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Method for the suppression of viral growth
RE40246 Method for the suppression of viral growth
Patent Drawings:Drawing: RE40246-2    Drawing: RE40246-3    Drawing: RE40246-4    Drawing: RE40246-5    Drawing: RE40246-6    Drawing: RE40246-7    Drawing: RE40246-8    Drawing: RE40246-9    
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Inventor: Huang, et al.
Date Issued: April 15, 2008
Application: 10/664,050
Filed: September 17, 2003
Inventors: Huang; Ru Chih C. (Baltimore, MD)
Gnabre; John N. (Baltimore, MD)
Assignee: John Hopkins University (Baltimore, MD)
Primary Examiner: Marx; Irene
Assistant Examiner:
Attorney Or Agent: Venable LLPHobbs; Ann S.
U.S. Class: 514/548; 514/371; 514/546; 514/551; 514/721; 514/734; 514/885; 514/934
Field Of Search: 514/371; 514/885; 514/934; 514/551; 514/721; 514/548; 514/546; 514/734
International Class: A61K 31/075; A61K 31/222; C07C 43/00
U.S Patent Documents:
Foreign Patent Documents:
Other References: Russell T. Jordan and Larry M. Allen, "Neoplasm Inhibitors Comprising Metal Salts and Phenol Derivatives," Chemical Abstracts, vol. 111, No.3, Jul. 17, 1989, p. 64. cited by other.
Perry et al, Synthesis of Lignans, I. Nordihydroguaiaretic Acid 1, J. Org. Chem, 1972, vol. 37, No. 26, p. 4371-4376. cited by other.
Giza et al., A self-inducing runaway-replication plasmid expression system utilizing the Ro protein, Elsevier Science Publishers B.V. (Biomedical Division), Gene 78, 1989, pp. 73-84. cited by other.
Staal et al., Antioxidants Inhibits Stimulation of HIV Transcription, Aids Research and Human Retroviruses, 1993, vol. 9, No. 4, pp. 299-306. cited by other.
Gnabre et al., Isolation of anti-HIV-1 lignans from Larrea tridentateounter-current chromatography, Journal of Chromatography A., 719, 1996, pp. 353-364. cited by other.
Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentateTetrahedrom, 1995, vol. 51, No. 45, pp. 12203-12210. cited by other.
Weislow et al., New Soluble-Formazan Assay for HIV-1 Cytopathic Effects: Application to High-Flux Screening of Symthetic and Natural Products for AIDS--Antiviral Activity, Journal of the National Cancer Institute, 1989, vol. 81, No. 8, pp. 577-586.cited by other.
Grabre et al., Inhibition of Human Immunodeficency Virus type 1 transcription and replication by DNA sequence-s-elective plant lignans, Proc. Natl. Acad. Sci. USA, 1995, vol. 92, pp. 11239-11243. cited by other.
Gisvold et al., Ligans from Larrea divaricata, Journal of Pharmecutical Sciences, 1974, vol. 63, No. 12, pp. 1905-1907. cited by other.
Ru Chih C. Huang et al., "Regulation of HIV Promoter Activities in Human Embryonal Carcinoma Cells, Ntera-2", Gene Regulation and Aids, T. S. Papas, ed. 1989, pp. 147-160. cited by other.
Paul E. Giza and Ru Chih C. Huang, "A Self-Inducing Runaway-Replication Plasmid Expression System Utilizing the RPO Protein," Gene, 78(1989) pp. 73-84. cited by other.
Chemical Abstracts No. 101816r, vol. 110, No. 11, issued Mar. 13, 1989, Jordan, "Compositions Containing Catecholic Butanes and Zinc For Treating Solid Tumors". cited by other.
Chemical Abstracts No. 118499d vol. 118499d vol. 112, No. 13, issued Mar. 26, 1990, Rao, "Regioselective Cleavage of the Methylendioxy Group: Conversion of(-)-Austrobailignan-5 To (-)-Dihydroguairetic Acid." cited by other.









Abstract: A method for suppressing undesired viral growth in a host which comprises administering to the host an effective amount of a compound of the formula: ##STR00001## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of HO--, CH.sub.3O-- and CH.sub.3(C.dbd.O) O--. The method is exemplified by inhibiting Tat transactivation of a lentivirus and in suppressing Herpes simplex virus.
Claim: What is claimed is:

.[.1. A method for suppressing viral growth in a host which consists essentially of administering to the host an effective viral growth suppressing amount of a compositionconsisting essentially of a compound of the formula: ##STR00024## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each selected from the group consisting of HO--, CH.sub.3O-- and CH.sub.3(C.dbd.O)O--, provided that R.sub.1, R.sub.2, R.sub.3 and R.sub.4are not each HO--..].

2. The method of claim .[.1.]. .Iadd.4, .Iaddend.wherein .[.said compound is.]. .Iadd.the .Iaddend.water-soluble .Iadd.substituent is --O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl.Iaddend..

3. The method of claim .[.1 for suppressing.]. .Iadd.4, wherein the host is infected with .Iaddend.Herpes simplex virus .[.in the host.]. .

.Iadd.4. A method for suppressing viral growth in a host infected with a virus comprising (a) providing a composition comprising a substantially purified compound and (b) administering said composition to the host in a dosage having aneffective amount of the compound to suppress viral growth, wherein the compound is a derivative of nordihydroguaiaretic acid (NDGA) having the formula: ##STR00025## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each selected from the group consistingof HO--, CH.sub.3O-- and CH.sub.3(C.dbd.)O--, or a water soluble substituent, provided that R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are not each HO--, wherein the water soluble substituent is selected from the group consisting of:--O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl, --O(C.dbd.O)CH.sub.2NH.sub.2, ##STR00026## ##STR00027## .Iaddend.

.Iadd.5. The method of claim 4, wherein the water-soluble substituent is --O(C.dbd.O)CH.sub.2NH.sub.2..Iaddend.

.Iadd.6. The method of claim 4, wherein the compound inhibits viral transcription..Iaddend.

.Iadd.7. The method of claim 4, wherein the compound inhibits transactivation of viral gene..Iaddend.

.Iadd.8. The method of claim 4, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane (4-O-methyl-NDGA)..Iaddend.

.Iadd.9. The method of claim 4, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.10. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,3',4-tri-O-methyl-NDGA)..Iaddend.

.Iadd.11. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,4,4'-tri-O-methyl-NDGA)..Iaddend.

.Iadd.12. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane (3',4-di-O-methyl-3-O-acetyl-NDGA)..Iaddend.

.Iadd.13. The method of claim 4, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane (3,3'-di-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.14. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane (4,4'-di-O-methyl-3-O-acetyl-NDGA)..Iaddend.

.Iadd.15. The method of claim 4, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane (3,4'-di-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.16. The method of claim 4, wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are not each CH.sub.3O-- or CH.sub.3(C.dbd.O)O-- simultaneously..Iaddend.

.Iadd.17. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 95 .mu.M..Iaddend.

.Iadd.18. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 62.7 .mu.M..Iaddend.

.Iadd.19. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 31.3 .mu.M..Iaddend.

.Iadd.20. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 25 .mu.M..Iaddend.

.Iadd.21. The method of claim 4, wherein the effective viral growth suppressing amount of the compound is less than 9.5 .mu.M..Iaddend.

