown). but, Directly incubating the polycyclic dione compound and Friend leukemia virus. The infectivity of the virus is completely eliminated. In addition, Ps (pseudohyvericin) and HY (hyvericin) directly inactivates viruses. It is thought to suppress the spread of virus infection.

The inventors have determined that the parenteral dosage ranges from about 0.1 ng to about 300 μq. PS and HY were tested on more than 800 mice; During the course of the treatment, no significant toxicity was observed in the administered mice, and the animals did not experience any pain or discomfort. The experiment did not reach a state where it would cause discomfort, that is, a state where the experiment should be stopped. .

The academic term "retrovirus" used here refers to RNA genome and RNA Denotes a virus with dependent DNA polymerase (reverse transcriptase) enzymatic activity It is something. All retroviruses share common morphological, biochemical and Because it has physical properties, it is classified as a family in virus taxonomy. It is treated as such. This property is summarized in Table 1 below.

(blank below) Table 1 General physical characteristics of known retroviruses (+)-heavy chain RNA (60s -70s): 5' structure (m'G'ppp'NmpNp); polyadenyl repeated sequences at the 3' and 5' ends; tRNA base-paired to genome complex; protein 60% by weight or more: gag, internal structure protein; pol, reverse transcriptase; env, envelope protein quality Lipids: Approximately 35% by weight; Carbohydrates extracted from cell membranes: Approximately 4% by weight; Physicochemical properties bound to envelope protein Density in sucrose 1.16-1. Density in 1897 m1, cesium chloride 1, 16-1.21 g/ml, sensitive to fat solvents and detergents; heat Inactivation by (56・C130 min); High resistance to UV and X-rays Morphological characteristics Spherical virus particles surrounded by an envelope (diameter 80- 120 nm); various surface protrusions (8 nm in diameter): with core shell (nucleoid) While the icosahedron contains the ribonucleoprotein complex, the HIV genome contains at least five It is coated with different proteins, and these proteins are TAT, ART/TR 3,3'-0RF.

They are called SOR and R. On the other hand, HTLV I has the pX gene, and this pX The gene encodes four proteins.

All retroviruses have similar chemical compositions and are generally proteinaceous. White matter: 60-70%, lipids = 30-40%, carbohydrates: 2-4%, and R NA: Consists of about 1%. The envelope of retrovirus particles is attached to the cell surface membrane. Almost all the lipids in the virus particle are extracted from the unit membrane envelope of the virus particle. It's in the rope. Examples of retroviruses include Friend leukemia virus (FV), radiation leukemia virus (RadLV), feline leukemia virus, coracoid bud bulbosis virus, and human) T-cell lymphotrophic virus family (HTLV I, II, III, and rv; HTLV III is human immunodeficiency virus or HI (also known as V). HTLV I causes adult T-cell leukemia It has become clear that HTLV III causes pilocytic leukemia.

HTLV IV is related to the monkey immunodeficiency virus, and this virus was introduced in Africa. It was discovered in an AIDS carrier among Kawa natives. This virus and HTLV III The relationship is currently under study.

The composition of the present invention contains an aromatic polycyclic dione compound and a nucleoside analog. diseases of mammals caused by retroviruses such as HIV. It can be used for treatment. Administration of nucleoside analogues that the amount of polycyclic dione compound is below the toxic concentration and that the dose used is Since it is not cytotoxic, the composition of the present invention is particularly effective against such diseases. It may also be a useful specific antiviral treatment. Retrovirus of the composition of the invention The effectiveness of the treatment against virus infection is demonstrated in the second example below, and it is non-toxic. Friend leukemia when treated with a mixture containing AZT and AZT within a range of concentrations. The survival rate of mice infected with the virus was 100% 65 days after infection. Ta. Conversely, when treated with the same dose of AZT alone, within 65 days post-infection, All mice died.

HIV can infect brain cells and central nervous system cells, which can cause , known to cause nervous system dysfunction and dementia. AZT blocks the blood-brain barrier You can pass. That is, preadministration of hibericin and pseudohyvericin to humans. When the central nervous system It is known because it acts on the nervous system. Therefore, nucleoside analogs Used with polycyclic dione compounds, it may be used in patients suffering from the neurological effects of AIDS. If given, it can be very effective. Another advantage of the composition of the present invention The point is that when administered orally, polycyclic dione compounds and nucleoside analogs Both are effective.

Furthermore, the effects of PS or HY treatment on Friend leukemia virus infection are Figure 4 shows that the nq amount for both PS and HY was determined by intraperitoneal injection 24 hours after infection. Treatment administered intravenously inhibited the enlargement of the spleen (megaphoria) caused by the virus. infected For mice (Ba1b/c), administer approximately 1 ng of ps or approximately 1100 n of HY. By doing so, the enlargement of the spleen caused by FV was inhibited by 50%.

