MXPA99001962A - Use of pkc inhibitors for the manufacture of a medicament for the treatment of aids - Google Patents

Abstract

A method for treating HIV infection is disclosed, particularly using the isozyme selective PKC inhibitor, (S)-3,4-[N,N'-1,1'-((2''-ethoxy)-3'''(O)-4'''-(N,N-dimethylamino)-butane)-bis-(3, 3'-indolyl)]-1(H)-pyrrole-2,5-dione or its acid salt.

Description

USE OF PKC'S I NH I BIORES FOR THE MANUFACTURE OF A N EDICAMENT FOR THE PROCESS OF AI DS This application claims the priority benefits of Serial US Provisional Application No. 60 / 024,873, filed on August 30, 1996.

BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION The present invention is broadly directed to a method for inhibiting the activation of latent human immunodeficiency virus (H IV). The invention is also directed to a method for inhibiting H IV replication. The present invention is particularly directed to the use of a particular class of isozyme-selective Protein Kinase C (PKC) inhibitors to treat H IV infection. 2. Description of the Related Art The H IV epidemic continues to grow at a rapid rate, and the clinical manifestations associated with this viral infection present increasingly complex socioeconomic and medical problems. The infection of H IV ag ua leads to a period of rapid viral replication, followed by vi remia that results in the infection of 1% or more of the circulating T lymphocytes, the primary target of the I virus. However, the viremia is transient, since the cells infected with HIV are removed from the circulation by means of an effective host immune response, which results in a 10 to 1000 fold decrease in the T cells infected with H IV. Unfortunately, there is still no effective therapy to prevent viral activation after exposure. Thus, although the response of the initial host is effective in reducing and controlling the number of cells infected with HIV, it is not sufficient to prevent latent postintegration or persistent low-level asymptomatic infections (LLP) of host reserve cells, such as circulating CD4 + T lymphocytes and monocyte / macrophages. Thus, the last pathogenic effects of H IV are not prevented and after the induction of the LLP or latent state, develops acquired immunodeficiency syndrome (AI DS). A cure for HIV infection is not yet found. Current treatments for H IV infection attempt to slow the progress of the disease or relieve its symptoms. The treatment that is in use today include certain dideoxynucleotides, such as azidothymidine (AZT or zidovudine, Burroughs Wellcome), dideoxyinosine (dd I, Bristol-Myers Squibb) or dideoxycytidine (ddC, Hoffman-LaRoche). These agents can be toxic. Its applicability is limited due to the appearance in some patients of expensive and sometimes lethal side effects. These side effects include myelosuppression, peripheral neuropathy, and pancreatitis. In some patients, AZT has lost its effectiveness after prolonged use. Although other medications have been proposed for the treatment of H IV infection. Including the recent introduction of several H IV protease inhibitors, one that is completely effective has not yet been shown. Therefore, there remains a need in the art to develop additional therapeutic agents to treat an H IV infection.

BRIEF DESCRIPTION OF THE INVENTION Therefore, an object of the invention is to provide a method for inhibiting the replication of human immunodeficiency virus in an infected cell. Another object of the invention is to provide a method for inhibiting the activation of immunodeficiency virus in an infected cell. Yet another object of the invention is to provide a method for treating a mammal infected with human immunodeficiency virus.

These and other objects of the invention are provided by one or more of the modalities described below. A method of the invention provides a method for inhibiting the replication of human immunodeficiency virus in an infected cell, which comprises contacting the cell with an inhibitory amount of viral replication of an inhibitor of the protein kinase C isozyme. Another embodiment of the invention provides a method for inhibiting the activation of human immunodeficiency virus in an infected cell, which comprises contacting the cell with an inhibitory amount of viral activation of an inhibitor of the proteinase kinase β isozyme. Yet another embodiment of the invention provides a method for treating a mammal infected with a human immunodeficiency virus. , which comprises administering to a mammal a therapeutically effective amount of a protein kinase C isozyme inhibitor.

The present invention thus provides the technique with the identification of effective compounds for treating H IV infection.

