JPH03504246A - Chemotherapeutic composition for AIDS - 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] Chemotherapeutic composition for AIDS This is a pending application filed May 16, 1988, which is incorporated herein by reference. This is a partial continuation of application number 07/194171.

Technical field The present invention is useful for treating or preventing AIDS or other similar viral diseases. related to things. More particularly, the present invention relates to the combination of antiviral agents and potentiating agents. However, this combination is more effective against viruses than each drug alone. Ru.

Background of the invention Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HI). It is thought to be caused by a retrovirus called V). the current , there is no treatment other than to slow the viral infection and disease progression. If valid AIDS is known to be fatal if there is no treatment.

Certain chain terminator compounds, such as azidothymidine (AZT), other Deoxynucleosides, dideoxynucleotides and their analogs are It has some effect against viruses [Mitsayx et al., 1 985, PNAS, USA, 82: 7096; Yarchon 1988, Lancet (i) 76-80; hlberg) et al., 1987, PNAS, USA, 84:2469:l. So They are phosphorylated within the cell and formed in the presence of viral reverse transcriptase. The DNA strand appears to be terminated in the triphosphate form. these drugs is an enzyme active in the phosphorylation of physiological nucleosides and nucleotides. They may also act by additional mechanisms, such as by antagonizing the elements.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindoly Gin) is a plant that alters glycosylation by inhibiting α-glycosidase I. It is a substance alkaloid. This drug has also been shown to have anti-HIV activity. Other compounds of the group (they are involved in glucose production during glycoprotein processing) They are called trimming glycosidase inhibitors because they inhibit the normal removal of residues. ) is dihydroxymethyldihydroxypyrrolidine (DMDP) and deoxy Nojirimycin (DNJ or DNM) C Gruters et al., 1 987, Nature 330 Near 4; Tymes et al., 198 7, Lancet (i) 1025; Walker et al., 198 7, PNAS, 84:8120].

Dipyridamole (DPM), also known as versantin, causes coronary artery vasodilation and It is commonly used in cardiovascular diseases to inhibit platelet aggregation. FitzGerild, 1987, New Engl, J .

Med, 316:1247), whose most studied mechanism of action is nucleoside transport. It is an inhibition of Through this mechanism, it can be used for nucleotide production [Reuse J In principle, it affects the route. It also inhibits cAMP phosphogesterase altering prostaglandin production, adenosine levels and C Altering D4 expression, altering cell surface properties, and interferosomal It has also been reported that it stimulates the production of cancer. DPM is also effective against some viruses. However, it has been reported that the AIDS virus or virus has activity. Activity against any of the toroviruses has not been reported [Toneu ) et al., 1977, Acta Virol, 21:146-150), and , DPM or similar potentiating agents and antiviral agents have not previously been known. It is not written, nor is it written.

Summary of the invention It is therefore an object of the present invention to provide effective treatment for HIV and other drugs that are equivalent to each drug used alone. present a combination of drugs with synergistic advantage as inhibitors of retroviral activity. It is to provide.

A further object of the invention is to convert HIV-infected cells to IIV replication (with the drug combination of the invention). A method of inhibiting HIV proliferation consisting of contacting the patient in sufficient quantities to prevent the production of HIV. The goal is to provide the following.

Yet another object of the invention is to treat infected or uninfected hosts with the AIDS virus. Administering a drug combination of the invention in an amount sufficient to primarily inhibit AIDS virus activity. to treat AIDS and other HIV-related syndromes consisting of is to provide a method of prevention.

An additional object of the invention is to treat retroviruses other than AIDS in humans and animals. To treat or prevent disease.

Other objects and advantages of the invention will become apparent from the following detailed description of the invention. Will.

Brief description of the drawing These and other objects, aspects, and attendant advantages of the present invention are described in the appendix. When considered in conjunction with the drawings, it is helpful to read the detailed description below. There will be: Figure 1 shows dipyridamole (DPM) alone and chain-terminating dideoxynucleotides. in human monocyte/macrophage (M/M) cultures in combination with leoside. Figure 3 shows effects on HIV-1 replication. Δ: AZT alone, DPM alone or AZ Adherent-purified cryopreserved monocytes/macrophages treated with T and DPM.

Cells were treated for 1 day and containing appropriate drug concentrations on days 6.11 and 14. The cells were re-fed with medium containing A 100 μl sample of supernatant containing HIV-1p24 was separated. was analyzed. The figure shows the results on day 14. AZTO only shows DPM processing Ru. Data show that DPM enhances the antiviral effects of AZT. 6 and The 11th day showed qualitatively similar results. B: Since washed monocytes were used Other tests are similar to those in A. Cells were treated with drugs at day 18 of culture, and Refed by replacing 100 μl of medium (without drugs) at intervals of 3-4 days. Ta. Data show p24 levels on day 17 and antiviral activity and and AZT's antiviral activity. The pattern of effects is on the 14th day. It was similar.