Such a low dose of PS and FU should be administered once intraperitoneally 24 hours after infection. Therefore, surprising effects can be obtained.

HY (hyvericin) and PS (busoid hibericin) are from the genus Hypericum (St , John's Wort) is an aromatic polycyclic dione compound that exists in plants. Ru. U.S. patent filed on August 10, 1987 by Davis et al. Application No. 084.000), HY and PS are in vitro and 1n It has been disclosed that it is a potent inhibitor of viral replication in vivo. ing.

As explained in the first example below, Fu and PS are from the genus Hypericum (St, Jo). It can be extracted from the hn's Wart plant. Also, in May 1955 U.S. patent filed by H. Plackman et al. on the 3rd (application number 2.707. No. 704) and Tetrohedoron Letters by H. Plaqueman σ Kyoko! ■Four By the method described in 上凪91本：1991-1994.1974, It is possible to synthesize the audio and PS. Hypericum genus (St, John ’s Wort) is distributed in places all over the world, and the extraction of previous and PS Because purification is relatively simple and inexpensive (as shown in the example below), small quantities (e.g. Extraction is preferable when large quantities (e.g. sample units) are required; In the case of production (in units of 1,000 gram or more), synthesis is preferred.

Furthermore, the compositions of the present invention contain hyvericin and pseudoviral which retain antiviral activity. It also contains derivatives or salts of hyvericin. In particular, salts are alkaline or Those having an amine type group fall under the scope of this application.

In the present invention, the number of nucleoside analogous compounds that can be used is 2.

3°-dideoxycytidine, 2°,3-deoxythymidine, 2°,3°-dideo oxyadenosine, 2°, 3°-dideoxyinosine, more preferably AZT These include, but are not limited to. AZT for implementing the present invention is Burroughs Welcome, Re5earch Triangle Park, NC) and 2゛, 3- Dideoxycytidine and 2゛,3゜-dideoxyadenosine are calbiochemical molecules. -ring (Calbiochem-Behring, SanDiega, C From A), 2°, 3°-xythymidine was obtained from Pharmacia Fine Chemicals (P harmacia Fine Chemicals, Piscataway, NJ).

Additionally, the present invention provides methods for treating mammals infected with retroviruses. the aromatic in an amount that exhibits antiviral efficacy in mammals in need of treatment. This method involves administering an aromatic polycyclic dione compound and a nucleoside-like compound. Group polycyclic dione compounds include hypericin, pseudohypericin, salts and their salts. A compound selected from the group consisting of mixtures is used.

When using the present invention to treat mammals infected with retroviruses, Polycyclic diionization, the most effective nucleoside analogue against virus The types of compounds and their compositions can be used, for example, to inhibit HIV in vitro. Example 4 concerning Friend Leukemia Virus in Laboratory Animals or Example 2 concerning Friend Leukemia Virus in Experimental Animals This can be confirmed through routine experiments using an appropriate system such as can.

AIDS1 viremia (presence of virus in the blood) caused by a virus (presence of virus in body fluids) or sepsis (presence of virus in body fluids) When carrying out the treatment in Preferably, parenteral administration, more preferably intravenous administration, The dosage is determined as follows with reference to hypericin.

Antiretroviral group containing an aromatic polycyclic dione compound as one of the active ingredients The composition is about 0.001 to 100,000 μg/kg body weight per treatment, preferably preferably about 2 to 50,000 μg/kg body weight, more preferably about 200 to 50,000 μg/kg body weight containing the dione in the range of g/kg body weight.

Using one or more other aromatic polycyclic dione compounds as active ingredients In this case, the dosage is approximately the same as for hibericin. However, the administered aromatic The polycyclic dione compound or composition thereof has twice the activity of, for example, hypericin. If so, the minimum effective dose is 1.5 times that of hibericin. more than two Active antiviral ingredients (e.g., antiviral ingredients other than the aromatic polycyclic dione compounds of the present invention) (including anti-inflammatory agents) is used as a therapeutic or prophylactic agent according to the present invention, it is necessary to If necessary or appropriate, the minimum dosage of the Aromatic Polycyclic Dione Compound is It may be decreased. Finally, more than one active ingredient may be used and the aromatic polycyclic When the antiviral compound interacts with one or more other antiviral components (or two or more aromatic polycyclic dione compounds and a nucleoside) (among similar compounds), the minimum effective dosage may be lower.