.Iadd.22. A method of inhibiting replication of an acyclovir-resistant virus in a cell comprising the steps of: (a) providing a substantially purified compound having a formula: ##STR00028## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 areeach selected from the group consisting of HO--, CH.sub.3O-- and CH.sub.3(C.dbd.O)O--, and a water soluble substituent, wherein the water soluble substituent is selected from the group consisting of: --O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl,--O(C.dbd.O)CH.sub.2NH.sub.2, ##STR00029## ##STR00030## (b) contacting the cell with the compound..Iaddend.

.Iadd.23. A method of treatment of acyclovir-resistant viral infection in a subject comprising the steps of: (a) providing a substantially purified compound having the formula: ##STR00031## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are eachselected from the group consisting of HO--, CH.sub.3O-- and CH.sub.3(C.dbd.O)O--, and a water soluble substituent, wherein the water soluble substituent is selected from the group consisting of: --O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl,--O(C.dbd.O)CH.sub.2NH.sub.2, ##STR00032## ##STR00033## (b) administering the substantially purified compound to the subject..Iaddend.

.Iadd.24. A method of treatment of a subject infected with a virus, wherein the virus is resistant to acyclovir comprising the steps of: (a) providing a composition comprising a substantially purified compound; and (b) administering saidcomposition in a dosage having a therapeutically effective amount of the compound to the subject, wherein the compound has the formula: ##STR00034## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each selected from the group consisting of HO--,CH.sub.3O-- and CH.sub.3(C.dbd.O)O--, and a water soluble substituent, wherein the water soluble substituent is selected from the group consisting of: --O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl, --O(C.dbd.O)CH.sub.2NH.sub.2, ##STR00035## ##STR00036##.Iaddend.

.Iadd.25. The method of claim 24, wherein the water-soluble substituent is --O(C.dbd.O)CH.sub.2NH.sub.2..Iaddend.

.Iadd.26. The method of claim 24, wherein the water-soluble substituent is --O(C.dbd.O)CH.sub.2NH(CH.sub.3).sub.2.Cl..Iaddend.

.Iadd.27. The method of claim 24, wherein the compound inhibits viral transcription..Iaddend.

.Iadd.28. The method of claim 24, wherein the compound inhibits transactivation of the viral gene..Iaddend.

.Iadd.29. The method of claim 24, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane (4-O-methyl-NDGA)..Iaddend.

.Iadd.30. The method of claim 24, wherein the compound is 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane (3-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.31. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,3',4-tri-O-methyl-NDGA)..Iaddend.

.Iadd.32. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbutane (3,4,4'-tri-O-methyl-NDGA)..Iaddend.

.Iadd.33. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane (3',4-di-O-methyl-3-O-acetyl-NDGA)..Iaddend.

.Iadd.34. The method of claim 24, wherein the compound is 1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane (3,3'-di-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.35. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane (4,4'-di-O-methyl-3-O-acetyl-NDGA)..Iaddend.

.Iadd.36. The method of claim 24, wherein the compound is 1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane (3,4'-di-O-methyl-4-O-acetyl-NDGA)..Iaddend.

.Iadd.37. A method of treatment of viral infection in a host comprising the steps of: (a) providing a composition comprising a compound; and (b) administering said composition in a dosage having a viral inhibitory amount of the compound to thehost, wherein the compound has the formula selected from the group consisting of: ##STR00037## ##STR00038## .Iaddend.
Description: BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the isolation, purification and characterization of derivatives of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (.[.nordihydroquaiaretic.]. .Iadd.nordihydroguaiaretic .Iaddend.acid, NDGA) The derivatives wereisolated from leaf and flower extracts of the creosote bush (Larrea tridentata, Zygophyllaceae) and together with NDGA can be used to suppress Tat trans- activation in lentiviruses, including the HIV virus. Other broader aspects of the invention,including use of the disclosed compounds for the treatment of Herpes simplex virus, will also be hereinafter apparent.

2. Description of the Related Art

Tat is a transactivator of human immunodeficiency virus (HIV) gene expression and is one of the two or more necessary viral regulatory factors (Tat and Rev) for HIV gene expression. Tat acts by binding to the TAR RNA element and activatingtranscription from the long terminal repeat (LTR) promoter.

The Tat protein stabilizes elongation of transcription and has also been shown to be involved in transcription initiation. Previous studies have shown that Tat mediates reduction of antibody-dependent T cell proliferation, contributingsubstantially to the failure of the immune response. Tat also directly stimulates Kaposi's cell growth.

Since Tat has no apparent cellular homologs, this strong positive regulator has become an attractive target for the development of anti-AIDS drugs (see FIG. 1). In contrast to currently available HIV reverse transcriptase inhibitors (AZT, DDI)or potential protease inhibitors that prevent new rounds of infection, an inhibitor which suppresses viral gene Tat regulated expression of integrated proviral DNA will arrest the virus at an early stage (Hsu et al., Science 254:1799-1802, 1991).

Efforts aimed at the elucidation of factors which control gene expression at transcriptional and post-transcriptional levels in host eukaryotes have recently made possible quantitative assessment of Tat function (Sim, Ann. N.Y. Acad. Sci. 616: 64-70, 1990, the entire contents of which are hereby incorporated by reference and relied upon). To screen for inhibitors for Tat regulated transactivation (Tat-TRS), the secreted placental alkaline phosphatase (SEAP) reporter gene is put under thecontrol of HIV-1 LTR promoter in the plasmid pBC12HIV/SEAP. The Tat-coded activity is supplied by a second plasmid construct pBC12/CMV/t2. Transient cotransfection of COS cells with these two plasmids leads to secretion of alkaline phosphatase into theculture medium which is analyzed by a simple colorimetric assay (Berger et al., Gene 66: 1-10, 1988, the entire contents of which are hereby incorporated by reference and relied upon) The SEAP assay, therefore, provides an indirect determination of Tattransactivation. An inhibitor should cause reduction of the SEAP activity which is due to inhibition of the expression of the SEAP mRNA via transactivation of the HIV-1 LTR promoter by Tat protein (Tat-TRS).

SUMMARY OF THE INVENTION

In the present application, we disclose Tat-TRS inhibitory activity of the desert plant Larrea tridentata. Among several plant extracts prepared from rain forest and desert medicinal plants used in traditional medicinal against viral affections,only the total extract from the leaves and flowers of the creosote bush (Larrea tridentata) showed Tat-TRS inhibitory activity. This extract also inhibits HIV cytopathic effects on human lymphoblastoid cells chronically infected with the virus asassessed by the newly developed soluble-formazan assay (Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and relied upon).

The present invention discloses compounds of the structural formula: ##STR00002## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each selected from the group consisting of HO--, CH.sub.3O-- and CH.sub.3(C.dbd.O)O--, provided that R.sub.1,R.sub.2, R.sub.3 and R.sub.4 are not each HO- simultaneously.

Each compound was isolated from leaf-flower extracts of the creosote bush Larrea tridentata and is a derivative of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (.[.nordihydroquaiaretic.]. .Iadd.nordihydroguaiaretic .Iaddend.acid, NDGA).

In addition, NDGA and derivatives can be used to suppress Tat transactivation of a lentivirus, including the HIV virus, in a cell by administering NDGA or a derivative thereof to the cell.

