WO2003059343A2

WO2003059343A2 - Drug compositions with indole alkaloids for the treatment of aids

Info

Publication number: WO2003059343A2
Application number: PCT/BR2002/000193
Authority: WO
Inventors: Dumith Cherquer Bou-Habib; Bernardo Galvão CASTRO; Edinete Melo Da Silva; Elvira Maria Saraiva Chequer Bou-Habib; Martin E. Kuehne
Original assignee: Fundacão Oswaldo Cruz - Fiocruz
Priority date: 2001-12-26
Filing date: 2002-12-26
Publication date: 2003-07-24
Also published as: BR0106261A; WO2003059343A3; AU2002357417A1

Abstract

The present invention refers to the use of iboga type indole alkaloids, especially coronaridine and its derivative 18-methoxy coronaridine, in the preparation of medications capable of inhibiting HIV virus action. One of the embodiments of the present invention is related to a pharmaceutical composition for the treatment of AIDS, containing coronaridine and a pharmaceutically acceptable vehicle. Another embodiment of the present invention refers to a pharmaceutical composition for the treatment of AIDS containing coronaridine analogues, especially 18-methoxy coronaridine and a pharmaceutically acceptable vehicle.

Description

"DRUG COMPOSITIONS WITH INDOLE ALKALOIDS FOR THE TREATMENT OF AIDS"

The present invention refers to drug compositions based on iboga-type indole alkaloids, specially coronaridine and/or its derivative 18-methoxy- coronaridine, used for the inhibition of the Human Immunodeficiency Virus (HIV) replication. INVENTION BACKGROUND

The Acquired Immunodeficiency Syndrome (AIDS) is related to a disease or condition resulting in a gradual annihilation of the immune system accompanied by a progressive deterioration of the central and peripheral nervous system. Since the early 1980' s when it was acknowledged, AIDS has been worldwide spread and has taken epidemic proportions.

The human immunodeficiency virus, AIDS etiological agent, infects and replicates in cells presenting the CD4 surface marker (monocytes/macrophages and T CD4+ lymphocytes) . Immediately after primary infection there is a plasma viremia peak, frequently associated to an acute symptomatic clinical picture (Fauci, A.S. Multifactorial nature of human immunodeficiency virus disease: implications for therapy. Science, 262:1011- 1018.1993). To this, a clinical latency period follows during which there is a persistent viral replication in lymphoid tissue inducing a progressive state of deterioration of the immune system functioning. The whole process culminates in a severe immunosupressi e clinical picture (Pantaleo, G. et al. HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease. Nature, 362:355- 358.1993).

Asymptomatic individuals show low plasma viremia and normal amount of T CD4+ cells, while the progression to the immunodeficiency state is associated to the increased peripheral viral titles and significantly decreased T CD4+ cell number (Weiss, R.A. How does HIV cause AIDS? Science, 260:1273-1279.1993).

HIV entry in target cells requires the presence of other cell surface molecules in addition to CD4 molecule. Several researchers described that chemokine receptors function as accessory factors during HIV-1 entry in target cells (Dimitrov, D.S. How do viruses enter cells? The HIV coreceptors teach us a lesson of complexity. Cell, 91:721-730.1997). The cofactor for T- tropic isolates is the alpha-chemokine receptor CXCR4 (Feng, Y. et al . HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science _r 212. -. 872-877.1996), whose natural binder, the SDF-1 alpha-chemokine, blocks infection by T-tropic isolates (Fauci,A. S. Host factors and the pathogeneses of HIV-induced disease. Nature. 384:529- 534.1996). Cofactors used by monocytotropic isolates are beta-chemokine receptors CCR2b, CCR3, and CCR5 (Alkhatib, G. et al . CC CKR5: ARATES, MIP-l^a, MlP-lb receptor as a fusion cofactor for macrophage-tropic HIV-1. Science.272:1955-1958.1996; Deng, H. et al . Identification of a major co-receptor for primary isolates of HIV-1. Nature.381: 661-666.1996 and Dragic, T. et al. HIV-1 entry into CD4⁺ cells is mediated by the che okine receptor CC-CKR5. Nature.381:667-673.1996) . The interaction between beta-chemokines RATES, MIP- lalpha and MlP-lbeta and CCR5 receptor prevents the fusion between the viral envelope protein (gpl20/41) and the cell membrane, in addition to inhibiting infection by HIV-1 monocytotropic isolates (Fauci, A.S., 1996).