DETAILED DESCRIPTION OF THE INVENTION It is a discovery of the present invention that therapeutic use gives a particular class of protein kinase C inhibitors, i.e., inhibitors of the protein kinase C isozyme, and especially selective inhibitors of PKC β isozyme, inhibits the activation and replication of H IV, especially such activation and replication associated with PKC signal transduction pathways. The particular class of PKC inhibitors can also inhibit the activation and replication of H IV, which are associated with cAMP signal transduction pathways. Accordingly, such compounds can be used therapeutically to treat patients infected with H IV. The course of H IV infection is characterized by a short peak of viremia followed generally by a long, albeit variable, period of latent or persistent infection without any intima of disease. The H IV provirus is hd in cells such as peripheral mononuclear cells and T lymphocytes. The activation of the latent H IV provirus in mononuclear cells and T lymphocytes is an important step to initiate the attack of clinical symptoms associated with the syndrome. of AI DS. The activation of H IV includes the states of productive infection and reactivation of latent infection. The replication of H IV includes the multiplication of the H IV genome during the productive infection and reactivation of latent infection. The reactivation of the latent, integrated H IV genome includes the replication of HIV, for example, forming multiple copies of the H IV genome, H IV expression, for example, the translation of virus-specific proteins such as p24, assembly of viruses, as well as releasing infectiHIV particles and H IV proteins. Applicants have shown that the compounds of the present invention can block the expression of H IV, the p24 production of HIV-1 induced by cAMP and PKC signal transduction pathway activators. While not wishing to be bound by any technical explanation, applicants believe that PCK affects viral activation via host cell transcription factors and viral trans-activating proteins. The induction of viral expression from the LLP or latent proviral state can be triggered by several cellular factors including mitogens, antigens and cytokines. The expression of virus depends on the activation state of the host cell and involves the activation of cellular PKC because the stimulation of cells latently infected with PMA, a known activator of PKC, induces virus replication in a dependent manner. the concentration (Laurence, er al., Biochem. Biophys., Res. Comm., 1 66: 349-357 (1 990); Kinter, et al., J. Virol., 64: 4306-431 2 (1 990) ). PKC inhibitors and cellular suppression of PKC by chronic phorbol ester treatment decrease the replication of H IV in chronically infected mononuclear cells induced by phorbol esters, tumor necrosis factor alpha, I L-6 or lipopolysaccharide ( Kinter et al., J. Virol. 64: 4306-431 2 (1990)).