It is also contemplated according to the invention that the compounds disclosed herein can be used to inhibit Herpes simplex virus and other undesired virus growth in a host as later described.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 graphically illustrates the life cycle of HIV-1 and different sites of action of potential therapeutic agents including Tat-TRS inhibitors. The basal transcription step is indicated by 1 and the viral regulatory protein dependenttransactivation step by 2.

FIG. 2 demonstrates the induction of the secreted alkaline phosphatase (SEAP) expression in the standard SEAP assay.

FIG. 3 shows the inhibition of Tat-TRS activity by creosote bush total extract in the secreted alkaline phosphatase (SEAP) assay.

FIG. 4 shows an analysis of plant-derived HIV Tat inhibitors in Component Lo by gas chromatography (GC) using an analytical non-destructive capillary cross-linked 5% phenyl-methylsiloxane (HP-5) column. Component Lo is a mixture of fourcomponents. The time (minutes) of elution appears on each peak.

FIG. 5 shows an analysis of plant-derived HIV Tat inhibitors in Component Gr by gas chromatography (GC) using an analytical non-destructive capillary cross-linked 5% phenyl-methylsiloxane (HP-5) column. Component Gr is a complex mixture. Thetime (minutes) of elution appears on each peak.

FIG. 6 illustrates the inhibition of Tat-induced SEAP expression by the plant-derived single compound Malachi 4:5-6 (Mal 4) and NDGA in the secreted alkaline phosphatase (SEAP) assay.

FIG. 7 depicts the quantitation of XTT formazan production as a measurement of viable cells in Mal 4 treated cultures of either HIV infected or uninfected CEM-SS cells in two separate experiments. The percent XTT formazan production for infectedcells without Mal 4 treatment for experiment 1 was 8.9% and for experiment 2 was 7.5%. For experiment 1, uninfected cells are represented by .cndot. - - - .cndot. and infected cells by x - - - x. In experiment 2, uninfected cells are represented by.cndot. - - - .cndot. and infected cells by x - - - x The EC.sub.50 was found to be 4.25 .mu.g/ml or 13.4 .mu.M. The IC.sub.50 was estimated to be 100 .mu.g/ml or 325 .mu.M.

FIG. 8 is a photograph providing a comparative showing of HSV-1 replication in a guinea pig which illustrates a further use of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention discloses the isolation, purification and characterization of derivatives of 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane or .[.nordihydroquaiaretic.]. .Iadd.nordihydroguaiaretic .Iaddend.acid (NDGA). Each derivativeof NDGA was isolated, purified and characterized according to the following procedures.

Materials and Methods

Cell line: COS-7 cells with SV40 origin of replication were maintained in Isocove's Modified Dulbecco's Medium (IMDM) supplemented with lo fetal calf serum (FCS) and antibiotics. Cells were incubated in a humidified 95% O.sub.2/5% CO.sub.2incubator at 37.degree. C.

Plasmids: The plasmid pBC12/HIV/SEAP containing the Tat-sensitive HIV LTR promoter with the SEAP reporter gene but no Tat-coded function was used to express SEAP basal activity; pBC12/CMV/t2 supplied the Tat-coded function, i.e., the induced SEAPlevel. pBC12/RSV/SEAP containing the constitutive Tat-insensitive LTR promoter of Rous Sarcoma Virus (RSV) served as a positive control. All plasmids were obtained from Dr. Bryan Cullen, Duke Medical Center. Plasmids pSEAP and pBKCMV and HIV LTR andTat DNA are commercially available from Clontech and Stratagene. Plasmid transformation was achieved in E. coli MC1061 strain, which was obtained from Dr. Barbara Bachmann, Department of Biology, Yale University. E. coli MC1061 strain can also bepurchased from Clontech. Plasmid DNAs were purified using Qiagen.RTM. purification kit (Qiagen).

Chemical Reagents: Diethanolamine (# 31589) and p-nitroph;enylphosphate (#71768) were purchased from Fluka BioChemika, and L-homoarginine (#H-1007) was purchased from Sigma Co. The lipospermine DOGS (Transfectam.RTM., #E123 A, Promega) was usedin DNA transfection studies.

Preparation of Plant Testing Materials: The leaves and flowers of the creosote bush were collected based on ethnopharmacological inquiries. Plant materials were dried and ground in a 3 mm screen Willy mill. In pilot studies, 1 g of the plantpowder was initially extracted by successive macerations using a mixture of chloroform:methanol. The extract was concentrated to residue. The entire 176 mg of the crude extract generated were treated 7 times with 3 ml of hexane. This step afforded 137mg of hexane-insoluble (HI) materials and 31 mg of hexane-soluble (HS) materials. All these extracts were monitored stepwise by SiO.sub.2 TLC with cerium sulfate charring, 2% CeSO.sub.4 (w/V) in 5.6% H.sub.2SO.sub.4 (v/v), and by the SEAP assay for antiTat-TRS activity.

For the SEAP assay, test materials were dissolved in 10% DMSO solution made in calcium/magnesium-free PBS. The suspension was centrifuged and the stock solution (10 mg/ml) was filter-sterilized using a Millex.RTM.-GS 22 .mu.m filter (Millipore). Appropriate dilutions of the stock solution were prepared in a final DMSO concentration of 0.2% in PBS to obtain the various concentrations of test compounds.

Differential Practionation and Purification of the Active Ingredient by Countercurrent Chromatography (CCC): Based on the preliminary results of the above fractions in the SEAP assay, further fractionation of the active HI fraction by CCC wasundertaken. This led to the identification of two major active fractions denoted "Green" and "Yellow" components. Identification of the most active fractions from these prospective studies prompted a full scale differential fractionation of plantpowder in the attempt to generate a large quantity of the Green and Yellow fractions. This fractionation was carried out on 101.4 g of plant powder, and started with a hexane treatment step, as outlined in Table 1.

Further fractionation of the major components from the organic phase (OG) obtained after chloroform:water partition was achieved by countercurrent liquid-liquid partition chromatography using the versatile cross-axis planet centrifuge (CPC) asdescribed by Ito and Conway, CRC Critical Reviews of Analytical Chemistry 17: 65 et seq., 1986, the entire contents of which are hereby incorporated by referenced and relied upon. The optimal solvent system was a mixture of hexane:EtOAC:MeOH:0.5% NaClin the ratio of 6:4:5:5, with the upper phase (organic layer) as the mobile phase. Five g of the organic fraction were dissolved in 23 ml of a mixture of the two phases and introduced into the coil via a loop valve. The mobile phase pumped through thecoil while rotating it at 800 rpm. At a flow rate of approximately 4 ml per minute, approximately 32% of-the stationary phase was initially lost (68%) retention). After the appearance of mobile phase in the elute, fractions of mobile phase werecollected, evaporated to dryness, monitored by TLC and pooled accordingly into 5 batches: Solvent front (SF), Green (Gr), Yellow (Ye), Red and Stationary phase (StP). All these fractions were then monitored by the SEAP assay for anti Tat-TRS activity.

Further purification of the Green and Yellow fractions was achieved by CCC using an epicyclic coil planet centrifuge known to the Ito multilayer coil separator-extractor (Ito and Conway, CRC Critical Reviews of Analytical Chemistry 17: 65 etseq., 1986, the entire contents of which are hereby incorporated by reference and relied upon). The solvent system was a mixture of hexane:EtOAC:MeOH:0.5% NaCl (7:3:5:5).