With regard to the anti-HIV-1 immunoresponse, this may be detected soon in the first weeks after infection (Poignard, P.A. et al . Antibody neutralization of HIV- 1. Immunol . Today.11. - 239-246.1996) . After the viral dissemination initial stage, the anti-HIV-1 primary immunoresponse limits virus replication, an effect mainly performed by CD8+ T-cytotoxic cells (Pantaleo,G. & Fauci, A.S. New concepts in the immunopathogenesis of HIV infection. Annu. Rev. Immunol .13:487-512.1995) . In addition to the specified cytotoxic activity, T-CD8+ cells produce soluble factors capable of inhibiting HIV-1 MT- isolates, such as the beta-chemokines RATES, MlP-lalpha and MlP-lbeta (Cocchi, F. et al . Identification of RANTES, MIP-la and MlP-lb as the major HIV-suppressive factors produced by CD8⁺t cells. Science.270: 1811-1815.1995) . However, the ongoing viral multiplication causes the slow and persistent decrease of T-CD4+ cell number and the progressive deterioration of the patient's immunoresponse. Finally, the plasma viral load considerably increases and infection advances to the AIDS clinical picture (Mellors, J.W. et al . Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science.272:1167- 1170.1996).

In spite of most HIV-1 infected individuals developing a strong humoral immunoresponse against the virus, no clear evidences were found until now that these antibodies neutralize viral dissemination and infection progression to AIDS final picture (Burton, D.R. A vaccine for HIV type 1: The antibody perspective. Proc. Natl . Read. Sci . USA, 94:10018- 10023.1997). During HIV-1 infection asymptomatic stage, the most frequent viral phenotype isolated in patients is the syncytium non-inducer monocytotropic (SNI) (Schuitemaker, H. et al . Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell- tropic virus population. J. Virol .66: 1354-1360.1992) , which is in vi tro resistant to neutralizing antibody blocking activity (AcN) (Poignard, P.A. et al . Antibody neutralization of HIV-1. Immunol . Today.17:239-246.1996 and Cohen, J. Jitters jeopardize AIDS vaccine trials. Science.262:980-981.1993) . Experimental evidences indicate that these viruses escape from humoral immunoresponse due to HIV-1 envelope glucoprotein immuno-dominant epitopes (gpl20/41) being not accessible to neutralizing antibodies (Bou-Habib, D.C. et al . Cryptic nature of envelope V3 region epitopes protects primary monocytotropic human immunodeficiency virus type 1 from antibody neutralization. J. Virol .68: 6006-6013.1994 and Sattentau, Q.J. & Moore, J.P. Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gpl20 oligomer. J. Exp.Med.182: 185-196.1995) . Several medications have been developed and used to fight AIDS. Nevertheless, side effects associated to these drugs make the search for new treatment alternatives to be intensified. An alternative that has been lately intensively researched is related to the medicinal properties of several plants whose active substances have been isolated, purified and, in many cases, synthetically reproduced. Many known drugs were originally derived from plants. Salicylic acid, the aspirin precursor, for instance, was originally isolated from plants.

Several other examples of well-known active substances originated from plants may be cited: glucosides for the treatment of heart diseases obtained from Digi talis pυrpurea L.; the antihypertensive and tranquilizer reserpine, originated from Rauvolfia serpentina (L.); quinine, anti-malaria agent, from Cinchona ssp.; the opiaceous analgesics codeine and morphine from Papaver somniferum L.; the anti-leukemia vinblastine and vincristine, from Catharanthus roseus (L.) (see Baker, J.T., Borris, R.P., Carte, B., Cordell, G.A. , Soejarto, D.D., Cragg, G.M., Gupta, M.P., Iwu, M.M., Madulid, D.R. and Tyler, V.E. "Natural product drug discovery and development: New perspectives on International Collaboration". Journal of Natural Products. 58 (9), 1325-1357. 1995).