To explain the above, nucleoside-like compounds with antiviral activity and We will also discuss therapeutic or preventive methods that involve administering aromatic polycyclic dione compounds. Ru. (Here, "together" means to administer at the same time or consecutively. (and each is not administered separately under the same Act).

One or more nucleoside analogs can be added to the aromatic polycyclic dione of the present invention. When administered with the compound, about 100 to 5 ooooo μq/k in - treatments q body weight is administered with the aromatic polycyclic dione compound of the present invention.

The duration of treatment or administration necessary to improve the condition will depend on the individual being treated. This varies depending on the severity, duration, and general condition of each individual. It is further determined by the specific antiviral activity and toxicity of the composition. . The total dosage required for each treatment may be administered in divided doses or all at once. anti-ui Lus treatment should be carried out daily, -at least twice a day, -at least once a week, or at least once a week. It depends on the condition of the individual being treated and the stage of the disease.

In addition, the present inventors have demonstrated that the antiviral activity of hyvericin increases with treatment frequency. I discovered that it depends on the cey of treatment. Ta. For example, studies using mice have shown that, as expected, a single dose of 10 μq It is also more effective to administer 100μq once. However, if 10μq is continued every day, Even after continuous administration for 10 days, the effect of even 10 μq administration was not shown.

In contrast, administering 10μ9 once a week is more effective than administering 10μq-times. It was effective. This means that the frequency of treatment affects its effectiveness. It is. Therefore, the same holds true for the composition of the present invention. mouse The findings obtained using this method are not applicable to other mammals or humans. can be determined by routine experimentation using methods known to those skilled in the art. example For example, create a matrix for dosage and frequency, and then All you have to do is assign the experimental population to these points. In such an experimental design, the preferred In addition, it is necessary to consider the tissue distribution characteristics of the composition of the present invention.

The present invention also provides pharmaceutical compositions and formulations for the treatment of retroviral infections. This is what we provide. Aromatic polycyclic dione compound and/or nuclei of the present invention Similar compounds of Oside are available in common solid and liquid forms (e.g. tablets, capsules, capsules). can be formulated in the form of liquids for injection, injection and oral use). The formulation of the present invention is , aromatic polycyclic dione compounds and nucleoside analogs of the present invention as active ingredients. Compound (disclosed above) in an effective amount. For example, parenteral therapeutic compositions , 0.001 to 100,000 μq of the aromatic polycyclic dione compound of the present invention and 100 A sterile isotonic solution containing ~50,000 μq of the above nucleoside analogs. Consists of pressure salt solution. The unit amount of active ingredient contained in the dosage for each dosage form is , need not be an effective amount on its own. This is because the necessary effective amount is , capsules, injections, or a combination thereof, each in a single dose This is because it can be obtained by changing .

The formulation according to the invention further comprises pharmaceutically acceptable carriers, diluents, fillers, salts, and an inert material containing substances known to those skilled in the art, and which are normally determined by those skilled in the art. Depending on the Ru. For example, tablets can be formed by conventional solid carrier manufacturing methods known to those skilled in the art. may be made. Examples of solid carriers include starch, sugar, bentonite, and silica. Mosquito, and other common carriers are used. Propylene glycol, benzylua Alcohol, Improper Tool, Ethanol, Dimethyl Sulfoxide (DMSO) , dimethylacetamide, or other biologically acceptable organic solvent, or water. The solution (e.g., pH 7 or higher, preferably pH 8) contains the antiretroviral agent of the present invention. In preparing solid and liquid pharmaceutical formulations containing compositions, carriers, diluents, and It is often used as a solvent. Furthermore, carriers and diluents are not limited. Examples include carbohydrates, albumin and other plasma proteins, such as low-tone lipoproteins. , high-density lipoproteins and lipids bound to these serum proteins. child Lipids such as phosphatidylcholine, phosphatidylserine, phosphatidyl Contains neutral lipids such as ethanolamine and triglycerides. Other lipids Contains copherol, retinoic acid and cyclodextran. Known semi-solid pharmaceuticals Agents may also include, for example, suppositories.

The preferred primary dosage form is an isotonic saline solution containing doses ranging from about 0.1 to 100,000 μq. The aromatic polycyclic dione compound of the present invention within the range of about 100 to 50000 μ9 nucleoside analogs in amounts within a range.

The capsules used in the present invention are made of pharmaceutically acceptable materials, such as gelatin. Or made of cellulose derivatives. Oral and transdermal methods that deliver drugs continuously Another administration method is also considered.

The compositions of the present invention may be incorporated into liposomes as specific drug carriers.