Two hundreds mg of Green fraction afforded 6.8 mg of a component termed Gr, i.e., =0.051% total yield based on the original plant powder. Similar studies on the Yellow fraction (Ye) generated 9.3 mg of a component denoted Lo. These purifiedcomponents (Lo and Gr) each consists of several compounds, and their respective "mother" fractions were stored at 4.degree. C. until tested for biological activity and used for further characterization.

TABLE-US-00001 TABLE 1 DIFFERENTIAL FRACTIONATION AND COUNTERCURRENT CHROMATOGRAPHY (CCC) OF NDGA DERIVATIVES FROM THE CREOSOTE BUSH, LARREA TRIDENTATA. ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007##

Cell Culture and DNA Transfection: COS cells were maintained as previously described (Cullen, Cell 46: 973-982, 1986, the entire contents of which are hereby incorporated by reference and relied upon). DNA transfection was performed using amodified procedure of the lipospermine (Transfectame, Promega #E123A) method originally described elsewhere (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993, the entire contents of which are hereby incorporated by reference and relied upon). Briefly, a day before DNA transfection, Linbro.RTM. 24 flat bottom well of 17-mm diameter plates were pretreated with 0.5 ml sterile solution of 0.1% gelatin. The plates were kept in the hood for 1 hour (all transfection steps were performed in thehood, unless otherwise stated). The gelatin solution was aspirated and the plates were washed with 0.5 ml of IMDM supplemented with 10% fetal calf serum and antibiotics (complete medium). COS cells were seeded at a density of.apprxeq.1.5.times.10.sup.5 cells per 17-mm plate and incubated in a humidified 95% O.sub.2/5% CO.sub.2 incubator at 37.degree. C. DNA transfection was performed at 30-50% cell confluency. The stock solution of the Transfectam reagent, DOGS, wasprepared according to the manufacturer's advice at 1 mg/0.380 ml (2.38 mg/ml or 3.4 mM) in 10% (v/v) ethanol in distilled water.

The transfection cocktail consisted of two solutions prepared in sterile tubes: a) Solution A contained a sterile 150 mM NaCl solution+plasmid DNAs (non selected/selected gene in 2:1 ratio): 0.35 .mu.g of pBC12/HIV/SEAP per well+0.175 .mu.g ofpBC12/CMB/t2 (coding for Tat function) per well. b) Solution B Contained an equal volume of 150 mM NaCl and a volume of Transfectame determined to be 6 times the total amount of DNAS. Solutions A and B were homogenized and immediately mixed.

Ten minutes were allowed for the reaction to proceed. Meanwhile, the growth medium was removed from the subconf luent COS cells and 300 .mu.l (100 .mu.l of complete IMDM+200 .mu.l serum-free medium) were added to each well. The transfectioncocktail was dispensed to the wells in equal volume. Control samples containing no DNA were similarly treated and received sterile 150 mM NaCl solution alone. All samples were incubated for 10 and 12 hours after which 700 .mu.l of complete growthmedium were added. Test compounds prepared in 5% DMSO/Ca-Mg-free PBS (for non-water-soluble materials) were immediately added at various concentrations to the wells. Drug-untreated control samples received 5% DMSO/PBS solution alone (final DMSOconcentration of 0.2%). All samples were then incubated for an additional 48 hours after which 300 .mu.l of each culture supernatant was removed for SEAP analysis.

The Secreted Alkaline Phosphatase (SEAP) Assay: The secreted alkaline phosphatase analysis was performed as originally described (Berger et al., Gene 66: 1-10, 1988, the entire contents of which are hereby incorporated by reference and reliedupon). Briefly, a 250-.mu.l aliquot was removed from COS cell culture supernatants, heated at 65.degree. C. for 5 minutes to selectively inactivate endogenous phosphatase (SEAP is heat stable) and centrifuged in a microfuge for 2 minutes. One hundred.mu.l of 2.times.SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl.sub.2; 10 mm L-homoarginine) were added to 100-.mu.l aliquot of the samples. The solution was mixed and transferred into a 96-well flat-bottom culture dish (Corning). Twenty.mu.l of pre-warmed substrate solution (120 mM p-nitrophenylphosphate dissolved in 1.times.SEAP assay buffer) were dispensed with a multipipeter into each well containing the reaction mixture. A.sub.405 of the reaction was read at 5-minute intervals at37.degree. C. for 60 minutes using an EL340i microplate reader (Bio-tek Instruments, Inc.) with 5-second automatic shakings before each reading. The change in absorbance was plotted against time in the standard assay of SEAP induction. In the drugscreening assay, the percent inhibition of SEAP expression was calculated at 30 minutes as follows: % Inhibition=100-[(CT.sup.+-C.sup.+).times.100] where:

C.sup.-: Control sample (no DNA, no drug)

CT.sup.-: Control sample (+DNA, no drug)

C.sup.+: Drug-treated sample (no DNA, +drug)

CT.sup.+: Drug-treated sample (+DNA, +drug)

Optimization of the Transfection Technique: Various techniques are utilized in the DNA transfection of eukaryotic cells. These procedures include DNA coprecipitation with calcium phosphate or cationic polymers, cell membrane weakening either bychemical means (detergents, solvents, enzymes, amphophilid polymers) or by physical means (thermic, osmotic or electric shocks, or particle bombardment). These techniques suffer, to some extent, variable efficiency and varying degrees of cytotoxicity.

Prerequisites for cells to be amenable to DNA uptake, i.e., to cross the intact cytoplasmic membrane, are "compaction and masking of DNA charges" (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). These requirements have beensuccessfully met with the newly developed Transfectam.RTM. procedure. The Transfectam reagent (dioctadecylamidoglycyl spermine) is a synthetic cationic lipopolyamine which contains a positively charged spermine headgroup with a strong affinity for DNA(K.sub.d=10.sup.5-10.sup.-7 M). This spermine headgroup is covalently attached to a lipid moiety by a peptide bond. The lipospermine molecules bind to DNA, coating it with a lipid layer. In the presence of excess lipospermine, cationic lipid-coatedplasmid DNA vesicles are formed and the lipid portion of the complex fuses with cell membrane. DNA internalization is-believed to occur by endocytosis.

Transfectam-mediated transfection has been shown to offer greater efficiency than existing methods (Barthel et al., DNA and Cell Biology 12 (6): 553-560, 1993). In addition, Transfectam.RTM. is a stable and virtually non-cytotoxic reagent. However, factors for optimization of transfection in the specific COS cell line had to be addressed. These factors include the duration of transfection, the ratio of the Transfectam reagent to DNA, DNA concentration and other dilution factors such asNaCl volume and strength. The results of optimization of transfection conditions are shown below.

a) Duration of transfection: COS cells were incubated with a fixed plasmid DNA concentration in time course studies. These studies aimed at the selection of the suboptimal incubation time point for inhibition studies of SEAP expression byvarious test compounds. The results of the time-course induction of SEAP expression (results not shown) indicate a gradual time-dependent increase in SEAR expression. The onset of this induction began at less than 4 hours and reached a maximum at 24hours. No significant difference was observed between the 10, 12 and 15-hour values. Therefore, the 12-15 hours endpoint was selected as the appropriate suboptimal incubation period for inhibition of SEAP expression in all subsequent drug screenstudies.