C and D: 2',3+-dideoxycytidine (ddC) and 21. al-di Same as in B except that deoxycytidine triphosphate (ddeTP) is used respectively. From the same exam. Again, DPM inhibits viral replication and ddC and dd Enhanced the antiviral effect of CTP. The pattern of effects was similar at day 14. . Mean of 4 wells ± S, E, M, (standard error): Some bars are too It is also so small that it is impossible to see it.

Figure 2. Time course study of HIV infection in washed M/M treated with DPM alone. The results are shown below. Same test as in Figures 1B-D. Cells were processed for 1 day. , and samples of culture supernatant were HI Sampled for V-1p24 assay. The sample is in a total volume of 200 μl in the well. 100 μl of the antigen removed at the previous time point, and the cumulative p24 production Calculated by correcting. DPM alone showed antiviral activity. be written. Mean of 4 wells ± S, E, M,.

Figure 3 shows the relationship between AZT and DPM in their cytotoxic effects on human bone marrow granulomonocyte precursors. It shows the interaction between.

Details of CFUG, verification, calculation of combination index (C,1,) and other tests are given below. Described herein below. Panels A and B represent mutually exclusive models, respectively. model (same site/mechanism of action) and mutually non-exclusive model (non-same site/mechanism of action). C calculated based on 1. Show value. Different symbols mean different healthy suppliers represents cells from. This result suggests a synergistic effect between AZT and DPM in bone marrow toxicity. Indicates that there is no use for it.

FIG. 4 shows T-lymphoblast-like cells (CEM-SS) infected with HIV-1. :oh DPM-AZT (panel A) and DPM-castanospermine (panel A) Figure 2 shows the effect of combination chemotherapy in LeB). Panel A shows that cells are infected with HIV-1 (I[ The results are shown when infected by co-culture with H9 cells harboring [ll]. process was started in one day. In this and other similar tests, the anti- The effect of DPM on viral potency was modest, but the cellular effects from AZT toxicity There was outstanding protection. Therefore, the in vitro therapeutic index has been significantly enhanced. Ta. Panel B shows the results of a similar study for DPM and castanospermine. vinegar. At high concentrations, DPM enhanced the antiviral effects of castanospermine. This conclusion As a result, a wide range of drugs are effective against HIV and other viruses. It emphasizes interaction.

Figures 5A and 5B show 6 cells at 37°C with washed human monocytes/macrophages. Intracellular release of phosphorylated derivatives of 3H-thymidine and 'H-AZT during incubation for hours. HPLC profiles showing the effect of DPM on accumulation are provided. DPM significantly reduced the appearance of phosphorylated thymidine (THY), but phosphorylated AZ The appearance of T was only slightly reduced.

Figure 6 shows nitrobenzene, an inhibitor of nucleoside transport that is similar to DPM. In human M/M with AZT in combination with luthioinosine (NBT I) Figure 2 shows inhibition of HIV-1. Attachment - Purified M/M cultures were freshly bled The cells in Figure 1A are prepared from donor blood and are not cryopreserved. The test was conducted in substantially the same way. At the concentrations tested, NBT I is a virus inhibited replication and enhanced the anti-HIV activity of AZT. 4 days on the 11th day mean±S, E, M,.

Figure 7 shows HIV in human M/M with AZT in combination with papaverine. -1 inhibition. Papaverine (PA) is a cellular cyclic AMP phosphogester The property of inhibiting the activity of enzymes matches that of DPM. The test was conducted in the same manner as in FIG.

At the concentrations tested, bapaverine inhibited viral replication and AZT's anti-HIV Enhanced activity. The pattern of effects was similar on days 13 and 21.

Mean ± S, E, M, of four wells on day 11.

Figure 8 shows H r in human M/M with AZT in combination with myoflagin. Inhibition of V-1 is shown. Like DPM, myofradin is an inhibitor of nucleoside transport. It is.

The test was conducted similarly to the test for FIG. At the concentrations tested, myofradin was inhibited viral replication and enhanced the anti-HIV activity of AZT. MF: Mioff Radin.

The pattern of effects was similar for 13 and 21 days. Mean ± S of 4 wells, E, M.

Detailed description of the invention The above and various other objects and advantages of the present invention provide for antiviral and potentiating agents. the combination produces higher virus inhibition than would be expected from the activity of each component alone, an effective amount of an antiviral agent and agent to inhibit viral reproduction in infected cells; Achieved by a pharmaceutical composition comprising a potency enhancer and a pharmaceutically acceptable carrier. . The compositions and methods of the present invention may be used for retroviruses or Diseases caused include HIV-1, HIV-2, HTLV-1, HTLV-n, etc. include. Of course, this composition may contain sterile agents, adjuvants, non-toxic sterile buffers, etc. may include those commonly used in such preparations. , and are well known to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein refer to The same as that commonly understood by a person of ordinary skill in the field to which the invention pertains. It has the meaning of Any method similar or equivalent to that described herein Although materials such as and materials can be used in the practice or testing of the present invention, preferred Methods and materials are now described. All publications mentioned below are incorporated by reference. It will be incorporated into the specifications. Unless otherwise specified, the techniques used herein are well known to those skilled in the art. This is the standard methodology.