Such posomes may contain other active drugs, such as HIV p120. such as p41 and p24 (including glycosylate derivatives of this protein) aHTV antibody specific for viral proteins that appear in Una virus-infected cells There is.

The invention is illustrated by, but not limited to, the following specific example.

1: Hypericin and pseudohypericin St., John’ hibericin (qoH+A, molecular weight 504.43, here referred to as liver) and pseudohybericin (qOH,60,, molecule t520 ．． 43, here referred to as PS) was obtained as follows.

St. John's Wort Plant It is harvested from the upper blade to the lower part of the hemisphere. This harvested hypericum is heated at 55oC. After drying, cut and crush the material for extraction, add acetone (approximately 5 per kilogram) (from lθ liters) to extraction.

That is, 1 kg of the material was extracted using a Tuxley extractor (manufactured by Kimax).

Fisher 5 scientific, New Brunswick, NJ ) and extract until the extraction solvent becomes colorless (approximately 5 to 10 hours). do. A solution containing an aromatic polycyclic dione compound exhibits a red fluorescent color, and be 5chenta, Ber, 75: 2019.1942. Bro ckmann, M.

Absorption spectra as described in Natureweiss 38:47.1951. and fluorescence spectrum.

The solvent containing the aromatic polycyclic dione compound is evaporated under reduced pressure, and the residue is completely removed. Upon drying, 95 grams of dry residue was obtained. Furthermore, this dried residue Silica gel 60 (0.06~0, 20 mm, Malinckrodt, American 5 scientific Products, McGawP Ark, IL) column chromatography. In other words, the dragon Using the icolumn method, 25 grams of the above dry residue was added to about 500 milliliters of acetic acid. To this, add an equal amount of silica gel 60 to make a mixture, and use a rotary evaporator. Porator (manufactured by Buchi, American 5 scientific Pro ducts) to homogenize the mixture while rotating the evaporator. The solvent was evaporated until the mixture was dry. Then, add this mixture to 1 onto a column of kilograms of silica gel 60 and eluted with chloroform. I set it. The elution continued until the chloroform fell to the bottom of the column. Next, Kuro Roform-acetone-methanol, 75:15:10 (Vol/V The solvent column was washed with a solvent column consisting of The red above is the original density of 115 , the concentration of chloroform decreases and the column becomes chloroform-acetone-method. Elution was performed with a solvent consisting of tanol, 55:15:10. In addition, the above column chroma The topography took about two days.

Further purification and separation of the two main components is GaSChrOmatOrah.

Pr1nci Ies Techni uesandA 1 cations , A, B, Littlewood, ed., Academic Press, Fra-Cush chromatography as described in New York 1970 This was done by using twice.

The two main components identified are: hypericin αIY), Rf value 0.45.0.19g; ) Rf value is 0.35, 0.73g. NMR spectral analysis of the two components is Brockmann, H.

et al, Tetrahedron Letters 1:37.197 It matched what was described in 5.

The compound thus obtained should be stored in a dry, dark place or in an evaporator until used. Stored at 4'c in Nord.

2: H's virus Antibiotics of compositions of the invention against Friend leukemia virus (FV') infection in mammals The virus effect was investigated.

Fr1end, C, J, Exp, Med, 105: 307-324. 1957 and Fr1end, C, et al.

Proc, Natl, Acad, Sci, U, S, A, 68: 37 g-383, 1971 and Fr1end, C, etal, Nat, Can cer In5t, Monogr, 22:505-522.1966 As previously mentioned, Friend leukemia virus (FV) is an aggressive retrovirus. This virus can be transmitted to susceptible strains of mice, such as the BALB/c strain and the NIH5 strain. It induces erythroleukemia in w155 strain mice. This malignant transformation is Pancreatic focus forming virus (SpleenFocus Forming; 5FFV) and Ecotropic Murine Fr1end Leulemia)h This is due to both activities of the lper virus (F-MuLv).

This acute erythroleukemia is caused by hepatomegaly (an extremely large increase in the size of the lungs and liver). ) and severe anemia.

Friend leukemia virus is used to treat infected patients 10 days after intravenous injection of the virus. Obtained by crushing (homogenizing) the enlarged pancreas of a mouse. That is, the applicable The lungs were homogenized in a phosphate buffer solution consisting of a volume equal to 10 times the weight of the lungs. I did it. BALB/c mouse (Jackson Labs, Bar Har HY and PS or a mixture thereof for pancreatic enlargement of The effects of AZT and AZT were investigated.