b) DNA concentration: The optimal DNA concentration for transfection was determined based on previous studies with Transfectam reagent (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). For contransfection, the ratio 2:1 (nonselectedgene/selected gene) was found to be the most appropriate as reported elsewhere (Hsu et al., Science 254: 1799-1802, 1991, the entire contents of which are hereby incorporated by reference and relied upon). The nonselected pBC12/HIV/SEAP plasmid wasutilized at a concentration of 0.35 .mu.g/well and pBC12/CMV/t2 plasmic coding for Tat function at a concentration of 0.75 .mu.g/well in Linbro.RTM. 24 flat bottom well of 17-mm diameter plates.

c) Ratio of Transfectam to DNA and Determination of Ionic Strength: The optimal ratio of Transfectam.RTM. (DOGS) to plasmid DNA and the ionic strength of NaCl used were a modification of the previously reported values (Loeffler and Behr, Methodsin Enzymology 217: 1799-1802, 1993) and determined as follows: From the original 1 mg/0.400 ml (2.38 mg/ml) stock solution of Transfectam.RTM. prepared in 10% (v/v) ethanol in distilled water, 6 times the volume (.mu.l) of stock solution was requiredfor each .mu.g DNA used. The optimal ionic strength of the solution was provided by an appropriate volume of 150 mM NaCl determined by the relation: Volume (.mu.l) of NaCl=Volume (.mu.l) Transfectam/0.6

The results of Tat-induced SEAP levels in the standard assay after optimization of these conditions, are illustrated in FIG. 2. Briefly, COS cells were maintained in Isocove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal calfserum (FCS) and antibiotics. Triplicate cell samples were seeded at a density of .apprxeq.1.5.times.10.sup.5 cells per well in Linbro.RTM. 24 flat bottom wells of 17-mm diameter and incubated in a humidified 95% O.sub.2/5% CO.sub.2 incubator at37.degree. C. until they reached 50% confluency. Subconfluent cells were transfected using the lipospermine procedure (Loeffler and Behr, Methods in Enzymology 217: 599-618, 1993). The medium of the subconfluent cells was aspirated and replaced by 300.mu.l of fresh minimum medium (IMDM supplemented with 3% FCS). COS cells were transfected with either pBC12/HIV/SEAP alone (0.35 .mu.g/well) or pBC12/CMV/t2 (coding for Tat function) at 0.175 .mu.g/well+pBC12/HIV/SEAP (0.35 .mu.g/well) or with bufferalone (no DNA control samples). The plates were incubated for 12 to 15 hours after which, 700 .mu.l of complete medium (IMDM containing 10% FCS) were added. Cells were then incubated for 48 hours after which, a 250-.mu.l aliquot was removed from COScell culture supernatants and heated at 65.degree. C. for 5 minutes to selectively inactivate endogenous phosphatases (SEAP is heat stable). The samples were then centrifuged in a microfuge for 2 minutes. One hundred .mu.l of 2.times.SEAP assay buffer(1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl.sub.2; 10 mM L-homoarginine) were added to 100-.mu.l aliquot of the samples. The solution was mixed and transferred into a 96-well flat-bottom culture dish (Corning). Twenty .mu.l of pre-warmed substratesolution (120 mM p-nitrophenylphosphate dissolved in 1.times.SEAP assay buffer) were dispensed with a multipipetter into each well containing the reaction mixture. A.sub.405 of the reaction was read at 5-minute intervals at 37.degree. C. for 60 minutesusing an EL340i microplate reader (Bio-tek Instruments, Inc.) with 5-seconds automatic shaking before each reading. The change in absorbance was converted in mU of SEAP expression as previously described (Berger et al., Gene 66: 1-10, 1988) and plottedagainst time.

These results indicate a nearly 65-fold increase in SEAP induction after 1 hour relative to the control (no DNA) levels or the induction of nonselected gene (HIV/SEAP) alone.

Assay-Guided Isolation of Creosote Bush Extract Active Component(s) by Countercurrent Chromatography: As stated labove, the differential fractionation and purification by countercurrent chromatography (CCC) of the creosote bush extractconstituents led to the isolation of two major components (Table 1). 6.8 mg of the component termed Gr was isolated from the Green fraction on SiO.sub.2 TLC. The total percent yield was .apprxeq.0.051% based on the original plant powder. 9.3 mg of thecomponent Lo was isolated from the Yellow fraction (Ye).

Inhibition of Tat-TRS Activity by Extracts from Creosote Bush Leaves and Flowers: In several plant extracts tested with the SEAP assay, only the extract from the creosote bush, Larrea tridentata, leaves and flowers showed significant inhibitoryactivity of HIV Tat protein. Creosote bush displayed a dose-response inhibition of SEAP expression as illustrated in FIG. 3. Briefly, triplicate samples of COS cells were transfected with a mixture of pBC12/HIV/SEAP and PBC12/CMV/t2 (coding for Tatfunction) in 2:1 ratio, using the lipospermine procedure as described above. Cells were incubated for 12-15 hours after transfection. Creosote bush extract stock solution (10 mg/ml) was made in calcium/magnesium-free PBS and 10% DMSO, andfilter-sterilized using a Millex.RTM.-GS 22 .mu.m filter (Millipore). The appropriate concentrations of creosote bush extract were added to the transfected cells at a final DMSO concentration of 0.2% and samples were incubated for 48 hours. For SEAPanalysis, a 250-.mu.l aliquot was removed from COS cell culture supernatants, heated at 65.degree. C. for 5 minutes to selectively inactivate endogenous phosphatases (SEAP is heat stable) and centrifuged in a microfuge for 2 minutes. One hundred .mu.lof 2.times.SEAP assay buffer (1.0 M diethanolamine, pH 9.8; 0.5 mM MgCl.sub.2; 10 mM L-homoarginine) were added to 100-.mu.l aliquot of the samples. The solution was mixed and transferred into a 96-well flat-bottom culture dish (Corning). Twenty .mu.lof pre-warmed substrate solution (120 mM p-nitrophenylphosphate dissolved in 1.times.SEAP assay buffer) were dispensed with a multipipetter into each well containing the reaction mixture. A.sub.405 of the reaction was read at 5-minute intervals at37.degree. C. for 60 minutes using an EL340i microplate reader (Bio-tek Instruments, Inc.) with 5-second automatic shaking before each reading. The percent inhibition of SEAP expression was calculated at 30 minutes as follows: %Inhibition=100-[(CT.sup.+-C.sup.+)/(CT.sup.--C.sup.-).times.100] where:

C.sup.-: Control sample (no DNA, no drug)

CT.sup.-: Control sample (+DNA, no drug)

C.sup.+: Drug-treated sample (no DNA, +drug)

CT.sup.+: Drug-treated sample (+DNA, +drug)

As seen in FIG. 3, the onset of this inhibition started at a concentration of 20 .mu.g/ml and reached a maximum inhibitory activity at 600 .mu.g/ml. The estimated EC.sub.50 (the. concentration exhibiting 50% of inhibition) for this crudematerial was 110 .mu.g/ml. As the purification of the active ingredients progressed, there was a stepwise increase in the activity of the active ingredient(s) which tripled (68%) with the organic phase (OG) fraction compared to 21% from the originaltotal crude extract.