It was suggested that the effect of PKC on the viral genome was mediated by modulation of host cell transcription factors, eg, NF-? B, and of the viral trans-activator protein tat (Gosh, er al., Nature, 344 : 678-682 (1990), Jakobovits, ef al., EMBO, 9: 1165-1170 (1990)). TPA has been shown to enhance the replication of HIV-1 in chronically infected HIV MOLT-4 cell lines and there is evidence that this works by induction of NF-? B, which binds to the HIV LTR enhancer region (Nabel). , ef al., Nature, 326: 711-713 (1987)). A specific role for the isoform of PKC-β in mitogen-stimulated and basal HIV replication activation is implicated by the ability of suppressed PKC cells that are reconstituted with PKC-β to induce transcriptional activation of HIV replication (Jakobovits, et al., EMBO, 9: 1165-1170 (1990)). The faf protein of HIV increases the expression of genes during productive infection up to 100 times. There is evidence that suppressed PKC cells exhibit a marked reduction in transactivation of HIV-1 without any significant effect on the synthesis of faf protein. Transactivation in these PKC deficient cells can be restored by transfection with a wild-type PKC expression vector (Jakobovits, et al., J. EMBO, 9: 1165-1170 (1990)). The state of phosphorylation of DNA topoisomerase II and activity also correlates well with the production of HIV. Inhibition of phosphorylation with inhibitors of PKC (analogues of O-alkylglucerophospholipid) results in the reduction of HIV production (Matthes et al. Antiviral Res., 13: 273-286 (1990)). Additionally, other activators of PKC, OAG and briostatin-1, induce the expression of H IV in chronically infected U 1 cells (Kinter, et al., J. Virol., 64: 4306-431 2 (1990)). Therefore, the PKC inhibitor compounds as described in the present invention can be used therapeutically to treat HIV infection both by suppressing viral activation and by inhibiting viral replication. PKC inhibitor compounds may also be therapeutically effective in the treatment of H IV infection by modulating the signal transduction pathways of PKC and / or cAMP, or by interacting with protein factors that regulate PKC and / or pathways. cAMP. The method of this invention preferably uses those protein kinase C inhibitors that effectively inhibit the β-isozyme. A suitable group of compounds are generally described in the prior art as bis-indolylmaleimides or macrocyclic bis-indolylmaleimides. The bis-indolylmaleimides well recognized in the prior art include those compounds described in U.S. Patents 5,621,098,5,545,636, 5,481, 003, 5,491, 242 and 5,057,614, all incorporated herein by reference. The macrocyclic bis-indolylmaleimides are particularly represented by the compounds of formula I. These compounds, and methods for their preparation, have been described in U.S. Patent 5, 552, 396, which is incorporated herein by reference. These compounds are administered in a therapeutically effective amount to a human to inhibit H IV replication of HIV-infected cells and activation of latent HIV, or to treat HIV infection. These compounds can also be administered to patients at risk of the disease conditions mentioned above as prophylactics. A preferred class of compounds for use in the method of the invention has the formula (I): wherein: W is -O-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) mO -, -heterocycle-, -heterocycle- (CH2) mO-, -bicyclic fused-, -cyclic fused- (CH2) mO-, -NR3-, -NOR3-, -CONH-, or -NHCO-; X and Y are independently C1-C4 alkylene, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; R1s are hydrogens or up to four optional substituents independently selected from halo, C1-C alkyl, hydroxy, Ci-C ^ haloalkyl alkoxy, nitro, -NR4R5, or -NHCO (C-, -C4 alkyl); R2 is hydrogen, CH3CO-, -NH2, or hydroxy; R3 is hydrogen, - (CH2) mariO, -alkyl of C1-C, -COO (aiquilo of d-C4), -CON R R5, - (C = NH) N H2, -SO (alkyl of 0, -04), -S02 (NR4R5), or -S02 (CrC4 alkyl); R4 and R5 are independently hydrogen, alkyl of 0, -04, phenyl, benzyl, or combine with the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2, or 3; and n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof. A most preferred class of compounds for use in this invention is represented by formula I, wherein the X-W-Y- moieties contain 4 to 8 atoms, which may be substituted or unsubstituted. More preferably, the -X-W-Y- portions contain 6 atoms. Other preferred compounds for use in the method of this invention are those compounds of formula I, wherein R 1 and R 2 are hydrogen; and W is a substituted alkylene, -O-, S-, -CONH-, -NHCO- or -NR3-. Particularly preferred compounds for use in the invention are compounds of the formula la: wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, d-04 alkyl, (CH 2) maryl, -N H (aryl), -N (CH 3) (CF 3), -N H (CF 3), or -NR 5 R 6; R5 is hydrogen or alkyl of 0, -04; R6 is hydrogen, alkyl of 0, -04 or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof. The most preferred compounds of formula I are those wherein Z is CH 2; and R 4 is -NH 2, -N H (CF 3), or -N (CH 3) 2, or a pharmaceutically acceptable salt, prodrug or ester thereof. Other preferred compounds for use in the method of the present invention are compounds wherein W in formula I is -O-, Y is a substituted alkylene and X is an alkylene. These preferred compounds are represented by the formula I b: wherein Z is - (CH2) P-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C, -C4 alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof. The most preferred compounds of formula Ib are those wherein p is 1; and R5 and R6 are methyl. Because they contain a basic portion, the compounds of the formulas I, la and Ib may also exist as pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inrogenic acids, such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids, as well as organic acids, such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulphonic, carbonic, succinic, citric acids. , benzoic, acetic, and related inorganic and organic acids. Such pharmaceutically acceptable salts include thus sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 2-butyn-1,4-dioate, 3-hexin-2,5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-I suifonate, naphthalene-2-sulfonate, mandelate and the like. Particularly mesylate and hydrochloric salts are used. In addition to pharmaceutically acceptable salts, other salts may also exist. They can serve as intermediates in the purification of the compounds, in the preparation of other salts, or in the identification and characterization of the compounds or intermediates. The pharmaceutically acceptable salts of the compounds of the formulas I, 1 a and I b may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the crystallization solvent, inherent in the preparation or crystallization solvent, or adventitious to such a solvent. It is recognized that there may be several stereoisomeric forms of the compounds of formulas I, I, and I b; for example, W may contain a chiral carbon atom in the substituted alkylene portion. The compounds are normally prepared as racemates and conveniently can be used as such. Alternatively, both n-individual enantiomers can be isolated or synthesized by conventional techniques if it is desired to do so. Such racemates and individual enantiomers and mixtures thereof form part of the compounds used in the methods of the present invention. The compounds used in this invention also encompass pharmaceutically acceptable prodrugs of the compounds of formulas I, la and Ib. A prodrug is a drug, which has been chemically modified and may be biologically inactive at its site of action, but which can be degraded or modified by one or more enzymatic processes or other processes in vivo to the mother's bioactive form. This prodrug may probably have a different pharmacokinetic profile than the parent, allowing easier absorption through the mucosal epithelium, better salt formation or solubility, and / or improved systemic stability (an increase in plasma half-life, for example). Normally, such chemical modifications include the following: 1) ester or amide derivatives, which can be cut by esterases or lipases; 2) peptides, which can be recognized by specific or non-specific proteases; or 3) derivatives that accumulate in a site of action through membrane selection of a form of prodrug or a modified form of averaging; or any combination of 1 to 3, supra. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in H. Bundgaard, Desig n of Prodrugs, (1 985).