In these experiments, BALB/c mice were injected with virus (106 focus format uniform). FFU) was intravenously inoculated, and 24 hours later, the antiretroviral composition of the present invention was administered. The indicated doses of the substances were administered intraperitoneally to the mice. 10 days later, the mice were The animals were sacrificed and their lungs were weighed.

At the time of virus inoculation, administer a mixture of PS and Fu once intravenously (mixed with virus). The long-term effects of AZT were investigated and compared with those of AZT. AZT In order to inhibit the high toxicity of (dated August 10, 2007) for polycyclic dione compounds. AZT was administered at 2, 5 and 16 hours after virus inoculation, respectively. Intraperitoneal administration was performed twice. For each group of mice (5 individuals per group), AZT (3 intraperitoneal injections) and PS or HY (1 intravenous injection) alone; As a result of administering these combinations, the survival rates of animals were as follows: . The results are shown in Table 2 and the graphs are shown in Figures 1-3.

(Hereafter, margin) Table 2 10Pg PS 40 50μg PS 80 150μg PS 10010μg HY 110 0501z BY 80 150μg HY 1000.2μg AZT 0 2μg AZT 0 O201L AZT 400.2 μg AZT + 50 PS 1002μg AZT + 50 PS 10020μg AZT + 5 0 PS 1000.2 μg AZT + 50 HY 1002 μg A ZT + 50 BY 10020μg AZT + 50 HY 100 As shown in Figure 1 and Table 2, no therapeutic agent was administered within 30 days after virus inoculation. ■ PS was administered even though all infected mice died The survival rate of mice 65 days after virus inoculation is 10 μq per mouse. 40% for 50 μq, 80% for 50 μq, and 100% for 150 μ9 Met. Furthermore, as shown in Figure 2 and Table 2, the wi The survival rate after 65 days of inoculation with M. 00%, 80% when administered at 10 μq or 50 μq. to this On the contrary, mice were given AZT in amounts ranging from 0.2 μq to 20 μq per individual. Survival rates of mice inoculated with ■ were only slightly increased. AZT Even at the lowest dose, the mixture with AZT h HY or PS is compatible. interacted (Figure 3 and Table 2). For example, mouse - 0.2 μ9 per individual In mice administered AZT, ■ 100% of inoculated mice developed by 30 days post-inoculation. At 50 μq PS per mouse, 20% of inoculated mice died. Mice died by 30 days post-inoculation, but 0.2 μq AZT and 50 Among the mice administered μq of n, the ■-inoculated mice survived even 65 days after inoculation.

Furthermore, it is noteworthy that when AZT is not mixed with PS or Regardless of the dose, all inoculated mice died by 65 days after inoculation.

From the above, in treatment combining AZT and Yari' or PS, AZT's It is possible to reduce the number of administrations and the dose, and it may also reduce the effectiveness of AZT. It has been found that it is possible to reduce side effects.

3: In vitro y, I was infected with EV on PS night We investigated the effect of Ps on HTV replication in HUT-78 cells and A comparison was made between the effect of AZT and a similar effect in the case of AZT. ■V whole thing antiserum (inactive serum obtained from an HIV carrier or AIDS patient) or Infection is monitored by indirect fluorescent antibody method using viral components purified from rabbits. I guessed it.

The effect of pseudohypericin on V infection of cells was determined by pseudohypericin of various strengths. ■V virus and uninfected cells are mixed with vericin at the same time, and these are heated. RPMI-1640 medium (GIE) supplemented with inactivated 10% calf fat serum CO, Grand Island, NY) 24.48.72 and 1 It was incubated for 44 hours and observed. After incubation, wash and fix cells and after further incubation with fluorescein-labeled anti-immunoglobulin. , and the percentage of infected cells was determined by incubation with anti-HIV antibody. hibericin and pseudohybericin must be dissolved in ethanol before use, but the final The alcohol concentration does not exceed 1-2%, and the target group has a ratio of virus to cells. The concentration was the same as that of the alcohol used to dissolve the alcohol drug. Why Alcohol is known to increase the proportion of cells that become infected with crabs. It is et al.

The spread of HIV infection is 1% of infected cells and 99% of uninfected cells. , was observed by culturing in the presence of an appropriate concentration of pseudohyvericin. Applicable Samples from the cultures were taken on 24.48.72 and 144 hours after the start of the experiment. The sample is then used to calculate the percentage of infected cells using the indirect fluorescent antibody method described above. It was. The results are shown in Table 3 below.