Inhibition of HIV cytopathic Effects: A compound inhibiting Tat transactivation should in principle block HIV replication. Consequently, creosote bush extract was tested at the National Cancer Institute (NCI) for inhibition of HIV-1 cytopathiceffects using the soluble-formazan assay (Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and relied upon). In principle, CEM-SS cells (ATCC, Rockville, Md.) are cocultivated with HIV-producingH9 cells. Viruses infect the host CEM-SS cells, replicate and kill most of the CEM-SS cells in a week. If the drug inhibits HIV production, CEM-SS cells are protected from HIV-induced cell death. The tetrazolium (XTT) reagent is thereforemetabolically reduced by the viable cells to yield a colored formazan product which is measurable by colorimetry at 450 nm.

In practice, triplicate samples of CEM-SS cells (5000) were plated in 96-well microtiter plate. Appropriate concentrations of test compounds were added in a final volume of 100 .mu.l calcium/magnesium-free PBS in 5% DMSO. Control samplesreceived the compound medium (PBS) alone. Five minutes later, 500 highly infectious HIV-1 producing II9 cells or normal H9 cells were added to the wells containing the appropriate drug concentrations. The microtiter plates were incubated at 37.degree. C. in 95% O.sub.2/5% CO.sub.2 for 6 days after which a 50-.mu.l mixture of XTT and N-methylphenazonium methosulfate (PMS) was added. The plates were reincubated for additional 4 hours for the color development (XTT formazan production). The plates weresealed, their contents were mixed by automatic shaking and the OD.sub.450 of samples was determined in a microplate reader. Each value represents the average of 3 determinations. No significant difference was found between the means of the duplicatevalues of the uninfected cells and HIV-challenged cells, in presence of test compounds. In contrast, there was a significant difference (p<0.05) between HIV-challenged samples in the presence or absence of test compounds.

The results of these studies are summarized in Table 2. At a concentration of 0.75 .mu.g/ml for component Gr, there was an average 58% protection (cell viability) against HIV as opposed to 15% viability in drug-free samples challenged with HIV. At a concentration as low as 0.187 .mu.g/ml, component Lo exhibited even stronger inhibitory activity of HIV cytopathic effects. The cell viability was 87%, very close to that of not treated control cells (89%), in contrast to 14% viability for thedrug-free samples challenged with HIV. These compounds were devoid of cytotoxicity at the concentrations used.

TABLE-US-00002 TABLE 2 INHIBITION OF HIV-1 CYTOPATHIC EFFECTS BY CREOSOTE BUSH EXTRACT COMPOUNDS IN THE SOLUBLE-FORMAZAN ASSAY. Concentration Percent of live cells at of the test day 6 as measured by XXT sample which Formazan production yieldedmax Un- HIV HIV protection infected infected infected against HIV plus plus minus without killing test test test Test Sample the cells .mu./ml sample sample sample Fraction Green 0.187 59 67 16 Component G.sub.r 0.75 80 67 16 -duplicates ComponentG.sub.r 0.75 70 48 14 Fraction 0.187 60 57 14 Yellow (Ye) Component L.sub.o 0.187 91 86 14 -duplicates Component L.sub.o 0.187 89 87 14

Structure elucidation of the active components of creosote bush extract: The chemical characterization of the purified plant active constituents was achieved mainly by mass spectroscopy and by H- and C- nuclear magnetic resonance (NMR). Component Lo was found to be a mixture of four related compounds (L1, L2, L3 and L4). Resolution and characterization of each peak of the mixture was accomplished by gas chromatography (GC) using an analytical non-destructive capillary cross-linked 5%phenylmethylsiloxane (HP-5) column attached to a mass spectroscope (MS). The GC studies revealed that the first compound (L1) represented 6% of the mixture; the second (L2) was 76% of the mixture (MW=316); the third (L3) was isomeric with L2 andrepresented 9% of the total mixture (MW=316); and the fourth compound (L4) represented 9% of the mixture (MW=358). The time (minutes) of elution of these compounds is indicated on the peaks (FIG. 4).

Component Gr consisted of fifteen compounds. Resolution and characterization of each peak of the mixture was accomplished by gas chromatography (GC) using analytical non-destructive capillary cross-linked 5% phenylmethylsiloxane (HP-5) columnattached to a mass spectroscope (MS). The GC studies revealed that four (G1, G2, G3 and G4) of the fifteen compounds. The time (minutes) of elution of these compounds is indicated on the peaks (FIG. 5).

The structures of these eight compounds (L1, L2, L3, L4, G1, G2, G3 and G4) are described as follows:

L1 has the composition C.sub.18H.sub.22O.sub.4 and has been identified as a previously known chemical, 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethylbutane (.[.nordihydroquaiaretic.]. .Iadd.nordihydroguaiaretic .Iaddend.acid, NDGA, Merck Index, 10thEdition, 16534). The structural formula for L1 is as follows: ##STR00008##

L2 has the composition C.sub.19H.sub.24O.sub.4 and has been identified as 3-O-methyl-NDGA or 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-hydroxyphenyl)-2,3-dimethylbutane. The structural formula for 3-O-methyl-NDGA is as follows: ##STR00009##

L3 also has the composition C.sub.19H.sub.24O.sub.4 and has been identified as 4-O-methyl-NDGA or 1-(3,4-dihydroxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-2,3-dimethylbutane. 4-O-methyl-NDGA is also known as Malachi 4:5-6 or Mal 4. The structuralformula for 4-O-methyl-NDGA is as follows: ##STR00010##

L4 has the composition C.sub.21H.sub.26O.sub.5 and has been identified as 3-O-methyl-4-O-acetyl-NDGA or 1-(3,4-dihydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylbutane. The structural formula for 3-O-methyl-4-O-acetyl-NDGA is asfollows: ##STR00011##

G1 has a molecular weight of 344, a composition of C.sub.21H.sub.22O.sub.4 and has been identified as 3,3',4-tri-O-methyl-NDGA or .[.1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dimethylbuta- ne.]. .Iadd.1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dime- thylbutane.Iaddend.. 3,3',4-tri-O-methyl-NDGA has the following structural formula: ##STR00012##

G2 has a molecular weight of 344, a composition of C.sub.21H.sub.22O.sub.4 and has been identified as 3,4,4'-tri-O-methyl-NDGA or .[.1-(3-methoxy-4-hydroxyphenyl)-4-(3,4-diiaethoxyphenyl)-2,3-dimethylbut- ane.]. .Iadd.1-(3-hydroxy-4-methoxyphenyl)-4-(3,4-dimethoxyphenyl)-2,3-dim- ethylbutane.Iaddend.. The structural formula for 3,4,4'-tri-O-methyl-NDGA is as follows: ##STR00013##

G3 and G4 each has a molecular weight of 372 and a composition of C.sub.22H.sub.28O.sub.5. G3 is either 3',4-di-O-methyl-3-O-acetyl-NDGA (as in G3a) or 3,3'-di-O-methyl-4-O-acetyl-NDGA (as in G3b)

3',4-di-O-methyl-3-O-acetyl-NDGA is also known as 1-(3-methoxy-4-hydroxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane and has the following structural formula:

G3a: ##STR00014##

3,3'-di-O-methyl-4-O-acetyl-NDGA is also known as 1-(3-methoxy-4-hydroxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane and has the following structural formula: ##STR00015##

Similarly, G4 is either 4,4'-di-O-methyl-3-O-acetyl-NDGA (as in G4a) or 3,4'-di-O-methyl-4-O-acetyl-NDGA (as in G4b). .[.4,4'-di-O-3-O-acetyl-NDGA.]. .Iadd.4,4'-di-O-methyl-3-O-acetyl-NDGA .Iaddend.is also known as1-(3-hydroxy-4-methoxyphenyl)-4-(3-acetoxy-4-methoxyphenyl)-2,3-dimethylb- utane and has the following structural formula:

G4a: ##STR00016##

3,4'-di-O-methyl-4-O-acetyl-NDGA is also known as 1-(3-hydroxy-4-methoxyphenyl)-4-(3-methoxy-4-acetoxyphenyl)-2,3-dimethylb- utane and has the following structural formula:

G4b: ##STR00017##

In addition to the above described isolation and purification procedures, each disclosed derivative of NDGA may be prepared by chemical synthesis following either methylation and/or acetylation of NDGA according to the procedure of Ikeya et al.,Chem. Pharm. Bull. 27(7): 1583-1588, 1979, the entire contents of which are hereby incorporated by reference and relied upon.