As used herein, a "strand extension terminator" refers to refers to a compound or its precursor that stops the elongation of the chain. These drugs are For example, for enzymatic activity in the phosphorylation of physiological nucleosides and nucleotides. They may also act by additional mechanisms, such as by antagonizing them. These transformations Compounds usually, but not always, have the general formula: (In the formula, "base" is treated with adenine, guanine, cytosine, thymidine and metabolic to obtain a molecule that can be incorporated into a DNA strand or a functional equivalent of said molecule. Analogs, derivatives that can be incorporated into the growing DNA strand to act as analogs or salts thereof, selected from the group consisting of: R represents H, an azide group or another group that does not allow for 3' bonding of the next nucleotide; thereby inhibiting retroviral DNA chain elongation under the influence of reverse transcriptase, :and X represents H, --, ni, tri- or other phosphate groups and analogs thereof . ).

Ribose-based dideoxy sugars have some functionally similar chain termination properties. by different organic moieties in compounds such as adenylphosphorus, citalurene and PMEM. Replaced.

The compound represented by general formula (1) is AZT or another 2',3'-dideoxy Preferably it is a synucleoside or a nucleotide. Chain extension terminator Nucle Typical examples of dideosides are azidothymidine, 21.31-dideo xycytidine, 21.31-dideoxyadenosine, 2T, 31-dideoxythi midine, 2',3'-dideoxyguanosine, 2',3'-dideoxyinosine etc. Dideoxynucleotides are m-, di-, and tri-nucleotides of dideoxynucleosides. Including phosphate salts. One (or more) phosphate group is a phosphorothioester methylphosphonate groups or other similar groups. Compounds similar to the above are also included. Such compounds (e.g. phosphorothioates and and methylphosphonate) are metabolically more stable than the parent nucleotides.

The term "enhancing agent" refers to divyridamole (DPM) and nitrobenzylthioi Nosine (NBTI): Dirazep: Lidoflazine: Hexabenzine: 7-Bromo -1゜5-dihydro-3,6-dimethylimidazole (2,1゜6) quinazoline -2(3H)-ones, myoflagin, etc., which foster overlapping mechanisms of action. Refers to similar drugs. These drugs are inhibitors of nucleoside and nucleobase transport. be. There is overlap in the effects on cyclic AMP phosphogesterase between papaverine and and mopidamol.

Among these compounds, DPM is preferred.

As used herein, the term "synergistic" or "enhanced J effect" refers to the and the potentiating agent showed higher antiviral activity than would be expected from the activity of the core component alone. This means that it produces a gas effect.

Various cell types such as monocytes, T lymphocytes, T lymphocyte tumors, macrophages etc. can be utilized as host cells for testing viral infection or replication. Wi virus activity, especially HIV activity, p24 production, reverse transcriptase activity, transactivator or other marker antigen production. Use of specific cell types or the method of measuring viral activity is not an essential part of the invention and the test Either intraluminal or in vivo systems can be used for testing viral activity.

An important part of the invention is that the enhanced antiviral efficacy is achieved by using non-toxic doses of DPM or When administered with one of the other reinforcing agents listed above, the chain elongation terminator causes and the discovery that it is caused by the α-glucosidase inhibitor castanospermine. be. DPM does not enhance the activity of AZT against human bone marrow cells in vitro. Both are also important. Bone marrow suppression is a dose-limiting toxicity of AZT in humans. It is known that there is. Chain terminators and/or reinforcing components may be used in combination. Conjugations either neat or carried by liposomes or other delivery vehicles It may further be stated that the drug may be administered separately. How to produce liposomes is up to those skilled in the art. are sufficiently known or such techniques are not an essential part of the present invention.

The reinforcing agent may be administered neat or in the form of a polymer.

Although preferred materials and methods are now described, it is for illustrative purposes only. However, it is not intended to limit the invention.

material and method ■And Diviridamol [2,6-bis-jetanol-amino-4,8-diviridinopyri mido-(5,4d)-pyrimidine] from Sigma (Missouri) St. Louis). In most tests, it was found that ethanol and the 0.1M stock solution was diluted to the test concentration (ETOH<a, (z%)). interpreted. 0.1 MDPM stock solution in O,1N HCl (pH readjusted to approximately 3) dilutions do not detectably affect the final pH of the medium and substantially gave identical results. AZT is Ash Stevens (Ash 5teven) s) (lot number HLRO221) and Development Therapeutics Department.

AIDS Program (Developmental Therapeutic Order CS B) Ranch.