(Hereafter, margin) Table 3 ! LLIJ!　　　　　　　　　　　　　　　　　　　　　 　 l! II! l　a　a　101 dishes comparison example　　　　　　　　　　　　　　2.7　 26.7 62.9 Control example + ethanol 2.9 30.9 　76.81! , LMAZT 1.8 0.75 0.610 μMAZT 1 0.4 0.32 μgps 3 52.8 77.910μg PS 3. 3 38 90.1 sop gps O, 21,218, 8M 2M & μ yo hill ■ 1 cold specimen AUMA 11 piece solid control example 2.7 26 681μM AZT 2 3 320μM AZT 2 22 5840μ9PS 2 24 4780μgps 2 5 29 From the above experiment, PS was found to have I (IV sensitivity) in HUT-78 cells in the culture medium. It can be seen that it is effective in preventing the spread of stains. On the other hand, 1 μM and 10 μM Concentrations of AZT inhibited the spread of HIV infection to uninfected cells by more than 95%. (Table 3), and 50 μq concentration of PS was found to be effective against HIV susceptibility at 144 hours after infection. The spread of staining was reduced by about 80% (Table 3, Experiment 1), and the ps at a concentration of 80 μq was The spread of HIV infection was reduced by about 70% (Table 3, 2nd experiment).

14: Human immunodeficiency syndrome (HIV) caused by H'IV virus (a) ) against hypericin, pseudohypericin, salts or mixtures thereof; The activity when combined with 2°,3-dideoxycytidine or AZT is as follows: can be researched in this way. ■V-infected 0KT4+ lymphoblastoid cells , for example, H9 clone (Popovic, M., et al., 5science 22 4:497-500.1984) or HUT 78 cells (Gazdar, A, F, et al., Blood 55: 409.1980) or Molt-78 Cells (% American Type Cu as ATCCCRL 1582 Available from 1ture Co11ection, Rockview, MD ), 20% calf fat serum (Flow Libraries, Ingl ewood.

CA) containing RPMI-1640 medium (manufactured by GIBCO, Grand l5land , NY). Inoculate cells at a concentration of approximately 4xlO5/ml cells. Prepare three culture media and mix them with polybrene (approximately 2 μg/ml, Sigma Ch. chemical Co, St, Louis, MO), 4xlO' infected with 2xlO' HTV virus particles per cell, as shown in Example 2 above. In this manner, the cells are cultured in the presence and absence of the composition of the present invention.

The antiviral activity of the composition of the present invention is determined by 5arin, P.S., et al., J., Na. t.

Those listed in Cancer In5t, 78: 663-665.1987 observation of reverse transcriptase activity and expression of HIV proteins p24 and p17 Determine by

HTva White 24 and 17 are 1゛HUT78 cells, Mo 1t-78 cells or gluteal cells (2x 10'), respectively, were not infected with HIV. Infected cells or infected cells were prepared, and these cells were diluted from 5 μg/ml. PS, HV or mixtures thereof at various concentrations ranging up to 100 μg/ml and 2° at various concentrations ranging from 0.2 μg/ml to 20 μg/ml, 3°-dideoxycytidine or AZT for 4 consecutive days. vinegar. Then, ■ Expression rates of V proteins p24 and p17gag were compared with HIV protein p2 Mouse monoclonal antibody against p17 gag (Abbot La bs, North Chicago, IL, Dupont, Wilmi ngton, DE.

Detection using indirect fluorescent antibody microscopy using an HIV serum antigen detection set manufactured by Co., Ltd. to be determined. Cells showing positive were treated with fluorescein-labeled sheep anti-mouse IgG. (Cappell Laboratories, Cochranville, PA). In this experiment, we conducted similar experiments at the same time. Repeated at least 3 times.

Reverse edition W As above, H9 cells, HUT78 cells or MOLT-78 infected with Cells (500 virus particles/cell) were treated with various concentrations of PS, HV, and their Expose the mixture to 2°,3-dideoxycytidine or AZ1' . On the fourth day, the medium supernatant was collected and virus particles were mixed with polyethylene glycol. Virus particles are obtained by coprecipitation and then centrifugation as shown in Example 2. like this The virus body obtained by of dithiothreitol, 250 mM potassium chloride, and 0.25% Triton. Suspend 300 μq in a buffer containing X-100. Then, reverse transcription of these samples Enzyme activity was determined using 50mM Tris-HCl (pH 7.5), 5mM dithiothreate. 100 mM potassium chloride, 0.01% Triton X-100, template 10 µi of dT, rA as target primer.

, 10mM magnesium chloride, 15μg [3H]dTrP (NewEn grand Nuclear, Boston, MA), and 10 μl of powder Analyze in 50-leaf reaction mixtures containing crushed virus suspensions. 37°C11 After incubation for 50 μq of yeast tRNA (SigmaChem ical, St, Louis, MO) to insoluble in cold trichloroacetic acid. Incorporation into fractions was investigated. In this experiment, similar experiments were conducted at the same time, and Repeated at least three times.