Large scale purification of component Lo and two pure compounds (L2 and L3) from component Lo: A large scale CCC fractionation of a batch of plant materials was initiated to generate a larger quantity of component Lo. A total of 110 g plantpowder was first treated with 700 ml hexane 5 times. The hexane soluble materials (1.17 g) were discarded. The hexane insoluble material (HI fraction) was dried and extracted 3 times by successive macerations with 800 ml chloroform:methanol. Thisafforded 20 g of total extract (Tex) which was combined with 7.6 g of HI fraction obtained from a previous batch differential extraction. A 27.6 g total of crude plant materials thus generated was divided into two batches of 10 g and 17.6 g. Thesebatches were initially run separately on the large-capacity versatile Cross-Axis CPC (Shinomiya et al., J. Chromatogr. 644:215-229, 1993, the entire contents of which are hereby incorporated by reference) using the solvent system hexane:EtOAc:MeOH:0.5%NaCl in 6:4:5:5 ratio with the upper phase (organic layer) as the mobile phase. The fractions were pooled exclusively according to the TLC patterns and four major fractions were identified from the two CCC operations and denoted fraction Green (Gr)(1.12g), fraction Lo (2.87 g), fraction End (1.78 g) and finally, the stationary phase SP (20.84). The entire 2.87 g of fraction Lo was further fractionated in the large model a triplet CPC with the hexane:EtOAc:MeOH:H.sub.2O system in 7:3:5:5 ratio, usingthe aqueous layer as mobile phase. Four fractions denoted LYI (0.375); LYII (0.113 g); LYIII (0.280 g) and LYIV (2.80 g) were identified according to the elution order and the TLC patterns. These fractions were assayed for anti Tat-TRS activity at 10.mu.g/ml. Based on the test results, LYI was selected for further purification.

Isolation of L2 and L3 pure compounds from LYI fraction of component Lo: The less hydrophobic fraction LYI was selected for further purification using the previously improved conditions, i.e., the triplet CPC and Hex:CHCl.sub.3:MeOH:10 mM NaClsolvent system in 1:4:4:2 ratio. This afforded preparation of 148 mg of homogeneous L3 and 109.3 mg of pure L2 as examined by NMR and mass spectroscopy. The structures of L3 (Malachi4:5-6) and L2 have been described above.

Compounds L2 and L3 are derivatives of a previously identified chemical, 1,4-bis-(3,4-dihydroxyphenyl)-2,3-dimethyl butane (.[.nordihydroquaiaretic.]. .Iadd.nordihydroguaiaretic .Iaddend.acid, NDGA, Merck Index, 10th Edition, #6534). Thestructural formula for NDGA, which is identical to that of L1 described above, is as follows: ##STR00018##

The anti-HIV activity (the inhibition of Tat regulated HIV transactivation) of NDGA and its derivatives was previously unknown. Comparative anti-HIV transactivation activity for NDGA and derivative Malachi4:5-6 (Mal 4) are illustrated in FIG. 6. Briefly, duplicate samples of subconfluent COS cells were co-transfected with plasmid pBC12/HIV/SEAP and pBC12/CMB/t2 (coding for Tat function) using the lipospermine procedure as described above. Cells were then incubated for 12-15 hours. The testcompounds were initially solubilized in 10% .[.DMSOicalcium-magnesium-free.]. .Iadd.DMSO in calcium-magnesium-free .Iaddend.PBS and added to the transfected cells in the appropriate concentrations at a final DMSO concentration of 0.2%. The samples wereincubated for 48 hours after which, a 250-.mu.l aliquot was removed from COS cell culture supernatants, and SEAP was analyzed as in the standard assay as in FIG. 3. The percent inhibition of SEAP expression was calculated at 30 minutes as follows: %Inhibition=100-[(CT.sup.+-C.sup.+)/(CT.sup.--C.sup.-).times.100] where:

C.sup.-: Control sample (no DNA, no drug)

CT.sup.-: Control sample (+DNA, no drug)

C.sup.+: Drug-treated sample (no DNA, +drug)

CT.sup.+: Drug-treated sample (+DNA, +drug)

Each point represents the average of two determinations. No significant difference was apparent between the EC.sub.50s of Mal 4 and NDGA which were 8 .mu.g/ml (25 .mu.M) and 6 .mu.g/ml (20 .mu.M), respectively. The EC.sub.50s are defined as theinhibitory concentration of the compound at which the Tat regulated HIV transactivation is reduced to 50% of that in untreated control cells.

The inhibition of transactivation of HIV promoter activity by Mal 4 and NDGA were compared in Table 3.

TABLE-US-00003 TABLE 3 INHIBITION OF TRANSACTIVATION OF HIV PROMOTER ACTIVITY BY NATURAL COMPOUNDS MAL 4 and NDGA. Inhibition of Tat-induced Scap Expression Test (% inhibition/concentration of test compound) Compound % .mu.m % .mu.m % .mu.m %.mu.m Mal 4 13.6 9.5 60.4 31.3 92 62.7 100 95 NDGA 17.0 9.9 73.8 32.6 88.1 65.2 92.9 99

The compounds NDGA and Mal 4 were assayed as described for FIG. 6. Control samples were run in quadruplicate. The percentage inhibition was determined after 30 minutes and the OD.sub.405 values were:

C.sup.-: Control sample (no DNA, no drug): 0.091

CT.sup.-: Control sample (+DNA, no drug): 0.805.

The quantitation of XTT formazan production as a measurement of viable cells in Mal 4 treated cultures of CEM-SS cells in shown in FIG. 7. Each figure shows infected or uninfected CEM-SS target cells (10.sup.4/M well) with serial dilutions ofMal 4. EC.sub.50 represents the concentration of Mal 4 (e.g. 13.4 .mu.M) that increases (protects) XTT formazan production in infected culture to 50% of that in uninfected, untreated culture cells. IC.sub.50 represents inhibitory or toxic concentrationof Mal 4 (e.g., 325 .mu.M, estimated) that reduces XTT formazan production in uninfected cultures to 50% of that in untreated, uninfected control cells. Levels of XTT formazan in untreated, infected control cells were 9% of those in untreated,uninfected control cells. The soluble-formazan assay for HIV-1 cytopathic effects was conducted according to the procedure described by Weislow et al., JNCI 81: 577-586, 1989, the entire contents of which are hereby incorporated by reference and reliedupon.