The synthesis of various bis-indole-N-maleimide derivatives is described in Davis et al. , U.S. Patent 5,057,614, and the synthesis of preferred compounds suitable for use in this invention are described in previously identified U.S. Patents 5, 552, 396 and in Faul ef al, EP 0 657 41 1 A1, all incorporated herein by reference. A protein kinase inhibitor-β for use in the method of this invention is the compound described in Example 5g. Hydrochloride salt of ((S) -3,4- [N, N'-1, 1, - ( (2"-ethoxy) -3 '" (0) -4' "- (N, N-dimethylamino) -butane) -bis (3,3'-indolyl)] - 1 (H) -pyrrole-2, 5 -dione of the aforementioned US patent 5, 552, 396. This compound is a potent inhibitor of protein kinase C. It is selective for protein kinase C on other kinases and is highly isozyme-selective, ie, it is selective for beta-1. and beta-2 isozymes Other salts of this compound would also be favored, especially the mesylate salts A preferred mesylate salt can be prepared by reacting a compound of formula II: with methanesulfonic acid in a non-reactive organic solvent, preferably an organic / water mixture, and most preferably water-acetone. Other solvents are also operable, such as methanol, acetone, ethyl acetate and mixtures thereof. The ratio of solvent to water is not critical and is generally determined by the solubility of the reactants. Preferred proportions of solvent to water are generally from 0.2: 1 to 1: 00: 1 solvent to water in volume. Preferably, the ratio is 1: 1 to 20: 1 and most preferably 5. 1 to 1 0: 1. The optimum ratio is dependent on the selected solvent and preferably it is acetone at a 9: 1 ratio of solvent to water. Usually, the reaction involves approximately equal amounts of the two reactants, although other proportions are operative, especially those in which the methanesulfonic acid is in excess. The rate of addition of methanesulfonic acid is not critical to the reaction and can be added quickly (<5 minutes) or slowly over 5 or more hours. The reaction is carried out at temperatures ranging from 0 ° C to reflux. The reaction mixture is stirred until the formation of the salt is complete, as determined by X-ray powder diffraction and can take from 5 minutes to 1 2 hours. The salts of the present invention are preferably and easily prepared as a crystalline form. The trihydrate form of the salt can be easily converted to the monohydrate upon drying or exposure to 20-60% relative humidity. The salt is substantially crystalline, demonstrating a defined melting point, bi-refrigerant, and X-ray diffraction pattern. Generally, the crystals have less than 10% amorphous solid and preferably less than 5% and most preferably less than 1% amorphous solid. The mesylate salt is isolated by filtration or other separation techniques appreciated in the art, directly from the reaction mixture in yields ranging from 50% to 1 00%. Recrystallization and other purification techniques known in the art can be used to purify the salt additionally, if desired. One skilled in the art will recognize that a therapeutically effective amount of the proteinase C inhibitor of the present invention is sufficient to inhibit the replication and / or activation of H IV or inhibit the effect of H IV. It is within the ability of a person skilled in the art to measure the activation and replication of H IV using well-known markers, such as T-cell count, viral count, viral specific protein and its activity, etc. The amount administered varies inter alia, depending on the concentration of the compound in the therapeutic formulation, and the patient's body weight. Generally, an amount of protein kinase C inhibitor to be administered as a therapeutic agent to treat HIV infection will be determined on a case-by-case basis by the attending physician. As a line of surgery, the degree of infection, the strength of the immune system, the body weight and age of the patient will be considered when establishing an appropriate dose.