5: Effect of blood “Virus control” For cats with positive feline leukemia virus (FeLV)emia, 5 mg/kg to 20 mg/kg Inoculate with PS or FU in doses ranging up to 1.5 kg.

For other cats, one dose of PS/HY plus 4-40 doses or 2°, 3°-di A combination treatment consisting of deoxycytidine (1 mg/kg) was administered at various time intervals. Leave it on and do it twice a day. Pursue the FeLV level in the serum and based on this, Either repeat the same therapy or adjust the treatment to the degree of viremia inhibition. Treatment (cats in the control group of this experiment were given a similar concentration of deoxynucleoside) (administer only similar compounds). The duration of pursuit of serum FeLV level is It will be determined based on the test conditions and will be pursued for at least 6 months.

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O-～　ω　　　　　φ　　Φ　　　−■　　　ψ　　　-の Photocopy and translation) Submission form (Article 184-7, Paragraph 1 of the Patent Act) ! 0 International Application Number PCT/US 89101211, Title of Invention Yoshi Antiviral composition containing aromatic polycyclic dione compound and nucleoside analog compound and methods for treating retroviral infections. 3. Patent applicant Address: United States of America, New York, 10012, New York, Washin Ton Square South 70 Name New York University Fee Presenter: Completion Nationality: United States of America - 4. Agent Address: 2-1-5-6 Yaesu, Chuo-ku, Tokyo List of attached documents Kasafu Old Joe 0 Rainbow Plate (1) A method for treating mammals sickened by retroviruses, A nucleoside analog compound and Hiberici in an amount that exhibits the antiviral effect necessary for the treatment. selected from the group consisting of a polycyclic dione compound, which is administered to a mammal. Treatment methods for retrovirus infections.

(2) Compounds similar to the nucleoside are 2°, 3°-dideoxycytidine, 2° , 3°-dideoxyadenosine, 2°, 3′-dideoxythymidine, 2′, 3° - the group consisting of dideoxyguanosine and 3°-azido-3°-deoxythymidine The method of treatment according to claim 1, characterized in that the treatment method is selected from:

(3) The effective amount described in claim 1 is characterized in that the effective amount is administered orally. Treatment methods listed.

(4) Claim 1, characterized in that the effective amount is administered parenterally. Treatment methods described.

(5) According to claim 1, the effective amount is administered intravenously. Treatment methods listed.

(6) A patent claim characterized in that the polycyclic dione compound consists of hibericin. The treatment method according to scope 1.

(7) The polycyclic dione compound is characterized in that it consists of pseudohypericin. The treatment method according to claim 1.

(8) Is the polycyclic dione compound a mixture of hypericin and pseudohypericin? The treatment method according to claim 1, characterized in that:

(9) The effective amount is about 1000μ9 per kg of body weight of the mammal. and similar compounds of said nucleosides ranging from 5ooooμ9 to said mammals. The above-mentioned polycyclic in a range of about 0.001 μq to 100,000 μq per 1 kg of body weight The method of treatment according to claim 1, characterized in that it consists of a dione compound of the formula .

(10) In pharmaceuticals for the treatment of mammals infected with retroviruses, Similar compounds of leoside, hibericin, pseudohyvericin, salts and their a polycyclic dione compound selected from the group consisting of a mixture of A retrovirus characterized by the fact that it is effective in treating viral infections. Pharmaceutical drugs against infections.

(11) The nucleoside analogue compound is 2°, 3°-dideoxycytidine, 2° , 3′-dideoxyadenosine, 2°, 3°-dideoxythymidine, 2′, 3 Consisting of '-dideoxyguanosine and 3'-azido-3'-deoxythymidine Pharmaceutical drug according to claim 1O, characterized in that it is selected from the group.

(12) A patent claim characterized in that the polycyclic dione compound consists of hibericin. The pharmaceutical agent according to item 10.

(13) The polycyclic dione compound is characterized in that it consists of pseudohybericin. The pharmaceutical agent according to claim 10.

(14) The polycyclic dione compound is a mixture of hypericin and pseudohypericin. The pharmaceutical agent according to claim 1O, characterized in that it consists of:

(15) The method according to claim 10, which is a parenteral administration form. Pharmaceutical drugs.

(16) be an oral administration form selected from the group consisting of tablets and capsules; The pharmaceutical agent according to claim 10, characterized in that:

(17) The effective amount is about 1000 μq to 5oooo1. Before Lg range in the range of about 0.001 μq to 100,000 μq. Claim 10, characterized in that it consists of the following polycyclic dione compound: pharmaceutical products.