AIDS Program), N.I.A.I.D. d dC and ddCTP were purchased from Moravek Biochemicals (Moravek Bi (Brea, California).

3H-ddeTP was purified again by HPLC as described below. nitrobenge Luthioinosine (NBTI; also abbreviated as NBMPR) is Obtained from Aldorich, Lewaukee, and Verin was obtained from Sigma, St. Louis, Missouri.

・　　　　　　　　Malov　-1 Cells were prepared and described by Gartner et al. (1986, Scig. icg, 233, 215-219). HIV-INIH/USA recovered from and grown in M/M /1985/HT LV m-AL was infected. In short, peripheral blood mononuclear white Ficoll from a healthy HI V-1 antibody negative donor whose blood cells were leukopherized. Obtained by high-bark separation.

- M/M with >95% non-specific esterase positive cells by late attachment purification M populations were obtained. The purified cells were incubated with 20% heat-inactivated fetal bovine serum (Fe2 ) and 10% (final concentration) DMSO in RPMI 1640. Stored frozen according to Prior to infection, cells were thawed, washed and treated with DMSO. and 10% pooled human serum, 20% FCS and antibiotics. was suspended in supplemented RPM11640.

Infection is 0.5-1. Virus inoculum containing Oxl O'cpa RT activity 1- by exposing 10' pelleted cells at 37°C for 45-60 minutes. It was carried out by After infection, cells were washed and cultured in a 96-well Coster-Mike. Rotiter Plate (Coster Mlcrotiter Plate) RPM11640 culture medium supplemented with 20% heat-inactivated FC5 and antibiotics. The cells were seeded in the ground (8 x 10'' cells/well).

′　　　　　　　Malovji　 Peripheral blood monocytes were tested as pyrogen-free phosphate buffer was used in the washing step. Standard countercurrent centrifugal washing [Wahl et al., 1984. CeLL I *munoL, 85.373-383; Wall et al., 1984. Ce LL Imwruno L, 85.384-395). new M/M prepared in 2008 was infected as described above. They are then washed and 10% FC3, 2sM glutamine and antibiotics in 96-well cosc plate 1c2X10' in Dulbecco's Modified Eagle's Medium (D-MEN) supplemented with Cells/well were seeded. After blating and washing, washed monocyte cultures are >99% positive for L e u M 3 and non-specific esterase I understand.

One day after infection, the cultured cells were completely Washed once with medium, and the drug to be tested is added to the microtiter wells. It was. Cultures were mixed with half of the cell supernatant (100 μi) in fresh medium (with drugs as indicated). (with or without drug) at 3 to 4 day intervals. Ta. The supernatant sample was prepared using an enzyme-linked immunoassay [ELISA] kit [Praue DuPont Co., Wilmington, AL; Catalog Number N HIv-1p24 antigen was analyzed using EKO41). NBTI, Bababeli Tests with myoplastine and myofradin were conducted similarly.

゛　　　　　　　　　.

ddeTP was incubated with uninfected washed M/M (5X10" cells/well). were kept warm under normal conditions in the Rus study. The Joname sample was sentry-free. Filter (Centrifree filter) C Massachusetts Da centrifuged through Asicon Co., Ltd. VYDAC303NT405 nucleotide column [Hesperia, California] Separations Group Metabolites of ddcTP were analyzed using HPLC. Column is 0.035 sM ammonium formate pH 4,65 and 0.5M IJ sodium formate pH 2 It was eluted with a 0-50% linear gradient of 8 (formed over 10 minutes).

Cell uptake of ddeTP was determined by adding 1 μM 'H-labeled ddeTP to uninfected washed M /M culture (5xlO' cells/well), followed by extensive washing of cells ( 4 times in water-cold saline), removed from the plate (0.5% Triton X-100 ) and were evaluated under similar conditions by measuring cell-associated radioactivity.

No ko - xl ゛ LJ-Zl The washed monocytes were placed in a 6-well microtiter plate (Costar) 5X. IO' cells/well were seeded. Uptake studies were started on the second day after cell preparation. It was.

The medium is either 8H-thymidine (Dupont) or 'H-AZT (Moravek). and DMEM-10% FC3l containing 0.5 μM unlabeled AZT. . Incubation was then continued at 37° C. in a CO7 thermostat. At various time points the supernatant was removed. , and the wells were quickly washed three times with ice-cold DMEM containing 20 μM DPM. purified (to prevent further transport). 0.5% Triton X-1000 ,9- was then added to each well, and the plate was rocked for 30 min at room temperature. . The contents were placed in a 1.5-polypropylene centrifuge tube [Eppendor f)) and 0.14 of 50% trichloroacetic acid was added. this far The heart tube was then placed in a microfuge and rotated at high speed for 10 minutes. I let it happen. The supernatant was collected and diluted with 20% tri-n-octylamine (trichlorotrifluoride). 1.2i in fluoroethane and vortex mixed. This extraction operation After cropping, the aqueous phase was transferred to another tube and freeze-dried. The sample is then vaporized. Dissolved in 120 μβ of distilled water and vortex mixed. The sample (100μl) is Verified by HPLC using a VYDAC column and the elution scheme described above. . Fifty aliquots from each run were counted in scintillation vials. in general Two wells were collected and separated at each time point.