Generally, a suitable dose is one which results in a concentration of the protein kinase C inhibitor at the treatment site in the range of 0.5 nM to 200 μM, usually 0.5 nM to 20 μM and more usually 0.5 nM to 200 nM. It is expected that serum concentrations of 0.5 nM to 20 nM will be sufficient under most circumstances. To obtain these treatment concentrations, a patient in need of treatment will probably be administered between approximately 0.001 mg per day per kg of body weight and 50.0 mg per day per kg. Usually, no more than about 10.0 mg per day per kg body weight of protein kinase C inhibitor should be necessary. As noted above, the above amounts may vary on a case-by-case basis. The effectiveness of the compounds of the invention can be tested in both in vitro and in vivo systems. For in vitro testing, populations of T-cell and mononuclear cells chronically infected with HIV can be employed, as described in Kinter et al., J. Virology 64: 4306-4312, 1990 and Sardoroski et al., Science 227: 171- 173, 1985. Both references are incorporated herein. The results obtained from the in vitro test system are predicted from the effectiveness of the compounds to reduce the replication of HIV in the basal state, as well as the effectiveness of the compounds to reduce the replication of HIV stimulated by esters of phorbol, factor -alpha of tumor necrosis, IL-6 and lipopolysaccharide, For in vivo testing, the humanized HIV-infected combined severe immunodeficiency (SCID) mouse model can be used (Mosier et al., Immunoiogy Today 15: 332-339, 1 994). In this model, SCI D mice are inoculated with human monocytic cells infected with H IV or CD4 + T lymphocytes. As a primary endpoint, the progression of the disease is monitored by assessing the suppression of CD4 + T cells. The ability of the compounds of the invention to decrease the decrease in this model either when infected with monocytes infected with H IV or CD4 + T cells is the one that predicts a positive response in HIV-infected humans either by prolonging the latency phase or retarding the clinical progression of AIDS.

The compounds of formula I, and the preferred compounds of formula la and Ib are preferably formulated before administration. Suitable pharmaceutical formulations are prepared by known procedures using well known and readily available ingredients. To make compositions suitable for use in the method of the present invention, the active ingredient will normally be mixed with a carrier, or diluted by a carrier or enclosed within a carrier which may be in the form of a capsule, sachet, paper or another container. When the carrier serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, capsules, ices, suspensions, emulsions, solvents, syrups, aerosols (as a solid or in a liquid medium), gelatin capsules. soft and hard, suppositories, sterile injectable solutions and powder sterile packs either for oral or topical application.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preservatives, sweetening agents or flavoring agents. The compositions of the invention can be formulated in order to provide quick releasesustained or delayed active ingredient after administration to the patient. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.05 mg to about 3 g, more usually about 750 mg of the active ingredient. However, it will be understood that the therapeutic dosage administered will be determined by the physician in light of the relevant circumstances including the severity of the condition to be treated, the choice of compound to be administered and the chosen route of administration. Therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutic carrier.