US patent 6,030,622 describes the use of a composition based on extracts of pomegranate, Hybiscus, arum and tea. Such compositions stimulate mediated cellular immunity in vitro, and inhibit gp 120 binding in vi tro, being potential candidates for the treatment of immune system disorder and infection by HIV. Another example may be seen in US patent 5,302,611, regarding indole compounds. This document describes the use of pentacyclic oxyindole alkaloids with formula C₂₁H₂₄O_{4 2}, capable of being used to stimulate immune system. Specifically in relation to coronaridine and its analogues, patents GB 924,041 and GB 924,042 referring respectively to: (i) process for the obtainment of isoquinuclidines such as 12, 13-dymethoxy-coronaridine from the extraction of roots and stem of Conopharingya durissima Stapf and (ii) process for indole alkaloid decarboxilation such as voacangine, isovoacangine and 12, 13-dymethoxycoronaridine.

US patent 3,681,362 referring to a process for chemical synthesis of isoquinuclidine-type alkaloids and the patent application BR PI 9804032, referring to the use of alkaloids voacangine and coronaridine for the treatment of leishmaniasis may also be cited.

Also, it must be reminded that despite the urgent need for the development of an efficient anti-HIV vaccine, results obtained until now are disappointing.

In this context, HIV characteristics such as for instance, (i) resistance to humoral immunoresponse

(Burton, D.R.,1997), (ii) high frequency of genetic mutations (Desrosiers, R.C. Strategies used by human immunodeficiency virus that allow persistent viral replication. Nature Med.5:723-725.1999) , and (iii) generation of chemotherapy-resistant infectious particles point to the need for a permanent search for prophylactic and therapeutic forms, capable of restricting virus dissemination in the population and inhibiting its multiplication in the infected patient. SUMMARY OF THE INVENTION

The objective of the present invention is the use of iboga-type indole alkaloids, in special coronaridine and its derivative 18-methoxy coronaridine, in the preparations of medications capable of inhibiting HIV virus action.

One of the embodiments of the present invention is related to a pharmaceutical composition for the treatment of AIDS containing coronaridine and a pharmaceutically acceptable vehicle.

Another embodiment of the present invention refers to a pharmaceutical composition for the treatment of AIDS, containing coronaridine analogues, in special 18- methoxy coronaridine and a pharmaceutically acceptable vehicle. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the inhibition of a HIV-1 replication by Coronaridine in PBMCs infected with Ba-L virus .

FIGURE 2 shows the inhibition of HIV-1 replication by Coronaridine in PBMCs infected with BA07 virus.

FIGURE 3 shows the inhibition of a HIV-1 replication by 18-methoxy coronaridine in PBMCs infected with Ba-L virus.

FIGURE 4 shows the inhibition of HIV-1 replication by 18-methoxy coronaridine in PBMCs infected with BA07 virus. FIGURE 5 shows the inhibition of HIV-1 replication in macrophages infected with Ba-L, by 18- methoxycoronaridine . DETAILED DESCRIPTION OF THE INVENTION

As aforementioned, due to the HIV particular characteristics, several chemotherapeutic agents used in AIDS treatment have shown to be poorly efficient and at the same time, highly toxic.

In this context, the search for vegetal-origin substances may be seen as a powerful source of new chemotherapeutics for the inhibition of HIV infection as far as broad chemical spectrum substances (non-iboga type alkaloids such as coronaridine and its derivatives, flavonoids, terpenes, quinones, phospholipids, among others) have already been reported as capable of inhibiting this virus replication (Vlietinck, A.J. et al . Plant-derived leading compounds for chemotherapy of human immunodeficiency virus infection. Planta Medica . 64:97-109.1998).

Indole alkaloids of the present invention are found in several genuses of Apocynaceae family such as Voacanga , Ervatamia, Tabernaemontana (syn. Peschiera) and others. Aiming at obtaining the compounds of the present invention, stems and/or roots are collected, for example, from Peschiera australis species individuals, and submitted to extraction as described in the Brazilian patent application by Fundacao Oswaldo Cruz no. BR PI9804032.