Super r・ O2-production in uninfected washed monocyte/macrophage cultures was According to Le et al. (1987, J. I. Munor, 139, 1342-1347). as described by measuring 01-dependent production of cytochrome C. was valued.

On day 144, the medium in each of the four wells was 160 μM ferricytochrome C. was replaced by 100 μl of Earle's balanced salt solution containing. Cells were then incubated at 37°C for 3 were incubated for hours and Ox-production was determined by measuring A,1. value is calculated from the molar extinction coefficient of reduced cytochrome C, and is calculated from the molar extinction coefficient of reduced cytochrome C. nanomole corrected for the presence of microorganisms and a vertical optical path of 3 μ through the well volume. Expressed as O3-/well. Base O! -Production depends on any Ol present - Add superoxide dismutase (final concentration 600u) to the wells to destroy g/+J): Phorbol ester stimulated production Add phorbol myristate acetate (final concentration 10 ng/++/). spontaneous production was determined without the addition of stimulants.

11”0[”1 Collection of bone marrow samples from healthy volunteers and granulocyte/monocyte colony formation (C For the FUc, ) test, Fine et al. (1987, C11n, 0 Standard procedures were used as described by Ta. Briefly, cells were separated on Ficoll-Hyberk and heated to 20% on inactivated FC3, 2mM glutamine and human myeloid leukemia lineage (P-38) 15% (v/v) HrV negative colony stimulating factor (C3F ) was suspended in Matsukoi's 5A medium supplemented with. Cells were prepared using the soft agar method. Pike et al., 1970. J, CeLL PhysioL.

0.3% with 20% FC3 and 15% C3F according to 76, 77-843. Blated with final concentration of agar. After 9-12 days of incubation, >40 cells colonies were counted. Approximately 10 cells from brated 2XlO' mononuclear cells 0 colonies were formed. Cells were collected from Kubota for morphological examination. a) etc.<1980. Exp, He*atoL, 8゜339-344) method stained by. For each supplier, the effects of AZT and DPM17) alone; and 2 to 10 different AZT/DPM ratios ranging from 0.16 to 20. The following combinations were tested.

1, above and below V' CD4”, OEM-SS and MT-27 lymphoblastoid cells were incubated with 10% FC3. The cells were grown in RPMI-1640 containing RPMI-1640. The cells are then free of HIV-1 (I[[, ) infecting either the stock or HIV-1 (RF) infected H9 cells, and A 96-well microtiter plate was inoculated. After 7 days of culture, tetrazo Lium salt "XTT" (1■/-) and electron acceptor phenazine methosulfate 50- of solutions containing (0.01-0.02 mM) were added to each well. medicine Uninfected cells or cells that are protected by the agent and continue to proliferate have a 0. D. produces soluble orange formazan which is read at 450n-. healthy cells and The data related are: % of untreated control formazan = (Test 0.D, 00) / (cell control 0.0.). This tetrazolium dye Tests and other assays such as p24 antigen capture, syncytium formation and tribabum It should be noted that there is a correlation with Roux exclusion.

1 hippa Mononuclear cells from healthy volunteers were treated with phytohemagglutinin (PHA) (5 μg). /yt) for 2 days and then 15% FC3, 1% L- IL2 in RPM11640 with glutamine and 0.1% gentamicin It was stimulated by After 3 days, cells were incubated at 37°C with various titers of HIv-t(I[[e)]. Infected for 0 minutes. After washing, cells were plated in 3X10' wells in microtiter wells. Cells were inoculated with 10.5+nl. The drug is added immediately after blating and then Their concentrations were then held constant in each re-batch. Place half of the supernatant on the cells. The cells were re-fed every 2-3 days by changing the solution. 10 days after infection, p24 and the cells were tested with Trivan blue exclusion.

Enie Mameoka The 50% inhibitory dose (IDS) of the drug tested is Log [f, / (1- f, )) versus ogD (intermediate effective plot), where f is the affected fractional value (i.e. percent inhibition/100) and D is the drug dose) Calculated by plotting [Chou et al., 1984. Ad v, Enzyme ReguL, 22.27-55; Chu et al., 19B? , New Avenues in Developmental Cancer Chemotherapy -J (New Avenues in Developmental Ca ncer Chemotherapy), Haratsubu, K.

R., and Connors, T. x I. (Harrap, R.).