In addition to the above formulations, most of which can be administered orally, the compounds used in the method of the present invention can also be administered topically. Topical formulations include ointments, creams and gels. The ointments are generally prepared using either (1) an oleous oil base, i.e., one consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or (2) an absorbent base, i.e., one consisting of one anhydrous substance or substances which can absorb water, for example, anhydrous lanolin. Commonly, following the formation of the base, either oily or absorbent, the active ingredient (compound) is added in an amount of the desired concentration. The creams are oil / water emulsions. They consist of an oil phase (internal phase), normally comprising fixed oils, hydrocarbons and the like, such as waxes, petrolatum, mineral oil and the like, and an aqueous phase (continuous phase), comprising water and any water-soluble substance. , such as added salts. The two phases are stabilized by the use of an emulsifying agent, for example, an active surface agent, such as sodium lauryl sulfate; hydrophilic colloids, such as, acacia colloidal clays, "veeg um", and the like. Upon formation of the emulsion, the active ingredient (compound) is commonly added in an amount to achieve the desired concentration. The gels comprise a selected base of an oleaginous base, water, or an emulsion-suspension base. To the base is added a gelling agent, which forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropylcellulose, acrylic acid polymers and the like. Commonly, active (compound) ing is added to the formulation at the desired concentration at a point preceding the addition of the gelling agent. The amount of compound incorporated in a topical formulation is not critical; the concentration should be within a sufficient range to allow easy application of the formulation to the area of affected tissue in an amount which will deliver the desired amount of compound to the desired treatment site. The amount of a topical formulation to be applied to an affected tissue will depend on the concentration of the compound in the formulation. In general, the formulation will be applied to the affected tissue in an amount providing from about 1 to about 500 μg of compound per cm 2 of an affected tissue. Preferably, the applied amount of the compound will vary from about 30 to about 300 μg / cm2, more preferably, from about 50 to about 200 μg / cm2, and most preferably, from about 60 to about 1000 μg / cm2. The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way.

Formulation 1 Hard gelatine capsules are prepared using the following ingredients: Amount (mg / capsule) Active agent 5 Suitable, dry 200 Magnesium stearate 1 0 Total 215 mg The above ingredients are mixed and filled into hard gelatin capsules in amounts of 460 mg.

Formulation 2 A tablet is prepared using the ingredients below: Quantity (mg / tablet) Active agent 1 5 Microcrystalline cellulose 10 Silicon dioxide, smoked 10 Stearic acid 5 Total 40 mg The components are mixed and compressed to form tablets, each weighing 665 mg.

Formulation 3 Tablets were made, each containing 60 mg of the active ingredient, as follows: Quantity (mg / tablet) Active agent 60 Starch 45 Microcrystalline cellulose 35 Polyvinylpyrrolidone 4 (as a 10% solution in water) Sodium carboxymethyl starch 4.5 Magnesium stearate 0.5 Talc 1 Total 1 50 mg The active ingredient, starch and cello slab are passed through a US sieve of No. 45 mesh and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a No. 14 mesh US sieve. The granules thus produced are dried at 50 ° C and passed through a US sieve. No. 1 8. Sodium carboxymethyl starch, magnesium stearate and talcum, previously passed through a No. 60 mesh US sieve, are then added to the granules, which, after mixing, compress in a tabletting machine to produce tablets, each weighing 150 mg.

EXAMPLES All of these examples demonstrate the use of (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3"' (0) -4 '"- (N, N ) -dimethylamino) -butane) -bis- (3,3'-indoiiI)] - 1 (H) -pyrrole-2,5-dione to inhibit the expression of HIV in U1 cells.

Example 1 In this example, the inhibitory effect of the compound noted on the expression of HIV stimulated with PKC activator was examined. The U1 cells were treated with either PMA or PMA in combination with the compound noted. HIV expression was measured via the production of HIV-1 p24. The results, as shown in Table 1, demonstrate that the compound noted has an inhibitory effect on HIV expression induced by PKC activator.

Table 1 Treatment Production of H IV-1 p24 (pg / ml) PMA (10 μg / ml) 3974 PMA (10 μg / ml) + PKC inhibitor (1 nM) 1899 PMA (1.0 μg / ml) + inhibitor of PKC (1 0 nM) 36 PMA (10 μg / ml) + PKC inhibitor (1 00 nM) 1 .8 PMA (1 0 μg / ml) + PKC inhibitor (500 nM) 9.1 Example 2 In this example, the inhibitory effect of the compound noted on the expression of H IV stimulated with cholera toxin was examined. It is known that cholera toxin increases the cellular level of cAM P. U 1 cells were treated with either cholera toxin or cholera toxin in combination with the compound noted. The expression of H IV was measured via the production of H IV-1 p24. The results, as shown in Table 2, demonstrate that the compound noted has an inhibitory effect on the expression of HIV induced in the cholera toxin.