(18) A patent claim characterized in that it consists of a pharmaceutically acceptable carrier or diluent. The pharmaceutical agent according to scope item 9.

(19) The method according to claim 9, characterized in that it consists of an injectable solution. Pharmaceutical drugs.

(20) The retrovirus is characterized by comprising a human immunodeficiency virus. The treatment method according to claim 1.

(21) The retrovirus is characterized by consisting of Friend leukemia virus. The treatment method according to claim 1.

(22) A patent application characterized in that the retrovirus consists of feline leukemia virus. Scope of Claims Item 1.

(23) an amount effective to inhibit the growth and replication of retroviruses; selected from the group consisting of syn, pseudohypericin, salts and mixtures thereof. a retrovirus, characterized in that the retrovirus is brought into contact with a compound A method of inhibiting the growth and reproduction of.

international search report

Claims (22)

[Claims] (1) A method for treating mammals sickened by retroviruses, A nucleoside analog compound and hypericycin in an amount that exhibits the antiviral effect necessary for the treatment. selected from the group consisting of salts, pseudohypericin, salts and mixtures thereof. a polycyclic dione compound, which is administered to a mammal. Treatment methods for retroviral infections. (2) Compounds similar to the above nucleoside are 2',3'-dideoxycytidine, 2' , 3'-dideoxyadenosine, 2',3'-dideoxythymidine, 2',3' - the group consisting of dideoxyguanosine and 3'-azido-3'-deoxythymidine The method of treatment according to claim 1, characterized in that the treatment method is selected from: (3) The effective amount described in claim 1 is characterized in that the effective amount is administered orally. Treatment methods listed. (4) Claim 1, characterized in that the effective amount is administered parenterally. Treatment methods described. (5) According to claim 1, the effective amount is administered intravenously. Treatment methods listed. (6) A patent claim characterized in that the polycyclic dione compound consists of hypericin. The treatment method according to scope 1. (7) The polycyclic dione compound is characterized in that it consists of pseudohypericin. The treatment method according to claim 1. (8) Is the polycyclic dione compound a mixture of hypericin and pseudohypericin? The treatment method according to claim 1, characterized in that: (9) The effective amount is based on 1 kg of the mammal's body weight. Approximately 1000μg per 50,000 μg of analogues of said nucleosides and of said mammals. Weight 1kg. from about 0.001 μg to 100,000 μg per polycyclic The method of treatment according to claim 1, which comprises a dione compound. (10) In pharmaceuticals for the treatment of mammals infected with retroviruses, Similar compounds of leoside, hypericin, pseudohypericin, salts and their a polycyclic dione compound selected from the group consisting of a mixture of A retrovirus characterized by containing an amount effective in treating viral infections Pharmaceutical drugs against infections. (11) The nucleoside analogous compound is 2',3'-dideoxycytidine, 2' , 3'-dideoxyadenosine, 2',3'-dideoxythymidine, 2',3' - the group consisting of dideoxyguanosine and 3'-azido-3'-deoxythymidine The pharmaceutical agent according to claim 10, which is selected from the following. (12) A patent claim characterized in that the polycyclic dione compound consists of hypericin. The pharmaceutical agent according to item 10. (13) The polycyclic dione compound is characterized in that it consists of pseudohypericin. The pharmaceutical agent according to claim 10. (14) The polycyclic dione compound is a mixture of hypericin and pseudohypericin. The pharmaceutical agent according to claim 10, characterized in that it consists of: (15) The method according to claim 10, which is a parenteral administration form. Pharmaceutical drugs. (16) be an oral administration form selected from the group consisting of tablets and capsules; The pharmaceutical agent according to claim 10, characterized in that: (17) The effective amount is in the range of about 1000 μg to 50000 μg. a creoside analogue compound and a range of about 0.001 μg to 100,000 μg of the above-mentioned polyamide. The product according to claim 10, characterized in that it consists of a cyclic dione compound. drug. (18) A patent claim characterized in that it consists of a pharmaceutically acceptable carrier or diluent. The pharmaceutical agent according to scope item 9. (19) The method according to claim 9, characterized in that it consists of an injectable solution. Pharmaceutical drugs. (20) The retrovirus is characterized by comprising a human immunodeficiency virus. The treatment method according to claim 1. (21) The retrovirus is characterized by consisting of Friend leukemia virus. The treatment method according to claim 1. (22) A patent application characterized in that the retrovirus consists of feline leukemia virus. Scope of Claims Item 1.