& Connors, T., A.) (Bristol? Years Symposium) um series (Bristol Myers Sy@posium 5eri es), Academic Press (Acade+sic Press), New York ), pp. 37-64]. Linearize the dose-effect relationship and intercept it on the x-axis ID as,. The plot giving was constructed (Chu et al., 1986). Dosage-effect on microcomputer Analysis: Apple ■ or IBM computer software for PC [UK Elisevier Btos of Cambridge oft)

Drug interactions in the CFUcv assay are determined by the “Combination Index (C.

1.) J, i.e. drugs when used alone and in combination is analyzed by calculating the parameters derived from the comparison of equi-effective doses of (Chu et al., ibid.: Chu et al., ibid.). of different fractional values between 0.1 and 0.9 Equi-effective doses of AZT alone and DPM alone at the level of efficacy are intermediate effective plots. Obtained from

Results Results As shown in Figure 1A, the AZ T inhibited virus replication in cryopreserved, adherently purified cells.

Dipyridamole in the range 0.08-10 μM has little effect on its own. However, unexpectedly, it significantly enhanced the antiviral effect of AZT. . TD of AZT, for example, in the presence of 2 μM DPM. and ID5s level (Table 1) were reduced by 5-fold and 10-fold more, respectively (i.e., AZT alone up to 18 and 8% of the value of ). Figure 1B shows the reinforcing effect of DPM in a relative sense. It is shown that it was further enhanced in washed M/M cultures. 2 or 108M D In the presence of PM, p24 expression is basal at each AZT concentration, including the lowest 1.6 nM. It was close to the level. This degree of inhibition is at the 1 μM level when AZT is used alone. Achieved only with Bell. Unexpectedly, dipyridamole also and the anti-HTV effects of 2',3'-dideoxycytidine triphosphate (ddCTP). Reinforced. ID*-values for these drugs are lower in the presence of ≧2 μM DPM. It decreased by at least 5 times (Fig. 1C-D). In the cleaning M/M system, DPM itself The body appears to be inhibitory, with a marked and unexpected decrease in p24 expression (Figure 2).

This effect of DPM alone was found to be concentration independent in the 2 to 20 μM range. won.

of 　 　 ゛ 　 Since nucleotides do not easily enter cells, ddCTP itself is an intracellular molecule. It was considered to be relatively ineffective against Lus RT. However, ddCTP Culture medium width approximately 12-14 hours, 7. Therefore, from dde-phosphoric acid to dde It was found by HPLC analysis that the This time course is “H-ddCT This corresponded to the time course of 3H accumulation in cells incubated with P. −Incorporated together These observations suggest that the conversion of ddcTP to ddC rate-limits uptake. , indicating that the preferential species entering the cells was probably < ddC.

To Marovji Cell counting in culture wells using an inverted microscope was performed after treatment with AZT and/or DPM. After 2 weeks of treatment, it was shown that there was no difference between the control group and the treated group on uninfected cells. . Cell viability of infected M/M cells was assessed by trypan blue exclusion. >95% in each group.

Super O S' Table 2 shows O7 production by uninfected M/M treated with AZT and/or DPM. shows. The principle importance of these data is that, at the concentrations to which they are applied, AZ There was no discernible DPM toxicity with or without T. deer However, some trends are numerically small but statistically significant. It was. Naturally occurring superoxide levels decreased with increasing AZT (p< 0.001: latter two types), while spontaneous generation (p=0.013) and PMA stimulation Both superoxides increased with increasing AZT (p < 0. OOl). Their relationship to the observed antiviral activity remains to be determined. ing.

m2 To evaluate the bone marrow toxicity of various combinations of AZT and DPM, CFUG, assay was performed on bone marrow samples from seven healthy donors. AZT average ID6. is a test Calculated from pooled values in the effective dynamic range (5-95% inhibition) of , this is 6 uM+:o, 1 uM (S, E, M, ;n-21) Ta. Even at high concentrations, DPM also inhibited colony formation (IDsa = 10.0 ±4.5 u M: n = 19) Shown in 3rd AFXJ C obtained from six suppliers over a wide range of inhibition levels, 1. value is" In the mutually exclusive model, they clustered around 1, suggesting substantial toxicity. do. In the “mutually non-exclusive” model, the value of,C,1,is well〉1 in Figure 3B. This may lead to an unexpected shift toward toxic antagonism rather than toward toxic synergy. This suggests a trend toward Pure granulocytes, pure monocyte colonies and mixed granulocytes - monocytes The relative frequency of colonies was not affected by treatment. Antiviral research and Together these results demonstrate that if AZT is combined with ≦2 μM DPM, then A Obtaining equivalent antiviral efficacy with lower myelotoxicity than with ZT alone and, unexpectedly, the in vitro therapeutic index was therefore high. This suggests that

In summary, the data presented herein demonstrate that DPM has a unique effect on HTV. and it has the antiviral effects of AZT, ddC and ddCTP in humans alone. clearly showing enhancement in globules/macrophages. In particular, treatment The relationship from this point of view is that in the combination of AZT/DPM, DPM is associated with the bone marrow toxicity of AZT. , and unexpectedly the therapeutic index in vitro was therefore increased. That was my discovery. Surprisingly, DPM is capable of inhibiting DPM-sensitive nucleoside transport. Mainly enters cells via [Zimmelmann et al., 19 87. J, BioL, Cohem, 262.5748-5754) It was also found that the anti-HIV activity of dde was enhanced. However, d Compared to dC, which is a physiological nucleoside, de does not have a hydroxyl group on one side. , which is more lipophilic. Therefore, the greater inhibition of DPM in the transport of dC effect may explain the enhanced dde activity.