Table 2 Treatment Production of HIV-1 p24 (pg / ml) Cholera toxin (CT) (10 ng / ml) 55 CT (10 μg / ml) + PKC inhibitor (1 nM) 21 CT (10 μg / ml) + PKC inhibitor (10 nM) 10 CT (10 μg / ml) + PKC inhibitor (100 nM) 6 CT (10 μg / ml) + PKC inhibitor (500 nM) 22 Example 3 This example demonstrates the effects of the compound noted on the expression of HIV induced by TNF. U1 cells were treated with either TNF or TNF in combination with the compound noted. HIV expression was measured via the production of HIV-1 p24. The results, as shown in Table 3, suggest that TNF can activate HIV expression through routes not associated with PKC.

Table 1 Treatment Production of HIV-1 p24 (pg / ml) TNF (10 U / ml) 176 TNF (10 U / ml) + PKC inhibitor (1 nM) 269 TNF (10 U / ml) + PKC inhibitor ( 10 nM) 176 TNF (10 U / ml) + PKC inhibitor (100 nM) 185 TNF (10 U / ml) + PKC inhibitor (500 nM) 167 Principles, preferred embodiments and modes of operation of the present invention have been described in the previous specification. However, the invention which is intended to be protected in the present, should not be construed as limited to the particular forms described, as they should be considered as illustrative rather than restrictive. Variations and changes can be made by those skilled in the art without departing from the spirit of the invention.

Claims (24)

1. A method for inhibiting the activation of the human immunodeficiency virus in an infected cell, which comprises contacting the cell with an inhibitory amount of viral activation of an inhibitor of the proteinase kinase β isozyme.

2. The method of the claim 1, wherein the inhibitor of the protein kinase isozyme C is a bis-indolylmaleimide or a macrocyclic bis-indolylmaleimide.

3. The method of claim 1, wherein the inhibitor is isozyme-selective and wherein isozyme selectivity is selected from the group consisting of beta-1 and beta-2 sozymas.

4. The method of claim 3, wherein the protein kinase inhibitor C has the following formula: wherein: W is -O-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) mO -, -heterocycle-, -heterocycle- (CH2) mO-, -bicyclic fused-, -cyclic-fused- (CH2) mO-, -N R3-, -NOR3-, -CON H-, or -N HCO-; X and Y are independently C, -C4 alkylene, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; R1 s are hydrogens or up to four optional substituents independently selected from halo, alkyl of 0, -04, hydroxy, C, -C4 alkoxy, haloalkyl, nitro, -NR4R5, or -NHCO (C, -C4 alkyl); R2 is hydrogen, CH3CO-, -NH2, or hydroxy; R3 is hydrogen, (CH2) maplo, C, -C4 alkyl, -COO (C, -C4 alkyl), -CONR4R5, - (C = NH) N H2, -SO (C, -C4 alkyl) , -S02 (NR4R5), or -S02 (C, -C4 alkyl); R 4 and R 5 are independently hydrogen, C 1 -C 4 alkyl, phenyl, benzyl, or combine with the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2, or 3; and n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 4, wherein the protein kinase C inhibitor has the following formula: wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R4 is hydroxy, -SH, alkyl C, -C4, (CH2) maryl, -NH (aryl), -N (CH3) (CF3), -NH (CF3), or -NR5R6; R5 is hydrogen or C, -C4 alkyl; R6 is hydrogen, C, -C, or benzyl alkyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

6. The method of claim 4, wherein the protein kinase inhibitor C has the following formula: (Ib) wherein Z is - (CH2) p-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C, -C alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 4, wherein the protein kinase C inhibitor comprises (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" (0) 4 '"- (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione or its pharmaceutically acceptable acid salt.

A method of claim 7, wherein the pharmaceutically acceptable acid salt is selected from the hydrochloride salt and the mesylate salt 9.

A method for inhibiting the replication of human immunodeficiency virus in an infected cell, which comprises in contact with the cell with an inhibitory amount of viral replication of an inhibitor of the protein kinase C isozyme 10.

The method of claim 9, wherein the inhibitor of the protein kinase C β isozyme is a bis-indolylmaleimide or a bis The macrocyclicindolylmaleimide 11.

The method of claim 9, wherein the inhibitor is isozyme-selective and wherein the isozyme selectivity is selected from the group consisting of beta-beta and beta-2.