It will be appreciated that the invention in its broadest method aspects contemplates the use of NDGA or any of its pharmaceutically acceptable derivatives for suppressing undesired viral growth in a host. These derivatives comprisemodifications of NDGA where one or more, and preferably all four, of the hydroxy groups are replaced by, for example, other substituents such as CH.sub.3O--, CH.sub.3(C.dbd.O)O--, or the like. Preferably, however, these substituents are selected toprovide water-solubility without affecting therapeutic properties. The active compounds to be used in the invention can, therefore, be structurally represented as follows: ##STR00019## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each independentHO--, CH.sub.3O--, CH.sub.3(C.dbd.O)O-- or other pharmaceutically acceptable equivalent thereof, with a preference for substituents which provide water-solubility. As representative water-soluble derivatives of NDGA suitable for use herein, there may bementioned compounds of the foregoing formula wherein each R.sub.1-R.sub.4 substituent is the group. ##STR00020## or the group ##STR00021##

Alternatively, the R.sub.1 and R.sub.2 substituents and the R.sub.3 and R.sub.4 substituents may be joined together with the adjacent carbon atoms of each phenyl ring to form cyclic structures as follows: ##STR00022##

As indicated, the foregoing are given only for purposes of illustration as those in the art will appreciate that other alternative pharmaceutically acceptable R.sub.1-R.sub.4 substituents may be employed to provide water-solubility or to obtainfunctionally equivalent pharmacological results.

While the foregoing disclosure is directed primarily to the suppression of HIV Tat transactivation, the compounds disclosed herein are also applicable to suppress other undesired viral gene expression. In other words, the invention is notlimited to use of the indicated compounds for the suppression of Tat transactivation of a lentivirus. The effectiveness of the compounds against other viral gene expression can be readily determined by testing the compounds in appropriate art-recognizedtest systems. Thus, for example, the compounds of the invention have been found to be effective for inhibition of the Herpes simplex virus. Without intending to be bound by any theory as to how the compounds function against this virus or other virus,it is believed that the compounds proceed by a common mechanism which includes inhibiting proviral transcription and transactivation essentially as earlier described herein.

This broader application of the invention is illustrated hereafter with data showing the suppression of Herpes simplex virus replication by inhibition of transcription and transactivation of HSV IE gene .alpha..sub.4 (ICP.sub.4).

By way of explaining this further aspect of the invention, it is generally known that manifestation of Herpes simplex virus (HSV-1, HSV-2) infection has frequently been observed in HIV infected patients. Initial treatments with anti viral agents(such as acyclovir for HSV and reverse transcriptase and viral protease inhibitors for HIV-1) have been found to be quite effective. However, drug resistance and high sensitivity to multiple drugs often developed in these patients with time. Antiviralstrategies aimed at controlling the replication of wild type and mutants of both viruses by drugs which can control both viruses thus are clinically important. Transcription of proviral HIV and HSV IE genes (.alpha..sub.0, .alpha..sub.4, .alpha..sub.22,.alpha..sub.27, and .alpha..sub.47) (Ref. Deluca, N. A. and Schaft, P., J. Virol. 14, 8, 1974) are essential for replication of these two respective viruses. The expressions are thoroughly host dependent, utilizing RNA polymerase II and cellulartranscription factors. The promoter activities of proviral HIV and HSV .alpha..sub.4 (ICP.sub.4) gene are regulated by host protein Spl in addition to other cellular factors (Ref. Jones, K. A. and Tjian, R., Nature 317, 179 1985).

The use of the invention for suppressing Herpes simplex virus (HSV-1, HSV-2) is illustrated hereinafter using tetramethoxyl nordihydroguaiaretic acid (CH.sub.3--O).sub.4 NDGA, 4N of the formula: ##STR00023## to inhibit HSV ICP.sub.4 genetransactivation and HSV replication in vero cells at a dosage range that is not toxic to the host cells (see Table 4).

In addition, in comparison with acyclovir (ACV), a commercially available HSV drug, HSV virus showed no drug resistance toward tetramethoxyl NDGA (4N) following ten viral passages in vero cells. The IC.sub.50 for 4N remained essentially the samefrom first passage to tenth passage (IC.sub.50 app 10 .mu.m) while the concentration of acyclovir required to inhibit HSV-1 increased from 7.5 .mu.m at first passage to 440 .mu.m at the 10th passage (see Table 5A) Consequently, tetramethoxyl NDGA, 4N,was able to also inhibit the acyclovir resistant strain (HSV-1 SM44-CV4r) very effectively (IC.sub.50 of 6.9 .mu.m) (see Table 5B).

It is also noted that tetramethoxyl NDGA 4N has been used in an animal system, i.e. to treat HSV infected skin of guinea pigs. At a concentration of 60 mg/ml, 4N eliminated HSV-1 growth completely in five days following daily topical treatmentof the drug at the infected skin areas (FIG. 8).

Two male guinea pigs (350 gm body weight) were used in the guinea pig experiment. The back skin of the animal was shaved and equally divided into six patches. Each skin patch was pinched with seven-pin needles and HSV-1 suspension was appliedtopically to infect each punched area. Twenty-four hours after infection, three agents (ABPS, 2 mg/ml, ABPS 4 mg/ml, tetramethoxyl NDGA, 4N 60 mg/ml and acyclovir 60 mg/ml) were applied to the infected areas five times per day for five days. Picture(FIG. 8) was taken 96 hours following infection.

Comparing with the two positive controls (HSV-SC, HSV-C, i.e. infection without drug treatment), the result indicated that agent ABPS is ineffective while tetramethoxyl NDGA and acyclovir are effective in suppression of HSV-1 replication. Differing from acyclovir, however, tetramethoxyl NDGA has the advantage in that it is a mutation insensitive drug (Table 5A and Table 5B).

TABLE-US-00004 TABLE 4 Tetramethoxyl NDGA Effect Concentration of (CH.sub.3O).sub.4 NDGA .mu.M % Inhibition IC.sub.50 I. Inhibition of HSV-1 Replication in Vero Cells 14 97.50 7.7 .mu.M 7 40 3.5 20 1.75 <0 0.875 <0 0.437 <0 II. Inhibition of HSV-1 ICP4 Transactivation in Vero Cells 80 78 33 .mu.M 70 68 60 64 50 52 40 35 30 30 20 8 III. Inhibition of Growth of Vero Cells 280 79.62 142 .mu.M 140 43.95 70 9.55 35 7.64 17.5 7.64 8.75 3.18 4.37 1.27

TABLE-US-00005 TABLE 5 Comparison Between the Drug Sensitivities of Acyclovir and Tetramethoxyl NDGA Against HSV-1 in Vero Cells A. IC.sub.50 at different passages IC50 .mu.M Passage ACV [CH.sub.3].sub.4 NDGA 1 7.5 11.7 2 37.8 4.4 3 >88.8 8.24 138.4 5.9 5 >220 9.9 10 440 10

Virus which recovered from medium of each passage at IC.sub.50 drug concentration were used to infect the subsequent passage of the cells.

TABLE-US-00006 B. Drug sensitivities of tetramethoxyl NDGA against ACV sensitive or ACV resistant strain of HSV-1 HSV-Sm44-ACVg HSV-18m44-CV4r Drug (IC50) (.mu.M) (IC50) ACV <6.9 117.6 [CH.sub.3].sub.4 NDGA 6.9 6.9

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on thecontrary is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Thus, it is to be understood that variations in the derivatives of NDGA and the method of suppression of Tat transactivation can be made without departing from the novel aspects of this invention as defined in the claims.

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