Preferably, however, compounds coronaridine and 18-methoxy coronaridine are obtained through chemical synthesis by means of processes known by those knowledgeable in this subject matter. Examples may be found in Bandarage et al (Bandarage, U.K.; Kuehne, M. E. e Glick, S.D. "Total syntheses of racemic albifloranine and its anti-addictive congeners, including 18- methoxycoronaridine" . Tetrahedron. 55. 9405-9424.1999) and Kuehne et al (Kuehne, M.E.; Wilson, T.E.; Bandarage, U.K.; Dai, W. e Yu, Q. "Enatioselective syntheses of coronaridine and 18- methoxycoronaridine". Tetrahedron. 57. 2085-2094. 2001) .

For oral administration, medication may be in the form of tablets, pills, capsules or as solution or suspension. The solid compositions contain the active ingredient mixed with non-toxic excipients proper for tablet manufacturing such as starch, milk sugar, certain carbonate and/or bicarbonate types, phosphates, etc. Tablets may be coated or not, depending on the gastrointestinal tract point where drug disintegration and absorption shall occur. In the case of suspensions or aqueous solutions, excipients such as methylcellulose, sodium alginate, acacia gum, lecithin, etc. may be used and one or more additives, such as preservatives, color, flavoring, thickening agents, etc.

The amount of coronaridine compound and/or its derivative 18-methoxy coronaridine that will be combined with the pharmaceutically acceptable vehicle such as to produce the proper dosage form will depend on the organism to be treated and the chosen route of administration. However, in the case of the present invention, the pharmaceutical compositions may preferably contain one or both compounds in an amount of up to 600 mg/g, to be used in the treatment of infections caused by HIV.

The present invention is described in detail through the examples presented below. It must be pointed out that the invention is not limited to these examples, but also includes variations and changes within the limits in which it functions, including the possibility of using other coronaridine analogues. EXAMPLE 1: OBTAINMENT OF CORONARIDINE AND 18-METHOXY CORONARIDINE

Coronaridine and 18-methoxy-coronaridine are obtained similarly to that described by Kuehne et al (Kuehne, M.E.; Wilson, T.E.; Bandarage, U.K.; Dai, W. e Yu, Q. "Enatioselective syntheses of coronaridine and 18-methoxycoronaridine". Tetrahedron. 57. 2085-2094. 2001) .

EXAMPLE 2: OBTAINMENT OF CELLS TO BE USED FOR HIV INFECTION Peripheral blood cells (PBMCs) of healthy individuals are obtained through density gradient in Ficoll-hypaque and suspended in RPMI 1640 culture medium, with 10% bovine fetal serum. Part of these cells is stimulated with mitogen phytohemagglutinin

(PHA) for two to three days and activated cells

(PBMC/PHA) are kept in culture medium supplemented with interleukin-2 (IL-2) for further viral infection assays. For the obtainment of macrophages, PBMCs are submitted to adhesion in dish of 24 wells in RPMI 1640 culture medium with no serum, for 1 hour at 37°C, 5%

CO2. Non-adherent cells are removed and remaining monocytes will be kept in culture for 7 to 10 days in DMEM medium with 10% human serum (37°C, 5% de C0₂) , for differentiation in macrophages. These cells are then used for HIV-1 infection. Purity of macrophages is assessed by flow cytometry by the amount of positive cells for CD14 molecule.

EXAMPLE 3: OBTAINMENT OF VIRAL ISOLATES.

Two viral isolates are being used, namely: (i) BA07 isolate and (ii) BA-L isolate.

BA07 isolate was obtained by the co-culture of PBMCs from positive HIV-1 patient with PBMCs from healthy donors, as described in WHO Guidelines for Standard Isolation Procedures (World Health Organization Guidelines for Standard HIV Isolation Procedures. WHO. Geneva. 1994) . This monocytotropic isolate, inducer of in vi tro syncytium formation, is capable of using chemokines CCR5 and CXCR4 receptors during cellular infection (Brazilian Network for HIV isolation and characterization. 2000. HIV diversity in Brazil: genetic, biological and immunological characterization of HIV strains in three potential HIV vaccine evaluation sites. J. Acquired Immune Defic. Syndr. 23:184-193) .