The method of claim 11, wherein the protein kinase inhibitor C has the following formula: wherein: W is -O-, -S-, -SO-, -SO2-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) mO -, -heterocycle-, -heterocycle- (CH2) mO-, -bicyclic fused-, -bicyclic fused- (CH2) mO-, -NR3-, -NOR3-, -CONH-, or -NHCO-; X and Y are independently C, -C4 alkylene, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; R1s are hydrogens or up to four optional substituents independently selected from halo, C, -C4 alkyl, hydroxy, C, -C4 alkoxy, haloalkyl, nitro, -NR4R5, or -NHCO (C, -C alkyl); R2 is hydrogen, CH3CO-, -NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, C-C4 alkyl, -COO (C-C4 alkyl), -CONR4R5, - (C = NH) NH2, -SO (C-C4 alkyl), - S02 (NR4R5), or -S02 (C, -C4 alkyl); R 4 and R 5 are independently hydrogen, C, -C 4 alkyl, phenyl, benzyl, or combine with the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently O, 1, 2, or 3; and n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 1, wherein the protein kinase inhibitor C has the following formula: wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, C, -C 4 alkyl, (CH 2) maryl, -N H (aryl), -N (CH 3) (CF 3), -NH (CF 3), or -NR 5 R 6; R5 is hydrogen or C, -C4 alkyl; R6 is hydrogen, C, -C4 alkyl or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 1, wherein the protein kinase inhibitor C has the following formula: wherein Z is - (CH2) P-: R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C? -C4 alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 12, wherein the protein kinase C inhibitor comprises (S) -3,4- [N, N'-1, 1, - ((2"-ethoxy) -3" '(0) 4 '"- (N, N-dimethylamino) -butane) -bis- (3,3'-indoIyl)] - 1 (H) -pyrrole-2,5-dione or its pharmaceutically acceptable acid salt.

A method of claim 15, wherein the pharmaceutically acceptable acid salt is selected from the hydrochloride salt and the mesylate salt

17. A method for treating a mammal infected with human immunodeficiency virus, which comprises administering to the mammal a Therapeutically effective amount of an inhibitor of the protein kinase C isozyme.

18. The method of claim 17, wherein the inhibitor of the proteinase kinase β isozyme is a bis-indolylmaleimide or macrocyclic bis-indolylmaleimide.

The method of claim 17, wherein the inhibitor is isozyme selective and wherein isozyme selectivity is selected from the group consisting of isozymes beta-1 and beta-2.

The method of claim 19, wherein the protein kinase C inhibitor has the following formula: wherein: W is -O-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, -aryl (CH2) mO -, -heterocycle-, -heterocycle- (CH2) mO-, -bicyclic fused-, -bicylic fused- (CH2) mO-, -NR; -NOR3-, -CONH-, or -NHCO-; X and Y are independently alkylene of 0, -04, substituted alkylene, or together X, Y and W combine to form - (CH2) n-AA-; R s are hydrogen or up to four optional substituents independently selected from halo, C, -C 4 alkyl, hydroxy, C, -C 4 alkoxy. haloalkyl. nitro, -NR4R5, or -NHCO (C, -C4 alkyl); R2 is hydrogen, CH3CO-, -NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, C1-C4 alkyl, -COO (d-C4 alkyl), -CONR4R5, - (C = NH) NH2, -SO (C-C4 alkyl), -S02 ( NR4R5), or -S02 (C, -C4 alkyl); R 4 and R 5 are independently hydrogen, C, -C, phenyl, benzyl, or combine with the nitrogen to which they are attached to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2, or 3; and n is independently 2, 3, 4, or 5, or a pharmaceutically acceptable salt, prodrug or ester thereof.

21. The method of claim 20, wherein the protein kinase C inhibitor has the following formula: gives) wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, C, -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3), -NH (CF 3), or -NR 5 R 6; R5 is hydrogen or C, -C alkyl; R6 is hydrogen, C, -C4 alkyl or benzyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

22. The method of claim 20, wherein the protein kinase inhibitor C has the following formula: wherein Z is - (CH2) P-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C, -C alkyl; p is 0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.

The method of claim 20, wherein the protein kinase C inhibitor comprises (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" (0) 4 '"- (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione or its pharmaceutically acceptable acid salt.

24. A method of claim 23, wherein the pharmaceutically acceptable acid salt is selected from the hydrochloride salt and the mesylate salt.