Hi 1゛Pa　　　　　　(Russ) The results shown in Figure 4A demonstrate that the human T lymphoblast-like cell line (CEM-3S) shows the beneficial effects of DPM upon treatment; It abolishes the toxicity of AZT, but its activity against HI V-1 (HTLV-1 [[Rv)] Enhance or have no effect. Consequently, the therapeutic index in vitro was not as expected. It was heightened in a way that was never seen before. MT-2, another human T lymphoblast-like cell line Nine cells were more susceptible to the toxic effects of both AZT and DPM. As shown in Figure 4B. The results presented also demonstrate that castanoin in CEM-5S cells at high concentrations of DPM. It shows a surprising enhancement of spermine's anti-HIV activity.

desecrated by A　　　　　　-4・ Table 3 shows the results of tests on PHA-stimulated T lymphocytes. The data is 0.074 An unexpectedly low DPM concentration of μM shows significant enhancement of AZT activity.

Cell counting and trypan blue exclusion only at the highest dose of AZT (10 μM) It showed major drug toxicity to cells.

These results indicate that DPM acts as a chain elongation arresting agent in T lymphoid cells as well as M/M. This is important because it shows that it can enhance the antiviral effect of. These are HIV infected These are the two most important cell lineages.

“6. - Shows the results obtained in tests on thymidine and 3H-AZT. DP M significantly reduced the appearance of phosphorylated thymidine species, whereas phosphorylated It had only a slight effect on the appearance of AZT. Two independent studies showed that phosphorus showed a qualitatively equivalent effect of DPM on the intracellular appearance of oxidized 3H-thymidine. Ta. In three separate studies, DPM was shown to induce intracellular phosphorylated “H-AZ” The amount of T was increased or left unchanged.

-hi”hiy”11”1 Certain analogs of DPM are also known for their inherent activity against HIV. and for their ability to enhance the activity of the chain elongation-terminating antiviral agent AZT. Evaluated. As shown in Figure 6, the nucleoside transport inhibitor NBT I has an inherent inhibitory effect on HIV-J replication in M/M, and also has an inhibitory effect on HIV-J replication in A Strengthened the inhibitory effect of ZT. As shown in Figure 7, bapaverine showed similar results. gave. As shown in Figure 8, myofradin also has an inherent inhibitory effect. , and enhanced the inhibitory effect of AZT. Similar to DPM, myofradin is inhibits oxidative transport, but crosses the blood-brain barrier more easily than DPM. [Deckerl et al., 1988. Life 5science 42゜1331-1345. Wauquier queer) et al., 1987. Psychophar Otori, jll, 434- 439]. Therefore, the unexpected and surprising antiviral effects of these DPs M analogs were obtained alone and in combination with AZT.

Without being tied to any theory, does DPM or its analogs strengthen? , or mechanisms that act synergistically to block nucleoside transport and/or to inhibit cyclic A Due to the well-established activity of DPM in inhibiting MP phosphogesterase activity It is expected that this is mostly due to this.

Plasma concentrations of DPM in excess of 10 μM can be maintained in humans. Therefore, the actual The study described in Example 1 showed that 2 μM (in 10%-20% fetal bovine serum) and Some studies have shown that lower DPM concentrations showed reduced dideoxynucleoside-mediated inhibition of HIV. It is important to demonstrate the enhancement of harm. DPM is mostly bound to proteins in the blood has been done. Therefore, at a given total concentration, free drug levels are Expected to be higher in tissue culture tests. Other strengthening agents are the same as DPM. used for.

The present invention now provides chemotherapy for treating AIDS or other viral diseases.

The method involves administering the disease to a host suffering from a viral infection, including an AIDS virus infection. administering an effective amount of the drug combination of the invention to inhibit the replication of the virus that causes It consists of giving. This drug combination can be administered by any suitable route, e.g. oral, parenteral. Orally, such as intrathecally, and may be administered as daily as tolerated. Not infected individuals or those exposed to the virus should be treated in a similar manner to prevent infection. It can be done. Antiviral agents, such as AZT, dde, etc., are effective against DPM and/or its The analogs may be administered in combination or on an alternating schedule.