BA-L isolate is monocytotropic, non-inducer of syncytium and tropic for CCR5. It was kindly donated by Dr. Michael A. Norcross, CBER/FDA, BETHESA,MD,USA.

It must be pointed out that both isolates^" shall be kept at temperature of -70°C.

EXAMPLE 5: VIRAL INFECTIONS AND INHIBITORY EFFECT MEDIATED BY CORONARIDINE AND 18-METHOXY CORONARIDINE ON HIV-1 REPLICATION. A. PBMCs These cells are infected by HIV-1 positive supernatants (10⁶ cells / 5 ng Ag p24 / ml) for a 2-hour period at 37°C and 5% C0₂, washed for residual virus removal and then incubated in 96-well culture dishes (2 x 10⁵ cells / 200 μl / well) with different concentrations of the drugs obtained in Example 1.

Infected cells are kept in culture for seven days in RPMI culture medium with 10% bovine fetal serum and supplemented with IL-2. After this period, viral replication is assessed by antigen p24 detection and/or reverse transcriptase activity in the culture supernatants. Inhibition level of HIV-1 replication may be verified by comparing viral replication of cultured cells in the presence of drugs with the replication of cells kept in culture medium only.

Figures 1 and 2 show the results obtained with coronaridine upon infection of PBMCs by Ba-L and BA07 isolates, respectively. Inhibition of viral replication mediated by Coronaridine was equal to or higher than

80%, in 20-μg/ml concentration.

In relation to compound 18-methoxy coronaridine (18M-Cor), results are shown in Figures 3 and 4. As can be seen, replication of both viral isolates was strongly inhibited by 20-μg/ml 18M-Cor in PBMCs of three different donors (infected by Ba-L virus) . B. Macrophages These cells are infected with HIV-1 monocytotropic strains (5 to 10 ng/ml Ag p24, for a 16- to 18-hour period at 37°C and with 5% C0₂) , washed for residual virus removal and cultured in DMEM medium with 10% human serum in the presence or not of different drug concentrations. Infected macrophages are fed back with these products each 7 days and cultures are followed-up for three weeks. Viral replication is evaluated by measuring p24 antigen and/or reverse transcriptase

(RT) enzyme activity in the culture supernatants . p24 antigen (Ag p24) is quantified in specific capture assays (ELISA) , in commercial kit, according to manufacturer's instructions. RT activity is checked by using (rA) (dT)₁₂-i8 poli-sequence as exogenous model as described by Oravecz et al (Oravecz, T., Norcross, M.A. B-Chemokine inhibition of monocytotropic HIV-1 infection: Interference with a postbinding fusion step. J. Immunol .157: 1329-1332.1996) . The comparison of viral replication in cultured cells in the presence of coronaridine or 18-methoxy coronaridine, with replication of cells kept in culture medium only, indicates the intensity of HIV-1 growth inhibition.

After three weeks in culture, concentrations of 5, 10 and 20 μg/ml of 18-methoxycoronaridine inhibited viral replication at levels around 80% in cells of three different donors . These results may be seen in

Figure 5.

Claims

1. Pharmaceutical composition to be used in the treatment of infections caused by HIV characterized by containing an efficient amount of one or more iboga- type indole alkaloids and a pharmaceutically acceptable vehicle.

2. Pharmaceutical composition according to claim 1 characterized by the fact of the active ingredient being present in a concentration of up to 600 mg/g.

3. Pharmaceutical composition according to claim 1 or 2 characterized by the fact of the alkaloid being coronaridine or its analogues.

4. Pharmaceutical composition according to claim 3 characterized by the fact of the analogue being 18- methoxy coronaridine.

5. Pharmaceutical composition according to claim 1 or 2 characterized by containing a coronaridine and 18- methoxy coronaridine mixture.