The examples and implementations described herein are illustrative only. , and various modifications and changes in this information will be suggested to those skilled in the art. and within the spirit and scope of this specification and the appended claims. It is understood that

Table 1. HI in adherent purified human M/M cultures in the presence and absence of DPM Levels of AZT that elicit 50% and 95% inhibition of V-1p24 antigen expression ID S. and ID□ values were calculated from the formula for the intermediate effective plot (see data analysis section). (see). Linear regression coefficients for rniblots. The percentage in parentheses is for AZT alone. ID. and regarding the value for the ID5s value.

AZT (μM) Processing ID5a IDes rTable 2. A to superoxide (0,-) production in washed human M/M cultures Effects of ZT and DPM Superoxide production (in healthy cells for 14 days in culture) It is adopted as a functional index. The details of the test are as follows: The cells are not infected with HTV-1. This was the same as in Figures 1B-D, except that Values are nanomole O3−/wa It is expressed as a file. Mean of 4 wells ± S, E, M,. data is processed For non-parametric tests of either trend, Jonck Table 3. PHA-stimulated human T cells Enhancement of AZT activity on HIV-1 replication by DPM in DPM (μM) 0.0 213 344 341 341 219 1620.156 313 93 8 8 0 00.625 125 3 0 0 0 0 02.5 17 0 0 0 0 0 010.0 7 0 0 0 0 Fractional effect fraction effect FIG, 4A LOG 2 Castanospermine concentration Cμg/ml FIG, 4B H-3H-3 p24 (ng/m1 Lolo■ p24 (ng/m1 Rota■ p24 (ng/m1 Rororo mother■ international search report

Claims (1)

[Claims] 1. The combination of an antiviral agent and a potentiating agent is more effective than expected from the activity of each component alone. to inhibit viral replication in infected cells, resulting in higher antiviral efficacy. a pharmaceutically acceptable carrier; agent composition. 2. The composition according to claim 1, wherein the antiviral agent is a chain elongation terminator. 3. Antiviral agents, potentiators or combinations thereof are encapsulated within ribosomes. The composition according to claim 1, wherein the composition comprises: 4. The chain terminator is a nucleoside or mono-, di- or triphosphorylated. The composition according to claim 2, which is a nucleoside. 5. The chain terminator is a ribose-based dideoxy sugar-free phosphorylated 5. The set according to claim 4, which is a nucleoside analog that is phosphorylated or non-phosphorylated. A product. 6. 11, 21 or triphosphate moieties are phosphonates or phosphorothioates 5. The composition of claim 4, wherein: 7. The chain extension terminator is azidothymidine, dideoxycytidine, dideoxyade Nosine, dideoxythymidine, dideoxyguanosine, dideoxyinosine or phosphorylated derivatives and mixtures thereof. composition. 8. The chain extension terminator is dideoxycytidine or dideoxycytidine triphosphate. The composition according to claim 7. 9. 8. The composition according to claim 7, wherein the chain extension terminator is azidothymidine. 10. The antiviral agent is castanospermine, dihydroxymethyl hydroxide. selected from the group consisting of cypyrrolidone, deoxynojirimycin and mixtures thereof; selected glucosidase inhibitors plant alkaloids or their derivatives or A composition according to claim 1, which is an analogue. 11. 11. The composition of claim 10, wherein the alkaloid is castanospermine. l2. The fortifying agent is nitrobenzylthioinosine; dirazep; lidoflazine; Hexobenzine; Myofrazine; 7-bromo-1,5-dihydro-3,6-dimethane tylimidazol-(2,1,6)quinazolin-2(3H)-one; or the like; The composition of claim 1 selected from the group consisting of derivatives and analogs of. 13. The fortifying agent is dipyridamol, mopidamol, papaverine and mixtures thereof. 2. The composition of claim 1, wherein the composition is selected from the group consisting of compounds, analogs or derivatives. 14. 14. The composition of claim 13, wherein the toughening agent is dipyridasol. 15. the chain extension terminator is AZT, ddC or ddCTP; A composition according to claim 1, wherein the toughening agent is DPM. 16. The chain terminator is AZT, and the reinforcing agent is NBTI, Papave. A composition according to claim 1, which is phosphorus or myoflagin. 17. to inhibit or prevent the proliferation of disease-causing viruses; An effective amount of the composition of claim 1 is administered to a normal host or a host affected by a viral infection. A method of treating or preventing a viral disease consisting primarily of administering. 18. Symptoms of the virus include HTLV1, HTLV2, HIV-1 and HI 18. Associated with a retrovirus selected from the group consisting of V-2. the method of. 19. an antiviral agent as the first agent, and the efficacy of the antiviral agent to the same extent; A second method that reduces the toxicity of antiviral agents to the host without reducing the A pharmaceutical composition consisting of a drug. 20. to inhibit or prevent the proliferation of disease-causing viruses; An effective amount of the composition of claim 19 is administered to a normal host or to a patient suffering from a viral infection. A method of treating or preventing a viral disease comprising administering to a host.